Helicobacter pylori : physiology and genetics 9781555812133, 1555812139, 9781555818005, 1555818005

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Helicobacterpylori P H Y S I O L O G Y AND GENETICS

Helicobacterpylori PHYSIOLOGY AND GENETICS

Edited by

Harry L.T. Mobley Department of Microbiology and Immunology University of Maryland School of Medicine Baltimore, Maryland

George L. Mendz School of Biochemistry and Molecular Genetics University of New South Wales Sydney, Australia

Stuart L. Hazell Faculty of Sciences University of Southern Queensland Toowoomba, Queensland Australia

ASM PRESS Washington, DC

Copyright © 2 0 0 1 ASM Press American Society for Microbiology 1 7 5 2 N Street N W Washington, DC 2 0 0 3 6 Library of Congress Cataloging-in-Publication Data Helicobacter pylori: physiology and genetics / edited by Harry L. T. Mobley, George L. Mendz, Stuart L. Hazell. p. cm. Includes bibliographical references and index. ISBN 1-55581-213-9 1. Helicobacter pylori infections. 2. Helicobacter pylori. I. Mobley, Harry L. T. II. Mendz, George L. HI. Hazell, Stuart L. QR201.H44 H465 2001 616'.01423—dc21 All Rights Reserved Printed in the United States of

2001022395

America

Cover photo: S-shaped Helicobacter pylori (see chapter 6). Photo courtesy of L. Thompson. Inset: Electron micrograph of negatively stained cells of H. pylori (see chapter 7). Photo courtesy of Paul O'Toole and Michael Lane, Massey University, and Doug Hopcroft, Hort Research, Palmerston, North New Zealand.

CONTENTS

Contributors • Preface • xv Acknowledgments

I.

9. Vacuolating Cytotoxin • 97 John C. Atherton, Timothy L. Cover, Emanuele Papini, and John L. Telford

ix •

xvii

Introduction HI. Energy Metabolism and Synthetic Pathways

1. Overview • 3 Harry L. T. Mobley, George L. Mendz, and Stuart L. Hazell 2. Epidemiology of Infection Hazel M. Mitchell

•

3. One Hundred Years of Discovery and Rediscovery of Helicobacter pylori and Its Association with Peptic Ulcer Disease • Barry J . Marshall

II.

10. Microaerobic Physiology: Aerobic Respiration, Anaerobic Respiration, and Carbon Dioxide Metabolism • 113 David J. Kelly, Nicky J . Hughes, and Robert K. Poole

7

19 11. Nitrogen Metabolism Hilde De Reuse and Stephane

Bacteriology

12. The Citric Acid Cycle and Fatty Acid Biosynthesis • 135 David J. Kelly and Nicky J . Hughes

4. Basic Bacteriology and Culture • 27 Lief Percival Andersen and Torkel Wadstrbm

13. Nucleotide Metabolism George L. Mendz

5.

Taxonomy of the Helicobacter Genus 39 Jay V. Solnick and Peter Vandamme 6. Morphology and Ultrastructure Jani O'Rourke and Giinter Bode 7. Cell Envelope • 69 Paul W. O'Toole and Marguerite

•

• 125 Skouloubris

•

147

14. Biosynthetic Pathways Related to Cell Structure and Function • 159 Partha Krishnamurthy, Suhas H. Phadnis, Cindy R. DeLoney, Raoul S. Rosenthal, and Bruce E. Dunn

53

Clyne

15.

Evasion of the Toxic Effects of Oxygen 167 Stuart L. Hazell, Andrew G. Harris, and Mark A. Trend

8. Molecular Structure, Biosynthesis, and Pathogenic Roles of Lipopolysaccharides 81 Anthony P. Moran v

vi

CONTENTS

IV. Physiology and Molecular Biology

29. Gene Regulation Nicolette de Vries, Arnoud Johannes G. Kusters

16. Urease • Harry L. T. Mobley

30. Mutagenesis • 335 Agnes Labigne and Peter J . Jenks

179

17.

Ion Metabolism and Transport 193 Arnoud H. M. van Vliet, Stefan Bereswill, Johannes G. Kusters

and

18. Metabolite Transport • 207 Brendan P. Burns and George L. Mendz 19. Protein Export Dag liver, Rino Rappuoli,

• 219 and John L.

Telford

20. Alternative Mechanisms of Protein Release • 227 Steven R. Blanke and Dan Ye

• 321 H. M. van Vliet, and

31. The cag Pathogenicity Island Markus Stein, Rino Rappuoli, and Antonello Covacci 32. Population Genetics Sebastian Suerbaum and Mark

•

• 355 Achtman

33. Heterogeneity and Subtyping • 363 Robert J . Owen, Diane E. Taylor, Ge Wang, and Leen-Jan van Doom

VI. Bacterial Virulence and Pathogenic Mechanisms

21.

Motility, Chemotaxis, and Flagella 239 Gunther Spohn and Vincenzo Scarlato

34. Adherence and Colonization Traci L. Testerman, David J . McGee, and Harry L. T. Mobley

22. Natural Transformation, Recombination, and Repair • 249 Wolfgang Fischer, Dirk Hofreuter, and Rainer Haas

35.

Lipopolysaccharide Lewis Antigens 419 Ben J . Appelmelk and Christina M. J . E. Vandenbroucke-Grauls

23. Chromosomal Replication, Plasmid Replication, and Cell Division • 259 Hiroaki Takeuchi and Teruko Nakazawa

36. Gastric Autoimmunity • 429 Mathijs P. Bergman, Gerhard Poller, Mario M. D'Elios, Gianfranco Del Prete, Christina M. J . E. Vandenbroucke-Grauls, and Ben J . Appelmelk

24.

Restriction and Modification Systems 269 John P. Donahue and Richard M. Peek, Jr.

37. Vaccines • 441 Jacques Pappo, Steven Czinn, and John

•

VII. Pathogenesis in the Host, Diagnosis, and Treatment

26. Transcription and Translation • 285 Sanjib Bhattacharyya, Mae P. Go, Bruce E. Dunn, and Suhas H. Phadnis

38. Pathology of Gastritis and Peptic Ulceration • 459 Michael F. Dixon

V.

39. Host Inflammatory Response to Infection • 471 Jide Wang, Thomas G. Blanchard, and Peter B. Ernst

27. The Genome • Richard A. Aim and Brian 28. Genetic Exchange Dawn A. Israel

295 Noonan •

313

381

Nedrud

25. Regulation of Urease for Acid Habitation • 277 George Sachs, David R. Scott, David L. Weeks, Marina Rektorschek, and Klaus Melchers

Genetics

345

40. Gastric Cancer • 481 Masahiro Asaka, Antonia R. Sepulveda, Toshiro Sugiyama, and David Y. Graham

CONTENTS

41. Markers of Infection • 499 David Y. Graham and Waqar A. Qureshi 42.

Antibiotic Susceptibility and Resistance 511 Francis Megraud, Stuart Hazell, and Youri Glupczynski

VIII. Animal Models and Other Helicobacter Species 43.

Enterohepatic Helicobacter 533 David B. Schauer

Species

44. Other Gastric Helicobacters and Spiral Organisms • 549 Stephen J . Danon and Adrian Lee 45. In Vivo Modeling of HelicobacterAssociated Gastrointestinal Diseases • Richard L. Ferrero and James G. Fox 46. 583

In Vivo Adaptation to the Host

Richard L. Ferrero and Peter J . Jenks Index

•

593

565

•

vii

CONTRIBUTORS

Mark Achtman Max-Planck Institut fur Infektionsbiologie, Berlin, Germany

Thomas G. Blanchard Department of Pediatrics, Rainbow Babies and Children's Hospital, Case Western Reserve University School of Medicine, Cleveland, OH 44106

Richard A. Aim Infection Discovery, AstraZeneca R & D Boston, Waltham, MA 02451

Steven R. Blanke Department of Biology and Biochemistry, University of Houston, Houston, T X 7 7 2 0 4

LiefPercival Andersen Department of Clinical Microbiology, National University Hospital, Copenhagen, Denmark

Giinter Bode Department of Epidemiology, University of Ulm, Ulm, Germany

Ben Appelmelk Department of Medical Microbiology, Vrije Universiteit Medical School, Amsterdam, The Netherlands

Brendan P. Burns Max von Petennkofer Institut fur Hygiene und Medizinische Mikrobiologie, Munich, Germany

Masahiro Asaka Department of Gastroenterology, Hokkaido University Graduate School of Medicine, Sapporo, Japan

Marguerite Clyne The Childrens Research Centre, Our Lady's Hospital for Sick Children, Crumlin, Dublin, Ireland

John C. Atherton Division of Gastroenterology and Institute of Infections and Immunity, University of Nottingham, Nottingham, United Kingdom

Antonello Covacci IRIS-Chiron SpA, Siena, Italy

Stefan Bereswill Department of Microbiology, Institute of Medical Microbiology and Hygiene, University of Freiburg, Freiburg, Germany

Timothy L. Cover Division of Infectious Diseases, Vanderbilt University, and Veterans Affairs Medical Center, Nashville, TN

Mathijs P. Bergman Department of Medical Microbiology, Vrije Universiteit Medical School, Amsterdam, The Netherlands

Steven Czinn Division of Gastroenterology, Case Western Reserve University, Cleveland, OH 4 4 1 0 6

Sanjib Bhattacharyya Departments of Pathology, Medical College of Wisconsin, Milwaukee, WI 5 3 2 2 6

Stephen J. Danon School of Microbiology and Immunology, University of New South Wales, Sydney, Australia ix

x

CONTRIBUTORS

Mario M. D'Elios Department of Internal Medicine and Immunoallergology, University of Florence, Florence, Italy

Wolfgang Fischer Department of Bacteriology, M a x von Pettenkofer Institut fur Hygiene und Medizinische Mikrobiologie, Munich, Germany

Cindy R. DeLoney Division of Biomedical Sciences, University of California, Riverside, CA 9 2 5 2 1

James G. Fox Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA 02139

Gianfranco Del Prete Department of Internal Medicine and Immunoallergology, University of Florence, Florence, Italy Hilde De Reuse Unite de Pathogenie Bacterienne des Muqueuses, Institut Pasteur, Paris, France Nicolette de Vries Departments of Gastroenterology and Medical Microbiology, Faculty of Medicine, Vrije Universiteit, Amsterdam, The Netherlands Michael F. Dixon Academic Unit of Pathology, University of Leeds, Leeds, United Kingdom John P. Donahue Division of Infectious Diseases, Department of Medicine, Vanderbilt University School of Medicine, Nashville, T N 3 7 2 3 2 Bruce E. Dunn Departments of Pathology, Medical College of Wisconsin, Milwaukee, WI 5 3 2 2 6 , and Pathology and Laboratory Medicine Service, Department of Veterans Affairs Medical Center, Milwaukee WI 53295 Peter B. Ernst Departments of Pediatrics and Microbiology and Immunology and The Sealy Center for Molecular Sciences, University of Texas Medical Branch, Galveston, T X 7 7 5 5 5 Gerhard Poller Department of Pathology, University Erlangen, Erlangen, Germany

Youri Glupczynski Laboratoire de Microbiologic, Clinique Universitaire de Mont-Godinne, Yvoir, Belgium Mae F. Go Gastrointestinal Section, Veterans Administration Health Care System, and University of Utah School of Medicine, Salt Lake City, UT 84148 David Y. Graham Baylor College of Medicine, Houston, T X , and Digestive Disease Section, Veterans Administration Medical Center, Houston, T X 77030 Rainer Haas Department of Bacteriology, M a x von Pettenkofer Institut fur Hygiene und Medizinische Mikrobiologie, Munich, Germany Andrew G. Harris School of Science, Food and Horticulture, College of Science, Technology and Environment, University of Western Sydney, Campbelltown, Australia Stuart L. Hazell Faculty of Sciences, University of Southern Queensland, Toowoomba, Queensland 4 3 5 0 , Australia Dirk Hofreuter Department of Bacteriology, M a x von Pettenkofer Institut fur Hygiene und Medizinische Mikrobiologie, Munich, Germany Nicky J. Hughes SmithKline Beecham Pharmaceuticals Research and Development Ltd, Anti-infectives Research, Collegeville, PA 19426 Dag liver IRIS, Chiron SpA, Siena, Italy

Richard L. Ferrero Unite de Pathogenie Bacterienne des Muqueuses, Institute Pasteur, Paris, France

Dawn A. Israel Division of Gastroenterology, Vanderbilt University School of Medicine, Nashville, T N 3 7 2 3 2

CONTRIBUTORS

Peter J. Jenks Institute of Infections and Immunity, University Hospital, Queen's Medical Centre, Nottingham NG7 2NH, United Kingdom David J. Kelly Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield, United Kingdom Partha Krisbnamurthy Departments of Pathology, Medical College of Wisconsin, Milwaukee WI 53226

xi

Anthony P. Moran Department of Microbiology, National University of Ireland Galway, Galway, Ireland Teruko Nakazawa Department of Microbiology, Yamaguchi University School of Medicine, Ube, Yamaguchi, Japan John Nedrud Department of Pathology, Case Western Reserve University, Cleveland, OH 4 4 1 0 6 Brian Noonan Infection Discovery, AstraZeneca R & D Boston, Waltham, MA 02451

Johannes G. Kusters Department of Medical Microbiology, Faculty of Medicine, Vrije Universiteit, Amsterdam, The Netherlands

Jani O'Rourke School of Microbiology and Immunology, University of New South Wales, Sydney, Australia

Agnes Labigne Unite de Pathogenie Bacterienne des Mugueuses, INSERM U389, Institut Pasteur, Paris, France

Paul W. O'Toole Institute of Molecular Biosciences, Massey University, Palmerston North, New Zealand

Adrian Lee

Robert J. Owen Helicobacter Reference Unit, Laboratory of Enteric Pathogens, Central Public Health Laboratory, London, United Kingdom

University of New South Wales, Sydney, Australia Barry J. Marshall Department of Microbiology, University of Western Australia, Nedlands, Western Australia David J. McGee Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland 2 1 2 0 1 Francis Megraud Laboratoire de Bacteriologie, Hopital Pellegrin, Bordeaux, France Klaus Melchers Byk Gulden, Konstanz, Germany George L. Mendz

Emanuele Papini Department of Biomedical Science and Human Oncology, Section of General Pathology, University of Bari, Bari, Italy Jacques Pappo Department of Immunology, Infection Discovery, AstraZeneca R & D Boston, Waltham, MA 02451 Richard M. Peek, Jr. Division of Gastroenterology, Department of Medicine, Vanderbilt University School of Medicine, Nashville, T N 3 7 2 3 2 , and Medical Service, Department of Veterans Affairs Medical Center, Nashville, T N 3 7 2 1 2

School of Biochemistry and Molecular Genetics, University of New South Wales, Sydney, Australia Hazel M. Mitchell School of Microbiology and Immunology, University of New South Wales, Sydney, Australia

Suhas H. Phadnis Departments of Pathology, Medical College of Wisconsin, Milwaukee, WI 5 3 2 2 6 , and Pathology and Laboratory Medicine Service, Department of Veterans Affairs Medical Center, Milwaukee WI 53295

Harry L. T. Mobley Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland 2 1 2 0 1

Robert K. Poole Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield, United Kingdom

xii

CONTRIBUTORS

Waqar A. Qureshi Baylor College of Medicine, Houston, T X , and Veterans Administration Medical Center, Houston, T X 77030 Rino Rappuoli IRIS, Chiron SpA, Siena, Italy Marina Rektorscbek Byk Gulden, Konstanz, Germany Raoul S. Rosenthal Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN 4 6 2 0 2 George Sachs University of California, Los Angeles, CA 90073 Vincenzo

Sebastian Suerbaum Institute of Hygiene and Microbiology, University of Wuerzburg, Wuerzburg, Germany Toshiro Sugiyama Department of Gastroenterology, Hokkaido University Graduate School of Medicine, Sapporo, Japan Hiroaki Takeuchi Department of Microbiology, Yamaguchi University School of Medicine, Ube, Yamaguchi, Japan Diane E. Taylor Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada John L. Telford IRIS, Chiron SpA., Siena, Italy

Scarlato

Department of Molecular Biology, IRIS, Chiron SpA., Siena, and Department of Biology, University of Bologna, Bologna, Italy David B. Schauer Division of Bioengineering and Environmental Health and Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA 0 2 1 3 9 David R. Scott University of California, Los Angeles, CA 90073

Tract L. Testerman Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland 2 1 2 0 1 Mark A. Trend School of Science, Food and Horticulture, College of Science, Technology and Environment, University of Western Sydney, Campbelltown, Australia

Antonia R. Sepulveda

Peter Vandamme Laboratoorium voor Microbiologic, Universiteit Gent, Gent, Belgium

Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, PA 15213 Stephane Skouloubris Unite de Pathogenie Bacterienne des Muqueuses, Institut Pasteur, Paris, France

Christina M. J. E. Vandenbroucke-Grauls Department of Medical Microbiology, Vrije Universiteit Medical School, Amsterdam, The Netherlands

Jay V. Solnick Department of Internal Medicine, Division of Infectious Diseases, and Department of Medical Microbiology and Immunology, University of California, Davis, CA 9 5 6 1 6 Gunther Spohn Department of Molecular Biology, IRIS, Chiron SpA., Siena, Italy Markus Stein IRIS-Chiron SpA, Siena, Italy

Leen-Jan van Doom Delft Diagnostic Laboratory, Delft, The Netherlands Arnoud H. M. van Vliet Departments of Gastroenterology and Medical Microbiology, Faculty of Medicine, Vrije Universiteit, Amsterdam, The Netherlands Torkel Wadstrom Department of Infectious Diseases and Medical Microbiology, University of Lund, Sweden

CONTRIBUTORS

Ge Wang Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada

David L. Weeks University of California, Los Angeles, CA 90073

Jide Wang Department of Pediatrics, University of Texas Medical Branch, Galveston, T X 7 7 5 5 5

Dan Ye Department of Biology and Biochemistry, University of Houston, Houston, T X 7 7 2 0 4

xiii

PREFACE

volumes of that book, the world literature on these related genera is beautifully summarized in what has become a frequently used reference. Although the text we proposed was smaller in size, we attempted to pro­ vide a similar comprehensive treatise. It was instructive for us to assemble a table of contents for the proposed book. We had to determine what was known to be fact from what was known only from anecdotal evidence. After long discussions and the addition and deletion of sections, we sought the experts to write the chapters. To our delight, all but very few colleagues were enthusiastic about par­ ticipating; thus, each chapter is authored by scientists who are active in their respective areas of expertise. It is not often in medical microbiology that an entirely new bacterial genus is discovered, as was the case with Helicobacter and its type species, H. pylori. The culture and study of this organism triggered a revolution in the treatment of gastritis and peptic ul­ ceration. Indeed, H. pylori is a model pathogen. The results of investigations on this bacterium, which is exquisitely well adapted for a lifelong colonization of the gastric mucosa of humans, revolutionized our understanding of upper gastrointestinal tract disease. The lessons we have learned about how it colonizes the gastric mucosa, avoids the immune response, and damages the host have been applied to the study of other mucosal pathogens. H. pylori was the first bacterial species for which the sequences of two complete genomes were deter­ mined and annotated. We reasoned that rapid devel­ opments in the understanding of H. pylori would re­ sult from the information derived from the DNA sequences. Thus, for researchers in specific fields, an up-to-date and comprehensive reference would be particularly valuable for placing new findings in the context of the overall knowledge of H. pylori and for stimulating original work in areas which had escaped proper attention.

In 1998, during a break at the Xlth International Workshop on Gastroduodenal Pathology and Helico­ bacter pylori in Budapest, Hungary, we sat in the back of the empty main auditorium at the Congress Center. The back doors opened out onto a concourse filled with row after row of posters on every possible H. pylori topic. We reflected that research on H. pylori had been truly global, more than for most organisms, and that its impact had been felt in every country. The bacterium was discovered in Australia, and much important work had been done "Down Under" and in the United States, Europe, and elsewhere. The workshop had been a forum for the announcement of recent salient discoveries, in the tradition of the annual European meetings which started 10 years be­ fore in Bordeaux, France. We noted that much experi­ mentation had been done and that many new concepts had been proposed. We also noted that "the wheel had been reinvented" a number of times, highlighting the need for a state-of-the-art reference that summa­ rized the field. At that moment, we decided with a handshake to approach ASM Press with the idea for a comprehensive text on H. pylori. The book would summarize and review the accumulated knowledge on this important human pathogen. The vast body of literature on this bacterium that has appeared over the last two decades is almost un­ paralleled in bacterial pathogenesis (see Fig. 1 of chap­ ter 1). In part, this was due to the interest of large numbers of investigators from many disciplines, in­ cluding bacteriologists, gastroenterologists, infectious diseases specialists, cancer biologists, epidemiologists, pathologists, and those in the pharmaceutical indus­ try. This body of knowledge needed a critical review that was comprehensive and systematic. We decided to use as a model the excellent reference Escherichia coli and Salmonella: Cellular and Molecular Biology, edited by F. C. Neidhardt and colleagues. In the two

X V

xvi

PREFACE

Why is this book different from other books al­ ready published on H. pylori? Most prior volumes concentrated on clinical or other special issues, and many are now out of date. While important findings about the physiology and genetics of H. pylori are summarized in this volume, it also includes sections on epidemiology, bacteriology, bacterial virulence and pathogenic mechanisms, pathogenesis in the host, diagnosis and treatment, animal models, and other

Helicobacter species. This book will have been pub­ lished less than six months after receipt of the final chapter and thus is up to date. We hope you enjoy reading it. Harry L.T. Mobley George L. Mendz Stuart L. Hazell February 2 0 0 1

ACKNOWLEDGMENTS

I thank Professor R. J . Doyle for teaching me the craft. H.L.T.M. I thank Emeritus Professor W. J . O'Sullivan who first drew my attention to this most intriguing organism and the present and past members of the Microbial Physiology Laboratory whose contributions made this work possible. G.L.M. I thank Professor Adrian Lee for his introduction into the word of "spiral bugs" and Darlene Williams for her valauble assistance on this book. S.L.H.

xvii

I. INTRODUCTION

Helicobacter pylori: Physiology and Genetics Edited by H. L. T. Mobley, G. L. Mendz, and S. L. Hazell © 2 0 0 1 ASM Press, Washington, D.C.

Chapter 1

Overview HARRY L . T . MOBLEY, GEORGE L . MENDZ, AND STUART L . HAZELL

more articles published on Helicobacter than on Sal­ monella and Bacillus, and the number of studies pub­ lished was comparable to those on Staphylococcus and Mycobacterium, which were behind only Esche­ richia coli, the most cited bacterial species. In retrospect, it is interesting to note that there were many references to the presence of H. pylori in the gastric mucosa before its culture by Marshall and Warren in 1 9 8 2 . Spiral-shaped bacteria were noted many times in the literature, but their presence was not properly correlated with gastroduodenal disease. After the successful culture of H. pylori, numerous investigators studied the epidemiology of transmis­ sion of the organism. Although all the factors have not been identified, it is safe to say that acquisition is most likely to occur at a young age and occurs more frequently in developing countries as opposed to de­ veloped countries.

Helicobacter pylori has been the subject of intense investigation since its culture from a gastric biopsy in 1982. From the beginning, this gram-negative bacter­ ium has provoked the interest of bacteriologists, gastroenterologists, infectious disease specialists, cancer biologists, epidemiologists, pathologists, and phar­ maceutical scientists. The possibility that a bacterium could cause gastritis, peptic ulcers, and, over time, cancer was a concept that was difficult to put forward. To convince colleagues and the public, Barry Mar­ shall drank a suspension of the bacterium and proved Koch's postulates for gastritis and made the idea that H. pylori is the etiologic agent of many gastric mala­ dies more easy to swallow. Owing to the unique characteristics of H. pylori, such as its microaerophily, nitrogen metabolism, and ecological niche, a sound understanding of its physiol­ ogy and genetics is of interest to fundamental and ap­ plied microbiology, taxonomy, molecular biology, microbial ecology, and medical, veterinary, and agri­ cultural microbiology. The wide interest from many disciplines has resulted in a steady increase in research on the bacterium. To quantify this interest, it is enough to look at the number of citations on the sub­ ject. Keeping in mind that the organism was first named Campylobacter pyloridis and then Campylo­ bacter pylori before taking its present moniker of Hel­ icobacter pylori in 1989, the literature bears evidence of the interest in Helicobacter from the frequency of research articles that have appeared in the scientific literature (Figure 1). The number of articles recovered from Medline by year using the keywords "Helico­ bacter" or "Campylobacter pylori" or "Campylo­ bacter pyloridis" shows that there has been a steadily increasing interest in Helicobacter from its discovery to the present day. From 1997 to 2 0 0 0 , there were

The bacteriology of this microaerophilic spiralshaped bacterium is fascinating. H. pylori is a member of a rapidly growing genus. New species are being isolated at a fast rate from many vertebrate hosts. Also, other Helicobacter species are being isolated from nongastric sites in humans and may be impli­ cated in diseases that previously had no assigned etio­ logic agent. H. pylori is motile via a tuft of polarsheathed flagella; these structures also carry a termi­ nal bulb, which perhaps makes it more adapted to swimming through mucus. Also, on the surface, the lipopolysaccharide has unique biological properties and the genes that control addition of the O-side chains can phase vary, a mechanism for avoidance of host responses. In addition, it has a unique peptidoglycan structure that differs from other gram-negative bacteria. The organism also secretes an autotransported vacuolating cytotoxin that exerts the unusual phenotype of vacuolation in host cells.

Harry L. T. Mobley • Department of Microbiology and Immunology, University of Maryland School of Medicine, 655 W. Baltimore St., Baltimore, MD 21201. George L. Mendz • School of Biochemistry and Molecular Genetics, The University of New South Wales, Sydney NSW 2052, Australia. Stuart L. Hazell • Faculty of Informatics, Science and Technology, University of Western Sydney, MacArthur, Campbelltown NSW 2560, Australia.

3

4

MOBLEY ET AL.

2000-j

-i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—r 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99

Year Figure 1. Helicobacter-related articles cited in Medline since the culture of H. pylori. The Medline database was searched by year for "Helicobacter" or "Campylobacter pylori" or "Campylobacter pyloridis." The total number of cited articles for all three categories is shown by year. In 1999, the last year for which a complete data­ base exists, 2,091 articles appeared on these topics.

Prior to the sequencing and annotation of the genomes of H. pylori strains 2 6 6 9 5 and J 9 9 , a large number of studies had elucidated central metabolic pathways, uptake and regulatory systems, responses to various stresses, and virulence factors. Neverthe­ less, the publication of the genomes has had a marked impact on our knowledge of the bacterium, and the data derived from these sequences have served to con­ firm experimental results, to provide insights into the biology of the bacterium, to deepen our understand­ ing of its diversity, and to suggest new areas of investi­ gation. Thus, it is not surprising that many chapters of this book discuss in detail the results of genomic analyses. The chapters on microaerobic physiology, nitro­ gen metabolism, and the citric acid cycle show excel­ lent correlations between the results of experimental investigations and genomic data. These chapters also illustrate eloquently how both approaches comple­ ment and support one another. Insights into the biol­ ogy of the bacterium are brought to light in the chap­ ters dealing with oxidative stress, urease, motility, chemotaxis and flagella, and the regulation of urease for acid habitation. Cogent explanations of the adap­ tation of H. pylori to the gastric environment and the regulation of its physiology by environmental factors are given for a substantial body of experimental knowledge by placing it in the framework of the ge­ nome of the organism. Survival and proliferation de­ pend intrinsically on the flux of nutrients. The chap­ ters on ion metabolism and transport, and metabolite

uptake show the considerable progress that has been made in understanding these processes in H. pylori. Importantly, the genomic data in these chapters also illuminate areas that still require exploration. The chapter on transcription and translation demonstrates the universality of some of the regula­ tion mechanisms present in H. pylori and, at the same time, shows important differences with other enterobacteria. The diversity of H. pylori is made clear also in the discussion of pathways related to cell structure and function, which relates its unique murein with the ability of the bacterium to colonize its niche. Many new areas of investigation are proposed in the book, and the chapters on protein secretion and alternative mechanisms of secretion describe lucidly that bacte­ rial protein secretion remains a fertile area of research. They point out specific adaptations of secretory path­ ways by H. pylori and suggest that protein secretion is a mechanism used by the bacterium to remodel its environment. The many similarities between H. pylori and archaeal enzymes, and the fact that genes encoding proteins that are part of operons in other proteobacteria are found at different loci in the H. pylori genome as discussed in the chapter on nucleotide metabolism, are two interesting characteristics of the bacterium that have emerged from metabolic experiments and genomic analyses and still lack proper explanations. Finally, there are areas of our current knowledge of the bacterium that depend strongly on genome analyses. This situation is well exemplified by the chapters addressing natural transformation, recombi­ nation and repair, restriction and modification sys­ tems, and replication and cell division. In contrast to these examples of the strong contri­ bution of genomics to understanding the physiology and genetics of H. pylori, it is important also to con­ sider that genome data provide very limited informa­ tion on topics such as protein posttranslational modi­ fication, structure, and subcellular location. Moreover, incomplete functional identification of genes encoding for enzymes of pathways for which there is experimental evidence, for example, the urea cycle and the de novo purine biosynthesis, emphasizes the need to exercise caution when attempting to re­ construct metabolic and regulatory networks from ge­ nome data. This naturally competent, transformable bacter­ ium was the first species for which two complete ge­ nome sequences were made available. The genome size of ~ 1 . 7 M b revealed a profile of an organism that was fine-tuned for its niche in the gastric mucosa, lacking many of the regulatory features found in the larger E. coli genome. Indeed, clever forms of regula­ tion such as the extensive use of slipped strand mis-

CHAPTER 1 • OVERVIEW

pairing allow the organism to present many faces to the host in terms of expression of outer membrane proteins and other surface structures. Numerous re­ striction-modification systems are present in this spe­ cies, but they differ between the two genomes ana­ lyzed. The presence of the cag pathogenicity island was identified prior to the sequencing of the whole genome, revealing a 40-kb stretch of DNA whose presence correlates with more virulent isolates. Most of the traditional protein secretion systems are used by H. pylori, including ABC transporters, sec-dependent (leader peptide) transport, flagellar assembly (a proto­ type of the type III secretion system), type IV homologs in the pathogenicity island, and autotransporters such as VacA. Mutagenesis is straightforward, and it is relatively easy to construct a double-crossover alle­ lic exchange mutant. But other genetic tools are lack­ ing, such as conjugation, transduction, and the ability to introduce transposons directly. Interestingly, the organism displays a great deal of heterogeneity with respect to nucleotide sequence. These differences, which are due to their ability to freely recombine, have been used as epidemiological tools to identify specific strains. Indeed, the population can be described as almost aclonal. H. pylori can colonize its human host for life. It is therefore well adapted for life in the stomach. While every H. pylori strain elicits at least some inflamma­ tion in the host, the organism must strike a balance so as not to provoke an immune response vigorous enough to clear itself from the host. Indeed, the bac­ terium has developed various strategies including mo­ lecular mimicry and a battery of adhesins to avoid clearance by the immune response. Since the host usu­ ally does not clear the organism, considerable efforts have been made to develop an oral vaccine to either prevent infection or eradicate an established infection. An added benefit to these efforts has been the develop­ ment of an understanding of the immune response in the stomach, a field that attracted little attention in the past.

5

Together with our understanding of the organ­ ism, much has been learned about the host response to H. pylori. Indeed, pathologists were among the first groups of scientists to reevaluate their data in the con­ text of the newly discovered bacterial etiological agent. Chronic inflammation elicited by the bacterium provided the missing link in the progression to gastric carcinoma; accordingly, H. pylori was named as a class 1 carcinogen by the World Health Organization. This fact provided a strong rationale to treat all who tested positive for H. pylori. Antibiotic regimens have been largely successful, but some agents such as met­ ronidazole and clarithromycin have been rendered in­ effective in several countries and geographical areas of the United States by the emergence of strains resis­ tant to these compounds. The mechanisms of resis­ tance have, in some cases, been worked out in part. From the beginning, animal models of infection have been useful in characterizing H. pylori virulence determinants, such as urease and flagella. Other spe­ cies of helicobacters, such as H. felis and H. mustelae, were employed as proxies for H. pylori in the mouse and ferret, respectively. The isolation of the Sydney strain of H. pylori (SSI), which could reproducibly infect mice, was a boon to the study of vaccines and additional virulence factors and vaccine development. In summary, we are approaching 2 0 years of re­ search on a bacterium proven to be the cause of gastri­ tis and indisputably correlated with the development of peptic ulcers and the progression to cancer. Pres­ ently, over 12,000 articles have been published on "Helicobacter," not counting articles under the previ­ ous classification of "Campylobacter." Researchers have established many important facts about H. py­ lori and are hot on the trail of many others. In this book, we have attempted to summarize the body of knowledge about this species. Internationally recog­ nized scientists, many of whom have made salient dis­ coveries, have summarized their respective topics. For these reasons, the editors are pleased to present the state-of-the-art knowledge in one volume.

Helicobacter pylori: Physiology and Genetics Edited by H. L. T. Mobley, G. L. Mendz, and S. L. Hazell © 2 0 0 1 ASM Press, Washington, D.C.

Chapter 2

Epidemiology of Infection HAZEL M .

MITCHELL

In 1982 when Barry Marshall and Robyn Warren first isolated the gastric pathogen Campylobacter pylo­ ridis, few if any gastroenterologists would have pre­ dicted that almost 2 0 years later, this bacterium would have been shown to be one of the most common bacte­ rial infections in humans and the etiologic agent of the majority of upper gastroduodenal disease. Today, Hel­ icobacter pylori, as it is now known, is firmly estab­ lished as the etiologic agent of acute or chronic gastritis and a predisposing factor in peptic ulcer disease, gastric carcinoma, and B-cell mucosa-associated lymphoid tissue (MALT) lymphoma ( 4 7 , 5 9 , 87, 111). Although over the past 18 years many important questions relating to the epidemiology of H. pylori have been defined, a number of issues, including the route of transmission of H. pylori, remain controver­ sial. This chapter aims not only to provide the reader with the most recent data in regard to the epidemiol­ ogy of H. pylori but also to review current areas of controversy.

to the rate of acquisition of H. pylori under the age of 10 years; the prevalence of infection in Australian children is 4 % in comparison with 2 7 % in Chinese children. Over the age of 10 years, however, the rate of acquisition of infection in both countries was simi­ lar (approximately 1% per annum) (99). Epidemiol­ ogical data from other developed and developing countries support this finding, with the prevalence of H. pylori infection in children under 10 years resident in developed countries being approximately 0 to 5 % compared with 13 to 6 0 % in children resident in de­ veloping countries. Over this age an increase in preva­ lence in the order of 0.5 to 2 % per annum is com­ monly observed (2, 4 5 , 6 0 , 9 0 , 114). It has been proposed that the increasing prevalence of H. pylori from younger to older subjects reflects the passage through the population of distinct cohorts. That is, all persons are infected in childhood and the decreased levels of H. pylori infection associated with younger age groups, particularly in developed countries, are due to gradual improvements in medical care, sanita­ tion, and/or living conditions (8, 2 3 , 1 2 1 , 133). In contrast to this view, a number of studies have argued that there is a continuous risk of acquisition of H. pylori of approximately 1% per year in adulthood ( 2 4 , 1 4 3 ) . Clarification of this issue will require large cohort studies that monitor the H. pylori status of approximately 1,000 subjects over a 5-year period. Given an acquisition rate of 0.5 to 2 % per annum, at the end of this period it would be expected that 25 to 100 subjects would have seroconverted (92).

PREVALENCE OF INFECTION H. pylori infection is ubiquitous and infects both males and females (42, 7 8 , 9 0 , 9 9 , 150). Although infection occurs worldwide, there are significant dif­ ferences in the prevalence of infection both within and between countries (42, 7 8 , 9 0 , 9 9 , 150). In general, the overall prevalence of H. pylori infection in devel­ oped countries is lower than that in developing coun­ tries (9, 4 5 , 9 9 , 1 1 4 ) . This difference in prevalence of infection has been attributed to the rate of acquisition of H. pylori in childhood (99). For example, in a study conducted in southern China, the overall prevalence of H. pylori infection in Chinese subjects was shown to be significantly higher than that in Australians (44.2 versus 2 1 % ) . Examination of the data for agerelated prevalence showed that this difference related Hazel M. Mitchell

NATURAL H I S T O R Y O F INFECTION Natural acquisition of H. pylori infection occurs, for the most part, in childhood. Once established within the gastric mucosa, the bacterium persists for life. Studies in children suggest, however, that in the

• School of Microbiology and Immunology, The University of New South Wales, Sydney 2052, Australia.

7

8

MITCHELL

early years of life prior to the establishment of infec­ tion, transient infection with H. pylori may be com­ mon. This is evidenced both by prevalence studies and a number of follow-up studies that have monitored H. pylori prevalence in the same children over a num­ ber of years (18, 4 9 , 6 4 , 82, 1 2 0 , 137). One of the first studies to suggest that loss of infection may occur in children was by Klein et al., who showed that 6month-old Peruvian children monitored for their H. pylori status at 6-month intervals over a 2-year period had an overall probability of acquiring H. pylori of between 0.28 and 0.38, and a probability of clearing the infection of between 0.22 and 0.45 in a given 6month period (64). Similar findings have been re­ ported by Granstrom et al., who monitored the preva­ lence of H. pylori infection in 2 9 4 Swedish children at the ages of 6, 8, 10, and 18 months and 2, 4 , and 11 years. This study showed that while at 2 years 1 0 % of children were H. pylori positive, by 11 years of age only 3 % of children remained seropositive (49). Although the above studies clearly demonstrate loss of H. pylori infection in children, unfortunately nei­ ther study controlled for antibiotic usage, a factor that clearly may affect H. pylori status. Consumption of antibiotics was, however, taken into consideration in a 2-year follow-up study of 48 H. pylori-positive Ital­ ian children in whom H. pylori status was monitored by the [ C]urea breath test at 6-month intervals over a 2-year period. In this study, 4 0 of the children were shown to remain persistently positive for H. pylori despite the fact that 10 had been treated for concomi­ tant infections with a short course of antibiotics. The remaining eight children were found to be negative for H. pylori after 2 years and, of these, two had been given antibiotics for concomitant infections (116). 13

Further indirect evidence for spontaneous clear­ ance of infection in children has come from a recent seroprevalence study of 365 primary school children aged 4 to 7 years from a low-income United States-Mexico border community. This study showed a sequential falloff in H. pylori prevalence from 3 6 % in 4-year-olds to 2 4 % in 5-year-olds, to 2 0 % in 6-year-olds to 1 4 % in 7-year-olds (120). The authors of the study concluded that the downward trend in prevalence observed in these children suggests that transient infection might be common in young children. Interestingly, in a recent study by Malaty et al. (82), acquisition and loss of infection were shown to differ in children who, although matched for socio­ economic class, were from different racial back­ grounds. In this large 12-year serological follow-up study, Malaty et al. found the rate of acquisition of infection among African American children to be four

times higher than that among Caucasian children. Loss of infection over the 12-year period was shown to be significantly higher ( 5 0 % ) among Caucasian children as compared with African American ( 4 % ) , with the latter group either remaining infected or be­ coming reinfected (82). Thus, based on current evidence, it appears that in the early years of life spontaneous clearance of in­ fection might occur. Further studies are required to determine factors that may lead to natural clearance of infection in children. Source of Infection A number of studies have proposed that acquisi­ tion of H. pylori occurs via a common environmental source. In particular, animals and water have been implicated as potential sources of infection. Animals as a potential source of H. pylori The possibility that H. pylori may be a zoonosis first arose following the publication of two seroepidemiological studies that showed that the prevalence of H. pylori infection in abattoir and meat workers was significantly increased as compared with that in sub­ jects not involved in handling animals or animal prod­ ucts (102, 141). These findings have subsequently been questioned, and it is now suggested that the in­ creased prevalence in these workers may have resulted from cross-reactivity between H. pylori and antibod­ ies to other gastrointestinal organisms such as Campy­ lobacter jejuni (39, 93). Although it has been shown that both germ-free and specific pathogen-free pigs can be experimentally colonized with H. pylori, at­ tempts to identify H. pylori in abattoir pigs with both serological and cultural techniques have failed (33, 3 4 , 5 0 , 1 2 3 ) . Dore et al. have reported a positive asso­ ciation between the prevalence of H. pylori in Sardin­ ian shepherds and contact with sheep and sheepdogs (26). In this study, 9 8 % of shepherds were shown to be infected with H. pylori, a prevalence significantly higher than that in their family members who did not have regular contact with sheep (73%) and blood do­ nors ( 4 3 % ) . These authors concluded that "the cycle of H. pylori infection might, in certain circumstances, include phases in the environment, animals (sheep or dogs) and human beings" (26). The subsequent recov­ ery of H. pylori from sheep's milk led Dore et al. to suggest that sheep may be the ancestral host of H. pylori (27). Although a number of groups have reported the isolation of H. pylori from rhesus monkeys, given the rare association between humans and monkeys, it is

CHAPTER 2 • EPIDEMIOLOGY OF INFECTION

doubtful whether this represents an important reser­ voir of H. pylori infection (30, 39, 5 4 , 105). Seroepidemiological studies examining the rela­ tionship between pet ownership and the prevalence of H. pylori have in general failed to support such a relationship (3, 14, 130, 148). The isolation of H. pylori from the stomachs of an entire colony of patho­ gen-free cats led Handt et al. to suggest that cats might represent an important reservoir of H. pylori (53). The validity of this conclusion, however, is questiona­ ble given that these cats were commercially reared and had been maintained in isolation. Although two studies have claimed that the do­ mestic housefly may provide a vector for the transmis­ sion of H. pylori (51, 52), the finding that H. pylori could not be recovered from houseflies fed human feces either naturally infected or artificially infected with H. pylori suggests that the domestic housefly is neither a vector for transmission nor a reservoir for H. pylori (110).

Water as a potential source of H. pylori One of the first reports to suggest that drinking water may be a source of H. pylori infection was pub­ lished by Klein et al., who showed that Peruvian chil­ dren whose homes had an external water supply were three times more likely to be infected with H. pylori than children whose homes had an internal water source (65). Although at that time attempts to culture H. pylori from water samples were unsuccessful, in a subsequent study Hulten et al. detected H. pylori DNA in drinking water samples collected from the same areas (58). In Colombia, acquisition of H. pylori infection in children has been associated with swim­ ming more than one time per year in rivers, streams, and pools and drinking stream water (44). Also in South America, Hopkins et al. found Chilean children who consumed uncooked vegetables contaminated with water containing raw sewage to have an in­ creased prevalence of H. pylori infection. This associ­ ation, however, was only shown in children over 5 years old, which led the authors to conclude that un­ known confounding factors may need to be consid­ ered (56). Interestingly, in their Colombian study, Goodman et al. found children who frequently ate raw vegetables to be more likely to be infected, al­ though this was at the limit of significance (44). In contrast to these studies in South America, seroepidemiological studies in southern China have failed to support the belief that water is important in the dissemination of H. pylori; no association was found between water source and the prevalence of H. pylori infection. Indeed, in this community, despite

9

the fact that the majority of subjects boil their water prior to consumption, the prevalence of H. pylori in­ fection is high ( 4 5 % ) (99). Studies in Korea and Bang­ ladesh have also found no association between H. py­ lori infection and a particular water source (22, 83). The presence of H. pylori-speciiic DNA in envi­ ronmental water sources has been reported by a num­ ber of studies (57, 5 8 , 129). For example, in a recent study that used primers based on the conserved region of ureH, Sasaki et al. reported H. pylori-specific DNA to be present in wells, springs, rivers, and ponds but not tap water of a region of Japan (129). In a second environmental study of water supplies conducted in Sweden, Hulten et al., using two different primers for their PCR assays (adhesin and 16S rRNA), showed 9 of 24 private wells, 3 of 25 municipal tap water sources, and 3 of 25 wastewater samples to be positive by PCR for H. pylori DNA (57). Although such stud­ ies may in some way support the presence of H. pylori in water, there are two important factors that must be considered; first, that the detection of H. pylori DNA does not indicate viable cells and, second, that the specificity of PCR in environments where as yet undiscovered Helicobacter spp. may be present is un­ known. Attempts to culture H. pylori from water samples have proven unsuccessful. It has been suggested that this failure may relate to the fact that when H. pylori is exposed to adverse environmental conditions, the organism takes on a viable but nonculturable coccoid form (13). Controversy exists, however, as to whether these coccoid forms of H. pylori exist in a viable form and hence are important in transmission (32, 3 5 , 3 6 , 6 7 , 1 4 6 ) . Although early studies reported noncultura­ ble coccoid forms of H. pylori to be metabolically active, more recent studies suggest that coccoid forms are not viable dormant forms but represent early stages of bacterial death (67). In conclusion, therefore, despite an extensive search for an environmental source of H. pylori, no significant reservoirs have been shown to exist outside the human stomach. This finding is perhaps not sur­ prising given that analysis of the genome sequence of H. pylori shows that this bacterium does not possess the full complement of enzymes required for an exclu­ sive aerobic or anaerobic metabolism (139) and hence its ability to survive in the natural environment seems less likely. Transmission of H. pylori Failure to consistently isolate H. pylori from res­ ervoirs other than humans suggests that direct personto-person contact is the most likely mode of transmis­ sion. The finding of an increased prevalence of

10

MITCHELL

H. pylori infection in institutionalized subjects sup­ ports this view and suggests that close personal con­ tact is important for the spread of H. pylori (11, 6 3 , 68, 147). The importance of close contact is further emphasized by the finding that the prevalence of H. pylori infection is significantly increased in family members of children infected with H. pylori as com­ pared with that in family members of children not infected with H. pylori (29, 9 4 , 1 0 1 , 1 2 5 ) . Such find­ ings have led to the view that transmission of H. pylori occurs mainly within the family setting. The relative risk of a child becoming infected with H. pylori has been reported to be approximately eight times greater if the mother is infected and approximately four times greater if the father is infected (125). The key role of infected mothers in the transmis­ sion of H. pylori within families has recently been confirmed by Malaty et al., who monitored longitudi­ nal changes in H. pylori status in 4 6 Japanese families with children and 4 8 Japanese couples without chil­ dren. This study showed that the relative risk of chil­ dren with H. pylori-positive mothers acquiring infec­ tion was 5.3 times that of children whose mothers were H. pylori negative. Confirming the importance of adult-child transmission, seroconversion only oc­ curred among children living with H. pylori-positive mothers over the period of the study (84). The finding in a number of studies of identical strains of H. pylori within family members further supports intrafamilial transmission (6, 2 0 , 9 5 ) . Although the majority of studies support interfamilial transmission, a case-control study conducted in Bangladeshi families has reported the prevalence of infection in parents of H. pylori-positive children to be the same as that in H. pylori-negative children. This finding may indicate that in some countries the source of H. pylori infection may lie outside the family (128). Family composition has also been shown to influ­ ence the transmission of H. pylori, the relative risk of infection being shown to increase according to the number of siblings within the household, the odds ratios for one, two, three, and four to five siblings being reported by Goodman et al. to be 1.4, 2.3, 2.6, and 4.3, respectively (43). This study also showed that transmission of infection occurred most readily among siblings who were close in age, transmission being most frequently from older to younger siblings (43). A similar finding has been reported by Rothenbacher et al. (126). Whether transmission occurs between spouses remains controversial. Although a number of early seroprevalence studies found no evidence to support such transmission (115, 117), a recent study of 110 employees of a health insurance company and their

partners showed a strong association between part­ ners' infection status and infection (adjusted odds ratio, 7.0), the risk of infection increasing with the number of years that the spouses had lived together (15). Further evidence that could support transmis­ sion between spouses is the finding that a significant number of couples are infected with the same strain of H. pylori ( 4 1 , 1 3 1 ) . For example, Georgopoulos et al., using ribotyping to compare strains, found 8 of 18 couples to carry an identical strain of H. pylori, the remaining 10 couples in the study being colonized with different strains (41). In contrast, Suzuki et al., who used PCR-restriction fragment length polymor­ phism electrophoretic patterns of amplified ureB to compare strains of H. pylori from 21 asymptomatic couples infected with H. pylori, found only 1 couple to harbor identical strains (135). Although such studies may suggest that in some cases transmission may occur between spouses, one cannot rule out the possibility that carriage of the same strain by spouses may have occurred due to a child infected by one parent subsequently infecting the second parent. Indeed, evidence that children may facilitate the spread of H. pylori has come from sev­ eral studies, some showing that the number of chil­ dren in a family is associated with an increased risk of infection in adult family members (16, 9 1 , 136, 148). Factors Influencing the Transmission of H. pylori Socioeconomic status Numerous studies conducted throughout the world have shown low socioeconomic status to be associated with an increased prevalence of H. pylori infection. In particular, the socioeconomic status of a subject during childhood is considered to be an impor­ tant determinant of the development of H. pylori in­ fection (80, 8 1 , 1 0 3 , 127, 142). The role of socioeconomic status per se is particu­ larly clear if one examines the prevalence of H. pylori infection in poorer racial groups living in developed countries. For example, in a study examining the rela­ tionship between socioeconomic status in childhood and the prevalence of H. pylori in African-American and Hispanic populations resident in the United States, Malaty et al. found the prevalence of H. pylori infection to be inversely related to social class during childhood, the prevalence of infection in the lowest social class ( 8 5 % ) being significantly higher than that in the highest social class ( 1 1 % ) (80). The importance of socioeconomic status in childhood has been further demonstrated in an elegant study of monozygotic twins reared apart and discordant for their H. pylori

CHAPTER 2 - EPIDEMIOLOGY OF INFECTION

status (81). In this study, Malaty and colleagues showed that the twins infected with H. pylori had been raised in homes under poorer socioeconomic conditions than those of their unaffected co-twins (81). Socioeconomic status is, however, a broad crite­ rion and encompasses factors such as level of hygiene, sanitation, density of living, and educational opportu­ nities, some or all of which have been reported to influence the level of infection within a population. Low levels of sanitation have been associated with an increased prevalence of H. pylori infection (2, 9 1 , 114). In particular, the absence of running water in the childhood home has been shown to be a signifi­ cant risk factor for H. pylori infection (91). Interest­ ingly, Irish soldiers exposed to poor living conditions and sanitation for 6 months showed no significant change in prevalence of H. pylori infection, a finding that further supports the view that acquisition of in­ fection primarily occurs in childhood (10). In both developed and developing countries high density of living has been consistently related to an increased prevalence of H. pylori infection (69, 8 1 , 88, 9 1 , 99). The importance of overcrowding in the acquisition of H. pylori is further accentuated by the finding that sharing a bed in childhood is associated with an increased prevalence of H. pylori infection (88). Educational level, also a surrogate marker of so­ cioeconomic status, has been shown in both devel­ oped and developing countries to be an important de­ terminant of H. pylori prevalence (38, 4 8 , 6 1 , 109, 1 1 3 , 1 2 4 , 1 4 0 ) . For example, in a large seroepidemio­ logical study that examined the prevalence of H. py­ lori infection in 3,194 asymptomatic subjects living in 17 different populations, Forman et al. showed an inverse relationship to exist between the prevalence of H. pylori infection and educational level, 3 4 % of subjects with a tertiary education being found to be infected compared with 4 7 % of those with a second­ ary education and 6 3 % of those with only a primary school education (38). The influence of living conditions on the preva­ lence of H. pylori infection is clearly illustrated in countries where socioeconomic conditions have sig­ nificantly improved over the last few decades. For ex­ ample, in Japan the fall in prevalence of H. pylori infection in subjects less than 4 0 years of age has been related to the significant improvement of the Japanese economy, and hence living conditions, following the Second World War (4). A similar trend has been noted in Korea, another country that has recently undergone substantial improvements in its standard of living (83).

11

Genetic predisposition To date, there have been few studies that have examined the role of genetic predisposition in relation to H. pylori infection. In an attempt to examine the importance of genetic factors on the acquisition of H. pylori infection, Malaty et al. compared the seroprevalence of H. pylori infection in 100 monozygotic and 169 dizygotic twins reared together and reared apart. The results of this study showed the correlation coeffi­ cient for the relative importance of genetic predisposi­ tion on acquisition of H. pylori infection to be ap­ proximately 0.66, with the remaining variance being accounted for by shared rearing environmental fac­ tors ( 2 0 % ) and non-shared environmental factors (23%) (77). As a result of this study, Malaty et al. concluded that genetic effects influenced the acquisi­ tion of H. pylori infection due to greater similarities within monozygotic twin pairs and that sharing of the same rearing environment also contributed to the familial tendency for acquiring H. pylori infection (77). Route of Transmission It is probably true to say that the most studied and certainly the most controversial area of H. pylori epidemiological research today is the route of trans­ mission of H. pylori. Given the location of H. pylori infection and the basic need of this bacterium for gas­ tric-type mucosa for in vivo proliferation, ingestion appears to be the most likely means of acquiring H. pylori. However, whether H. pylori reaches the oral cavity via the gastro-oral, oral-oral, or fecal-oral route remains open for conjecture. One of the major diffi­ culties in attempting to culture H. pylori from feces or the oral cavity is the presence in these sites of the autochthonous microbiota. These bacteria tend to grow much more rapidly than H. pylori and hence, even if H. pylori is present, they will often mask its presence (58). Evidence for gastro-oral transmission The presence of H. pylori in the gastric juice of up to 5 8 % of patients infected with H. pylori raises the possibility that refluxed gastric juice may repre­ sent a vehicle of transmission for this organism (145). Indeed, direct contact with gastric secretions has been implicated in the higher prevalence of H. pylori infec­ tion reported in gastroenterologists (74,97) and in the reported epidemics of Helicobacter gastritis following gastric intubation experiments (46, 119). The possibility that the gastro-oral route may be an important route of transmission of H. pylori in

12

MITCHELL

childhood has been postulated by a number of re­ searchers (5, 9 6 , 100). For example, an early report postulated that the most likely route of transmission of H. pylori was via stomach secretions or vomitus (96). Although at that time there was no evidence to support this view, vomiting and regurgitation of gas­ tric material into the mouth are fairly common in childhood and may represent an important route of transmission (96). Evidence to support the view that gastro-oral transmission via contaminated vomitus may represent an important mode of transmitting H. pylori, espe­ cially in children, has recently been published by Leung et al. (70). In this study, four children present­ ing with gastroenteritis-associated vomiting were shown serologically to be infected with H. pylori. In one of these children H. pylori was isolated from the vomitus and from two others H. pylori DNA was de­ tected in vomitus by PCR. Interestingly, an 18-monthold girl, negative by serology for H. pylori but in whom H. pylori DNA was detected in vomitus 6 months later, showed seroconversion for H. pylori (70). Support for the view that vomitus may be an important vehicle in the spread of H. pylori has come from a recent study by Parsonnet et al. (112). In this study, H. pylori was cultured from the vomitus of 1 0 0 % of adult subjects who had been given an emetic to induce vomiting (112). Interestingly, air sampled in an area 0.3 meter away from these subjects during vomiting grew H. pylori in 6 of 16 (37.5%) instances, but air samples collected 1.2 meters away from sub­ jects failed to yield H. pylori (112). Indirect evidence of the importance of vomiting in the transmission of H. pylori has also recently been shown by Luzza et al. (76). In this study, vomiting siblings and siblings of 100 vomiting index children were screened by means of the [ C]urea breath test for H. pylori. A high rate of active H. pylori infection was shown to be present in both vomiting siblings (60%) and siblings ( 6 7 % ) of H. pylori-infected vomit­ ing index children, with a history of vomiting in sib­ lings being shown to be positively associated with ac­ tive H. pylori infection in index children (multivariate odds ratio 2.4) (76). 13

Evidence for and against oral-oral transmission of H. pylori Attempts to culture H. pylori from the oral cavity have proved in many cases to be fruitless. There have, however, been a limited number of studies where H. pylori has been isolated from dental plaque and saliva (19, 37, 66). In an early study Krajden et al. isolated H. pylori from the dental plaque of 1 of 29 patients whose stomach biopsies were shown to be positive

for H. pylori (66). Comparison of the strains isolated from the stomach and dental plaque of this patient with restriction endonuclease analysis subsequently showed one of three strains isolated from dental plaque to be indistinguishable from that isolated from the stomach (132). Cellini et al. also reported the iso­ lation of H. pylori from the dental plaque of 1 of 20 H. pylori-positive endoscopy patients. In this case, comparison of the protein patterns as well as the re­ striction endonuclease pattern of H. pylori isolated from the stomach biopsy and from dental plaque again showed these to be identical (19). The isolation of low numbers of H. pylori from the saliva of one of nine H. py/on'-positive subjects has been reported by Ferguson et al. (37). Again this group showed, using restriction fragment length polymorphism, that the H. pylori strain isolated from saliva was identical to that in gastric tissue (37). In contrast to the low detection rate in the above studies, Desai et al. found H. pylori to be present in the dental plaque of 9 8 % of Indian dyspeptic patients; however, in this study, identification of H. pylori was based solely on the urease test. Given the presence of other urease-positive organisms in the mouth, it is possible that the identification of isolates as H. pylori in this study may have been false (25). The possibility of falsely identify­ ing normal flora from the oral cavity as H. pylori has been reported by Namavar et al., who showed that organisms isolated from the tongue and palate of one patient and considered to be phenotypically identical to H. pylori were in fact negative by an H. pylorispecific PCR (104). In an important study recently published by Parsonnet et al., H. pylori was success­ fully cultured from the saliva of three subjects ( 1 9 % ) . Following the induction of vomiting in these subjects with an emetic, Parsonnet and colleagues were able to culture H. pylori from nine (56%) subjects (112). The ability to detect H. pylori-specific DNA from the oral cavity has varied significantly (7, 12, 17, 86, 107). Although a number of studies have failed to detect H. pylori DNA in the dental plaque of any H. py/on'-positive patients (12, 17), Banatvala et al., using an H. pylori species-specific ureA (urease) gene internal sequence, showed 7 2 % of dental plaque sam­ ples taken from 54 patients attending for endoscopy to be positive for H. pylori DNA (7). In a smaller study, Mapstone et al., using a 16S rRNA probe, de­ tected H. pylori DNA in 3 8 % of dental plaque sam­ ples obtained from 13 H. pylori-positive patients (86). Using nested PCR, Dowsett et al. detected H. pylori DNA in periodontal pockets as well as the dorsum of the tongue in 8 7 % of subjects examined (28). No association was shown, however, between periodon­ tal pocket depth and the detection of H. pylori (28). The presence of H. pylori DNA in the subgingival

CHAPTER 2 • EPIDEMIOLOGY OF INFECTION

plaque of patients with adult periodontitis has also been reported (122). It has been suggested that the differences in detec­ tion rate of H. pylori in the oral cavity with PCR may relate to the specificity of the primers used (89). This point may be particularly relevant to a recent study by Song et al., who using "a highly sensitive and specific PCR" detected H. pylori DNA in the saliva of 5 5 % (23/42) of patients and the dental plaque of 9 7 % of patients. Given that only 11 of these patients were positive for the presence of gastric H. pylori, Song et al. suggested that H. pylori might belong to the nor­ mal oral microflora (134). Although it is possible that these findings are correct, given that there may be as yet undiscovered Helicobacter species present in the oral cavity, some caution may be required in the inter­ pretation of these data. Epidemiological data suggest that a number of cultural habits may enhance the oral transmission of H. pylori. For example, premastication of food by African mothers prior to feeding their children has been shown to be a risk factor for H. pylori infection (1). The use of chopsticks and communal eating have also been associated with transmission of H. pylori within Chinese communities outside of China (21). However, in a more recent study in Chinese subjects resident in Hong Kong, H. pylori was rarely detected in chopsticks after eating (71). The finding that the prevalence of H. pylori infec­ tion in dentists or dental workers is not increased has been used to argue against the oral-oral transmission of H. pylori (73, 79, 106). Evidence for and against fecal-oral transmission of H. pylori Although there is some supportive evidence for the passage of H. pylori through the intestine (31), this bacterium is not well adapted for such passage. Indeed, several groups have shown that H. pylori is sensitive to the lethal effects of bile (98, 118); hence, survival of H. pylori after transit through the intes­ tinal tract seems unlikely. In an attempt to examine the role of the fecaloral route in the transmission of infection, a number of studies have investigated the association between the prevalence of H. pylori infection and hepatitis A virus, an organism known to be transmitted by the fecal-oral route. In a study using paired sera from the same individuals, the prevalence pattern of hepatitis A was compared with that of H. pylori in an urban and rural southern Chinese population. Although ini­ tial examination of the seroprevalence data from rural areas supported a correlation between H. pylori and hepatitis A, when the prevalence data from the urban

13

area were examined, it became evident that no such correlation existed. Although in this urban area the prevalence of H. pylori infection in subjects < 1 0 years was high (approximately 3 2 % ) , not one of these sub­ jects was shown to be infected with hepatitis A. As a result of this study, it was concluded that communitywide fecal-oral spread of H. pylori might be of limited importance (55). This lack of association between the prevalence of H. pylori and hepatitis A has been re­ ported by a number of other studies conducted in both developed and developing countries (40, 7 5 , 130, 149). Attempts to culture H. pylori from feces have by and large been unsuccessful. In 1 9 9 4 , however, the first report of the isolation of H. pylori from human feces appeared in the literature. In this study Thomas et al. isolated H. pylori from the feces of 1 infected adult and 9 of 23 randomly selected children living in a Gambian village (138). In the same year, Kelly and colleagues using the same isolation technique as Thomas's group also claimed to have isolated H. py­ lori from the feces of 12 of 2 5 H. pylori-positive sub­ jects with dyspepsia (62). Definitive proof that the organisms cultured in this study were H. pylori was, however, unsubstantiated. Attempts by other groups to isolate H. pylori from patient populations using these methods have failed, and it has been suggested that the ability of Thomas et al. to culture H. pylori from Gambian children may relate to the fact that these children were malnourished and had an ex­ tremely short fecal transit time (89). This view is sup­ ported by a recent study by Parsonnet et al. that showed that although H. pylori could not be cultured from the stools of 16 H. py/on'-positive adult subjects, if patients were given a cathartic to induce diarrhea and the stools were then tested, the bacterium could be cultured from the stools of 7 of 14 ( 5 0 % ) patients (112). Attempts to detect H. pylori DNA in feces by PCR have resulted in variable outcomes. Whereas some studies have reported the detection of H. pylori DNA in the feces of 25 to 9 0 % of subjects known to be infected with H. pylori (72, 85, 108), others have reported less than 1 0 % of H. py/on'-positive subjects to have H. pylori DNA in their feces (104, 144). Al­ though detection of H. pylori DNA in feces may add to the evidence supporting the fecal-oral route of transmission, it is again essential to remember that the finding of H. pylori DNA does not necessarily mean that viable H. pylori is present in the feces. CONCLUSION Given the association between H. pylori and pep­ tic ulcer disease, gastric cancer, and B-cell MALT lym-

14

MITCHELL

phoma, there is an urgent need for the development of intervention strategies to prevent the spread of this bacterium. Although development of a vaccine against H. pylori is progressing well, it is highly likely that it will be 5 to 10 years before such a vaccine becomes available. Given the increasing levels of resis­ tance to current antimicrobial therapies used against H. pylori and the high cost of such an approach, mass programs to treat H. pylori-'miected individuals is clearly out of the question. In many other diseases with an infectious etiol­ ogy, public health measures based on epidemiological data have been extremely successful in preventing the spread of pathogenic agents. Before such measures can be implemented, clarification of the route of transmission of H. pylori will be essential. REFERENCES 1. Albenque, M., F. Tall, F. Dabis, and F. Megraud. 1990. Epi­ demiological study of Helicobacter pylori transmission from mother to child in Africa. Enferm. Digest. 78:48. 2. Al-Moagel, M. A., D. G. Evans, M. E. Abdulghani, E. Adam, D.J.J. Evans, H. M. Malaty, and D. Y. Graham. 1990. Preva­ lence of Helicobacter (formerly Campylobacter) pylori infec­ tion in Saudi Arabia, and comparison of those with and with­ out upper gastrointestinal symptoms. Am. J. Gastroenterol. 85:944-948. 3. Ansorg, R., E. H. Vonheinegg, and G. Vonrecklinghausen. 1995. Cat owner's risk of acquiring a Helicobacter pylori infection. Zentralbl. Bakteriol. Int. ] . Med. Microbiol. Vir. Parasitol. Infect. Dis. 283:122-126. 4. Asaka, M., T. Kimura, and M. Kudo. 1992. Relationship of Helicobacter pylori to serum pepsinogens in an asymptomatic Japanese population. Gastroenterology 102:760-766. 5. Axon, A. T. R. 1995. Is Helicobacter pylori transmitted by thegastro-oral route? Aliment. Pharmacol. Ther. 9:585-588. 6. Bamford, K. B., J. Bickley, J. S. A. Collins, B. T. Johnston, S. Potts, V. Boston, R. J. Owen, and J. M. Sloan. 1993. Heli­ cobacter pylori—comparison of DNA fingerprints provides evidence for intrafamilial infection. Gut 34:1348-1350. 7. Banatvala, N., C. R. Lopez, R. J. Owen, A. Hurtado, Y. Abdi, G. R. Davies, J. M. Hardie, and R. A. Feldman. 1994. Use of the polymerase chain reaction to detect Helicobacter pylori in the dental plaque of healthy and symptomatic individuals. Microb. Ecol. Health Dis. 7:1-8. 8. Banatvala,N.,K.Mayo, F.Megraud,R.Jennings,J.J. Deeks, and R. A. Feldman. 1993. The cohort effect and Helicobacter pylori. J. Infect. Dis. 168:219-221. 9. Bardhan, P. K. 1997. Epidemiological features of Helico­ bacter pylori infection in developing countries. Clin. Infect. Dis. 25:973-978. 10. Basso, L., S. Beattie, S. Lawlor, J. Clune, and C. O'Morain. 1994. A descriptive follow-up study on Helicobacter pylori infection before and after exposition to a war area. Eur. J. Epidemiol. 10:109-111. 11. Berkowicz, J., and A. Lee. 1987. Person-to-person transmis­ sion of Campylobacter pylori. Lancet ii:680-681. 12. Bickley, J., R. J. Owen, A. G. Fraser, and R. E. Pounder. 1993. Evaluation of the polymerase chain reaction for detecting the urease c gene of Helicobacter pylori in gastric biopsy samples and dental plaque. / . Med. Microbiol. 39:338-344.

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vouring the gastro-oral route in the transmission of Helico­ bacter pylori infection in children. Eur. J. Gastroenterol. Hepatol. 12:623-627. Malaty, H. M., L. Engstrand, N. L. Pedersen, and D. Y. Gra­ ham. 1994. Helicobacter pylori infection: genetic and envi­ ronmental influences. Ann. Intern. Med. 120:982-986. Malaty, H. M., D. G. Evans, D. J. Evans, and D. Y. Graham. 1992. Helicobacter pylori in Hispanics—comparison with blacks and whites of similar age and socioeconomic class. Gastroenterology 103:813-816. Malaty, H. M., D. J. Evans, K. Abramovitch, D. G. Evans, and D. Y. Graham. 1992. Helicobacter pylori infection in dental workers: a seroepidemiology syudy. Am. J. Gastroen­ terol. 87:1728-1731. Malaty, H. M., and D. Y. Graham. 1994. Importance of childhood socioeconomic status on the current prevalence of Helicobacter pylori infection. Gut 35:742-745. Malaty, H. M., D. Y. Graham, I. Isaksson, L. Engstrand, and N. L. Pedersen. 1998. Co-twin study of the effect of environ­ ment and dietary elements on acquisition of Helicobacter py­ lori infection. Am. J. Epidemiol. 148:793-797. Malaty, H. M., D. Y. Graham, W. A. Wattigney, S. R. Srinivasan, M. Osato, and G. S. Berenson. 1999. Natural history of Helicobacter pylori infection in childhood: 12-year followup cohort study in a biracial community. Clin. Infect. Dis. 28:279-282. Malaty, H. M., J. G. Kim, S. D. Kim, and D. Y. Graham. 1996. Prevalence of Helicobacter pylori infection in Korean children—inverse relation to socioeconomic status despite a uniformly high prevalence in adults. Am. J. Epidemiol. 143: 257-262. Malaty, H. M., T. Kumagai, E. Tanaka, H. Ota, K. Kiyosawa, D. Y. Graham, and T. Katsuyama. 2000. Evidence from a nine-year birth cohort study in Japan of transmission pathways of Helicobacter pylori infection. /. Clin. Microbiol. 38:1971-1973. Mapstone, N. P., F. A. Lewis, D. S. Tompkins, D. A. F. Lynch, A. T. R. Axon, M. F. Dixon, and P. Quirke. 1993. PCR identification of Helicobacter pylori in faeces from gastritis patients. Lancet 341:447. Mapstone, N. P., D. A. F. Lynch, F. A. Lewis, A. T. R. Axon, D. S. Tompkins, M. F. Dixon, and P. Quirke. 1993. Identifi­ cation of Helicobacter pylori DNA in the mouths and stom­ achs of patients with gastritis using PCR. /. Clin. Pathol. 46: 540-543. Marshall, B. J., D. B. McGechie, P. A. Rogers, and R. J . Glancy. 1985. Pyloric Campylobacter infection and gastroduodenal disease. Med. J. Aust. 142:439-444. McCallion, W. A., L. J. Murray, A. G. Bailie, A. M. Dalzell, D. Oreilly, and K. B. Bamford. 1996. Helicobacter pylori infection in children—relation with current household living conditions. Gut 39:18-21. Megraud, F. 1995. Transmission of Helicobacter py­ lori—faecal-oral versus oral-oral route. Aliment. Pharmacol. Ther. 9:85-91. Megraud, F., M. P. Brassens Rabbe, F. Denis, A. Belbouri, and D. Q. Hoa. 1989. Seroepidemiology of Campylobacter pylori infection in various populations. /. Clin. Microbiol. 27:1870-1873. Mendall, M. A., P. M. Goggin, N. Molineaux, J. Levy, T. Toosy, D. Strachan, and T. C. Northfield. 1992. Childhood living conditions and Helicobacter pylori seropositivity in adult life. Lancet 339:896-897. Mitchell, H. M. 1999. The epidemiology of Helicobacter py­ lori. Gastroduodenal Disease and Helicobacter pylori: Patho­ physiology, Diagnosis and Treatment 241:11-30.

CHAPTER 2 • EPIDEMIOLOGY OF INFECTION

93. Mitchell, H. M. 1993. The epidemiology of Helicobacter py­ lori infection and its relation to gastric cancer, p. 95-114. In C. S. Goodwin and B. W. Wormsley (ed.), Helicobacter py­ lori: Biology and Clinical Practice. CRC Press, Boca Raton, Fla. 94. Mitchell, H. M., T. Bohane, R. A. Hawkes, and A. Lee. 1993. Helicobacter pylori infection within families. Zentralbl. Bakteriol. Int. J. Med. Microbiol. 280:128-136. 95. Mitchell, H. M., S. L. Hazell, T. Kolesnikow, J. Mitchell, and D. Frommer. 1996. Antigen recognition during progression from acute to chronic infection with a cagA-positive strain of Helicobacter pylori. Infect. Immun. 64:1166-1172. 96. Mitchell, H. M., A. Lee, and T. D. Bohane. 1989. Evidence for person-to-person spread of Campylobacter pylori, p. 197-202. In B. J. Rathbone and R. V. Heatley (ed.), Campy­ lobacter pylori and Gastroduodenal Disease. Blackwell Sci­ entific Publications, Oxford, United Kingdom. 97. Mitchell, H. M., A. Lee, and J. Carrick. 1989. Increased inci­ dence of Campylobacter pylori infection in gastroenterologists: further evidence to support person-to-person transmis­ sion of C. pylori. Scand. J. Gastroenterol. 24:396-400. 98. Mitchell, H. M., Y. Li, P. Hu, S. L. Hazell, G. Du, D. J. Byrne, and A. Lee. 1992. The susceptibility of Helicobacter pylori to bile may be an obstacle to faecal transmission. Eur. /. Gastroenterol. Hepatol. 4:S79-S83. 99. Mitchell, H. M., Y. Y. Li, P. J. Hu, Q. Liu, M. Chen, G. G. Du, Z.J. Wang, A. Lee, and S. L. Hazell. 1992. Epidemiology of Helicobacter pylori in Southern China—identification of early childhood as the critical period for acquisition./. Infect. Dis. 166:149-153. 100. Mitchell, J. D., H. M. Mitchell, and V. Tobias. 1992. Acute Helicobacter pylori infection in an infant, associated with gastric ulceration and serological evidence of intra-familial transmission. Am. J. Gastroenterol. 87:382-386. 101. Miyaji, H., T. Azuma, S. Ito, Y. Abe, F. Gejyo, N. Hashimoto, H. Sugimoto, H. Suto, Y. Ito, Y. Yamazaki, Y. Kohli, and M. Kuriyama. 2000. Helicobacter pylori infection occurs via close contact with infected individuals in early childhood. /. Gastroenterol. Hepatol. 15:257-262. 102. Morris, A., G. Nicholson, G. Lloyd, D. Haines, A. Rogers, and D. Taylor. 1986. Seroepidemiology of Campylobacter pyloridis. N. Z. Med. ] . 99:657-659. 103. Murray, L. J., E. E. McCrum, A. E. Evans, and K. B. Bamford. 1997. Epidemiology of Helicobacter pylori infection among 4742 randomly selected subjects from northern Ireland. Int. J. Epidemiol. 26:880-887. 104. Namavar, F., R. Roosendaal, E. J. Kuipers, P. Degroot, M. W. Vanderbijl, A. S. Pena, and J. Degraaff. 1995. Presence of Helicobacter pylori in the oral cavity, oesophagus, stomach and faeces of patients with gastritis. Eur. J. Clin. Microbiol. Infect. Dis. 14:234-237. 105. Newell, D., M. J. Hudson, and A. Baskerville. 1987. Natu­ rally occurring gastritis associated with Campylobacter pylori infection in the rhesus monkey. Lancet ii:1338. 106. Nguyen, A., F. Elzaatari, and D. Y. Graham. 1995. Helico­ bacter pylori in the oral cavity. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endodontics 79:705-709. 107. Nguyen, A. M. H., L. Engstrand, R. M. Genta, D. Y. Graham, and F. A. K. Elzaatari. 1993. Detection of Helicobacter pylori in dental plaque by reverse transcription polymerase chain reaction. /. Clin. Microbiol. 31:783-787. 108. Notarnicola, M., F. Russo, A. Cavallini, M. Bianco, E. Jirillo, S. Pece, C. Leoci, G. Dimatteo, and A. Dileo. 1996. PCR identification of Helicobacter pylori DNA in faeces from pa­ tients with gastroduodenal pathology. Med. Sci. Res. 24: 785-787.

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109. Olmos, J. A., H. Rios, and R. Higa. 2000. Prevalence of Heli­ cobacter pylori infection in Argentina—results of a nation­ wide epidemiologic study. /. Clin. Microbiol. 31:33-37. 110. Osato, M. S., K. Ayub, H. H. Le, R. Reddy, and D. Y. Gra­ ham. 1998. Houseflies are an unlikely reservoir or vector for Helicobacter pylori. J. Clin. Microbiol. 36:2786-2788. 111. Parsonnet, J., S. Hansen, L. Rodriguez, A. B. Gelb, R. A. Warnke, E. Jellum, N. Orentreich, J. H. Vogelman, and G. D. Friedman. 1994. Helicobacter pylori infection and gastric lymphoma. N. Engl. J. Med. 330:1267-1271. 112. Parsonnet, J., H. Shmuely, and T. Haggerty. 1999. Fecal and oral shedding of Helicobacter pylori from healthy infected adults. JAMA 282:2240-2245. 113. Peach, H. G., D. C. Pearce, and S. J. Farish. 1997. Helico­ bacter pylori infection in an Australian regional city—preva­ lence and risk factors. Med. ] . Aust. 167:310-313. 114. Perez-Perez, G. I., D. N. Taylor, L. Bodhidatta, J. Wongsrichanalai, W. B. Baze, B. E. Dunn, P. D. Echeverria, and M. J. Blaser. 1990. Seroprevalence of Helicobacter pylori infec­ tions in Thailand./. Infect. Dis. 161:1237-1241. 115. Perez-Perez, G. I., S. S. Witkin, M. D. Decker, and M. J . Blaser. 1991. Seroprevalence of Helicobacter pylori infection in couples. /. Clin. Microbiol. 29:642-644. 116. Perri, F., M. Pastore, R. Clemente, V. Festa, M. Quitadamo, G. Niro, P. Conoscitore, P. Rutgeerts, and A. Andriulli. 1998. Helicobacter pylori infection may undergo spontaneous erad­ ication in children—a 2-year follow-up study./. Pediatr. Gas­ troenterol. Nutr. 27:181-183. 117. Polish, L. B., J. M. J. Douglas, A. J. Davidson, G. I. Perez Perez, and M.J. Blaser. 1991. Characterisation of risk factors for Helicobacter pylori infection among men attending a sex­ ually transmitted disease clinic: lack of evidence for sexual transmission. /. Clin. Microbiol. 29:2139-2143. 118. Raedsch, R., S. Pohl, J. Plachky, A. Stiehl, and B. Kommerell. 1989. The growth of Campylobacter pylori is inhibited by intragastric bile acids, p. 409-412. In F. Megraud and H. Lamouliatte (ed.), Gastroduodenal Pathology and Campylo­ bacter pylori. Elsevier, Amsterdam, The Netherlands. 119. Ramsey, E. J., K. V. Carey, W. L. Peterson, J. J. Jackson, F. K. Murphy, and N. W. T. Read. 1979. Epidemic gastritis with hypochlorhydria. Gastroenterology 76:1449-1457. 120. Redlinger, T., K. O'Rourke, and K. J. Goodman. 1999. Age distribution of Helicobactor pylori seroprevalence among young children in a United States/Mexico border community: evidence for transitory infection. Am. ] . Epidemiol. 150: 225-230. 121. Replogle, M. L., W. Kasumi, K. B. Ishikawa, S. F. Yang, T. Juji, K. Miki, G. C. Rabat, and J. Parsonnet. 1996. Increased risk of Helicobacter pylori associated with birth in wartime and post-war Japan. Int. J. Epidemiol. 25:210-214. 122. Riggio, M. P., and A. Lennon. 1999. Identification by PCR of Helicobacter pylori in subgingival plaque of adult perio­ dontitis patients./. Med. Microbiol. 48:317-322. 123. Rocha, G. A., D. M. M. Queiroz, E. N. Mendes, A. M. R. Oliveira, S. B. Moura, and R. J. A. Silva. 1992. Source of Helicobacter pylori infection: studies in abattoir workers and pigs. Am. J. Gastroenterol. 87:1525. 124. Rosenstock, S. J., L. P. Andersen, C. V. Rosenstock, O. Bonnevie, and T. Jorgensen. 1996. Socioeconomic factors in Helicobacter pylori infection among Danish adults. Am. J. Public Health 86:1539-1544. 125. Rothenbacher, D., G. Bode, G. Berg, U. Knayer, T. Gonser, G. Adler, and H. Brenner. 1999. Helicobacter pylori among preschool children and their parents: evidence of parent-child transmission. /. Infect. Dis. 179:398-402.

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126. Rothenbacher, D., G. Bode, and H. Brenner. 2000. Helico­ bacter pylori among siblings. Lancet 355:1998. 127. Rothenbacher, D., G. Bode, T. Winz, G. Berg, G. Adler, and H. Brenner. 1997. Helicobacter pylori in out-patients of a general practitioner—prevalence and determinants of current infection. Epidemiol. Infect. 119:151-157. 128. Sarker, S. A., M. M. Rahman, D. Mahalanabis, P. K. Bardham, P. Hildebrand, C. Beglinger, and K. Gyr. 1995. Preva­ lence of Helicobacter pylori infection in infants and family contacts in a poor Bangladesh community. Dig. Dis. Sci. 40: 2669-2672. 129. Sasaki, K., Y. Tajiri, M. Sata, Y. Fujii, F. Matsubara, M. G. Zhao, S. Shimizu, A. Toyonaga, and K. Tanikawa. 1999. Helicobacter pylori in the natural environment. Scand. J. In­ fect. Dis. 31:275-280. 130. Sathar, M. A., E. Gouws, A. E. Simjee, and A. M. Mayat. 1997. Seroepidemiological study of Helicobacter pylori infec­ tion in South African children. Trans. R. Soc. Trop. Med. Hyg. 91:393-395. 131. Schutze, K., E. Hentschel, B. Dragosics, and A. M. Hirschl. 1995. Helicobacter pylori reinfection with identical organ­ isms—transmission by the patients' spouses. Gut 36: 831-833. 132. Shames, B., S. Krajden, M. Fuksa, C. Babida, and J. L. Penner. 1989. Evidence for the occurrence of the same strain of Campylobacter pylori in the stomach and dental plaque. /. Clin. Microbiol. 27:2849-2850. 133. Sipponen, P. 1995. Helicobacter pylori: a cohort phenome­ non. Am. J. Surg. Pathol. 19:S30-S36. 134. Song, O., T. Lange, A. Spahr, G. Adler, and G. Bode. 2000. Characteristic distribution pattern of Helicobacter pylori in dental plaque and saliva detected with nested PCR. /. Med. Microbiol. 49:349-353. 135. Suzuki, J., H. Muraoka, I. Kobayasi, T. Fujita, and T. Mine. 1999. Rare incidence of interspousal transmission of Helico­ bacter pylori in asymptomatic individuals in Japan. /. Clin. Microbiol. 37:4174-4176. 136. Teh, B. H., J. T. Lin, W. H. Pan, S. H. Lin, L. Y. Wang, T. K. Lee, and C. J. Chen. 1994. Seroprevalence and associated risk factors of Helicobacter pylori infection in Taiwan. Anti­ cancer Res. 14:1389-1392. 137. Thomas, J. E., A. Dale, M. Harding, W. A. Coward, T. J. Cole, and L. T. Weaver. 1999. Helicobacter pylori coloniza­ tion in early life. Pediatr. Res. 45:218-223. 138. Thomas,J. E., G. R. Gibson,M. K. Darboe,A.Dale, andL.T. Weaver. 1992. Isolation of Helicobacter pylori from human faeces. Lancet 340:1194-1195. 139. Tomb, J. F., O. White, A. R. Kerlavage, R. A. Clayton, G. G. Sutton, R. D. Fleischmann, K. A. Ketchum, H. P. Klenk, S. Gill, B. A. Dougherty, K. Nelson, J. Quackenbush, L. X. Zhou, E. F. Kirkness, S. Peterson, B. Loftus, D. Richardson,

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Helicobacter pylori: Physiology and Genetics Edited by H. L. T. Mobley, G. L. Mendz, and S. L. Hazell © 2 0 0 1 ASM Press, Washington, D.C.

Chapter 3

One Hundred Years of Discovery and Rediscovery of Helicobacter pylori and Its Association with Peptic Ulcer Disease BARRY J . MARSHALL

Today's understanding of Helicobacter-telated gas­ tric diseases in humans stems from an explosion in research, which occurred after the first culture of the organism by Marshall and Warren in 1982 (47). This event may have been the culmination of over 100 years of study of helicobacters and their epiphenomena. This chapter is a concise overview of major highlights during that time following four storylines of spiral bacteria, epidemic gastritis with hypochlorhydria, urease, and bismuth therapy.

The organisms were noted in the gastric mucosa of macaques by Doenges in the United States (7), and in resected gastric specimens by Freedberg and Baron in 1940 (11). Freedberg and Baron found "spirochetes" in about 4 0 % of resected specimens. In retrospect, the population of the United States was probably more than 4 0 % infected with Helicobacter pylori at that time, but because patients undergoing gastric sur­ gery had such severe disturbances of their physiology, the helicobacters such as H. pylori may have actually regressed or disappeared, as is believed to happen in late gastric carcinoma cases today. Nevertheless, Freedberg and Baron presented their findings in 1940 and generated a vigorous discussion in which some members of the audience, used to treating syphilis with heavy metals such as mercury, arsenic, and bis­ muth, commented on anecdotal cases of complete re­ mission of peptic ulcer disease while treating the syph­ ilis spirochetes.

SPIRAL BACTERIA The first well-known report of gastric helicobac­ ters was by Bizzozero in Turin in 1893 (1). Bizzozero was a well-known anatomist, famous already for his proof that all dividing cells required cell nuclei (4). In his anatomical observations of the gastric mucosa of dogs, Bizzozero reported "spirochetes" inhabiting the gastric glands (9) and even the canaliculi of the pari­ etal cells. In hand-drawn color illustrations, Bizzozero showed gram-negative organisms with approximately 10 wavelengths within the parietal cells and gastric glands. We now know these organisms variously iden­ tified as Helicobacter canis, Helicobacter felis (24), and/or Helicobacter heilmannii (18). Bizzozero's work was extended by Salomon, who was able to propagate these spiral organisms in mouse stomachs after feeding ground-up gastric mucosa from cats and dogs to his mouse colony (42). Salomon's work was a precursor to current studies where the H. felis-'mfected mouse is an important model in vaccine and therapeutic studies of helicobacter eradication (5). In the 20th century, anatomists and pathologists noticed spiral organisms in the human mucosa from time to time, initially adjacent to carcinomas (21).

Doenges and Freedberg were challenged in the early 1950s by Palmer, who found no evidence of spi­ rochetes in more than 1,000 gastric biopsies taken with a blind suction biopsy instrument (37). To this day, no one knows how such an incorrect conclusion could have been made at a time when more than 5 0 % of the population was bound to be positive for H. pylori. One can only guess that the appropriate stains were not performed or that the investigators were looking for something other than H. pylori and dis­ counted the presence of black, silver-stained prolific organisms. In the 1950s and 1960s, Susumu Ito of Harvard Medical School made some of the first detailed ana­ tomic descriptions of the gastric mucosa appearance under the electron microscope. Ito's photographs and drawings of the structure of the parietal cell and acid-

Barry J. Marshall • Department of Microbiology, University of Western Australia, L Block, QEII Medical Centre, Hospital Avenue, Nedlands 6009, Western Australia.

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MARSHALL

EPIDEMIC GASTRITIS W I T H HYPOCHLORHYDRIA

of the answers. In their Principles and Practice of Medicine volumes published between 1900 and 1920, Osier and McCrae described an acute form of gastritis with hypochlorhydria (36). They were not the first to observe this syndrome, since this had been reported in several earlier medical texts. Because endoscopy was not possible, the exact pathophysiology of this disorder was unknown, but it was known to be a com­ mon syndrome in young children, characterized by a transient vomiting illness with neutral pH of the gas­ tric fluids. Various treatments were advised, including bismuth, and the occasional patient who developed a chronic dyspeptic syndrome was described. This epidemic-gastritis-with-hypochlorhydria syndrome re­ mained in the medical textbooks until about the mid1960s, when its rarity led to its deletion from most chapters on gastritis or gastroenterology. It was, therefore, a surprise when investigators began en­ countering epidemics of hypochlorhydria in the 1970s when it again became popular to study gastric acid secretion by passing nasogastric tubes in volunteers (39). Basil Hirschowitz reported this illness from Bir­ mingham, Ala., where acute mucosal changes were thought to be due to the effect of cortisone on the gastric mucosa (19). More recently, an epidemic was reported by Richard Hunt that occurred in several volunteers undergoing gastric analysis in the British Navy (13). The most notable of these reports came from the laboratory of Fordtran, where Ramsay and colleagues noticed that more than half of their volun­ teers undergoing gastric analysis developed hypo­ chlorhydria associated with gastritis (39). The hypo­ chlorhydria lasted several months and was associated initially with a vomiting illness and even abnormal liver function tests in a few individuals. In retrospect, this was acute H. pylori infection transmitted from one volunteer to the next by the nasogastric tubes or by contamination within the laboratory itself. Reex­ amination of these individuals more than 15 years after the event revealed that they still had H. pylori, but few had developed peptic ulcer disease; in fact, most of them were asymptomatic (17). One other no­ table episode reported by Wiersinga and Tytgat in Holland (49) occurred in a patient with ZollingerEllison syndrome who developed acute gastritis after endoscopy and subsequently was hypochlorhydric. Retrospective examination of those sections revealed H. pylori from what was probably a case of endoscope-transmitted H. pylori. Wiersinga's observations are of interest because he suggested that the infectious agent, whatever it might be, could be used as therapy for patients with Zollinger-Ellison syndrome.

As with the spiral organism literature, the medi­ cal literature from the late 19th century contains some

Thus, the acute syndrome of H. pylori had been well described in the literature before the presence of the organism in the hypochlorhydric syndrome was

secreting glands of the gastric corpus are well known and have been copied in many medical texts. How­ ever, he also observed "spirilli" in some of his mate­ rial. He published an excellent photograph of one of these organisms in 1967, showing a greatly enlarged H. pylori within a parietal cell gland, complete with several sheathed flagella and typical spiral morphol­ ogy (20). In subsequent years, Lockard and Bolar found these organisms once more in the stomach of cats and dogs and also published electron mi­ crographs of them (25). In the mid-1970s, the spiral bacteria were the subject of a paper by Steer and Colin-Jones (43). Their research occurred at the threshold between old and new therapies for peptic ulcer, i.e., carbenoxolone or bismuth versus the new drug, cimetidine. Steer and Colin-Jones studied the presence of bacteria and in­ flammation during gastric ulcer healing with carbe­ noxolone. They found that the drug healed the ulcers but had no major effect on the histology, because the inflammation was just as severe after ulcer healing. They noted that numerous spiral bacteria were pres­ ent in 8 0 % of their gastric ulcer specimens. Unfortu­ nately, they were unable to culture the organism, or, at least, only cultured pseudomonas. It was several more years before the microaerophilic Campylobacter isolation techniques were well known, so their mis­ take is understandable. Nevertheless, they published excellent photographs of the gastric mucosal histol­ ogy, including H. pylori in the mucous layer and even phagocytosed within neutrophils. Further papers fol­ lowed, and some of the best illustrations of H. pylori on duodenal mucosa at ulcer borders subsequently appeared in a publication in Gut a year or so after the Australian culture and rediscovery of the organ­ ism (44). The interest of pathologists and anatomists in the spiral bacterium remained rather focused, however, and divorced from clinical observations on gastric dis­ eases. For this reason, persons studying gastritis and clinicians studying peptic ulcer disease were generally unaware of the spirochete literature, and never con­ sidered bacterial causes of gastric diseases. Neverthe­ less, after H. pylori was cultured and observed to be present in so many people, both symptomatic and healthy, the question was asked: where did these or­ ganisms arrive from, and what happened during the acute infection with helicobacter?

CHAPTER 3 • H. PYLORI AND PEPTIC ULCER DISEASE

noted in the 1980s. Further studies in this vein con­ tinue, so that even volunteer experiments have been described (28, 34) as well as sporadic accidental re­ ports of laboratory-acquired infections (32, 4 6 ) , all confirming the earlier observations.

T H E ORIGIN OF GASTRIC UREASE Without regard to the spirochete and hypochlorhydria literature, other scientists studied gastric urease enzyme from the 1920s. Urease was an impor­ tant enzyme because it was the first enzyme purified into crystalline form whereby analysis could identify that it was actually a protein. Prior to Sumner's obser­ vations on jackbean urease (45), the exact composi­ tion of enzymes was not known, and whether they were proteins or merely cofactors was highly contro­ versial. In 1924, Murray Luck and colleagues studied the urease enzyme of the gastric mucosa of dogs (26). They observed that many carnivorous animals, partic­ ularly cats and dogs, had high levels of gastric urease present. They equated strength of the enzyme with that present in raw jackbeans (Canavalia ensifortnis), i.e., a very high amount. Since Luck could not separate the urease from the gastric mucosa, he presumed it was intrinsic to the epithelial cells and was secreted into the mucous layer. The purpose of this urease was unknown, but he postulated that it acted as a safety valve, whereby animals with uremia could destroy ex­ cess blood urea with gastric urease and vomit up the ammonia-laden secretions. Thus, uremic vomiting could be explained by the presence of the urease en­ zyme. Gastric urease in humans was the subject of a thesis by Fitzgerald and Murphy published in the Irish Journal of Medical Science in 1950 (10). They could not perform endoscopy, however, so most of the spec­ imens again came from patients who were having par­ tial gastrectomies for peptic ulcer disease or gastric cancer. Thus, almost all gastric specimens had urease present. Fitzgerald and Murphy went a step further, measuring urea levels in the gastric juice and propos­ ing that urea could be used as an antacid, since it was broken down into alkaline, ammonia, and bicarbon­ ate. Again, Fitzgerald made the mistake of assuming the urease was part of the gastric mucosa, probably because he had inadequate control material. Fitzger­ ald's research resulted in trials of urea in patients with refractory peptic ulcer disease, but the amounts of urea required to be taken by the patients were in the range of several grams and tended to cause severe side effects such as vomiting. A milestone in the study of gastric urease came in the late 1950s when Charles Lieber and his colleague

21

Lefevre in Belgium and subsequently in New York studied the gastric urease of alcoholics. The interest was that this urease could contribute to blood ammo­ nia levels and therefore induce hepatic encephalopa­ thy in persons with cirrhosis. They measured the gas­ tric urea and ammonia concentrations by aspirating gastric juice from their subjects and showed that tetra­ cycline caused reversal of the normal ratio. That is, whereas "normal" persons had urea nitrogen present mainly as ammonia, treatment with antibiotics re­ sulted in urea nitrogen being present in gastric juice, mainly as urea. Thus, they had evidence for a bacterial origin of gastric urease and justification for the treat­ ment of encephalopathic persons with antibiotics, in­ cluding neomycin, kanamycin, and other antibiotics. Their work was largely forgotten, because subsequent studies of hepatic encephalopathy and antibiotic use focused mainly on the presence of urease in the colon. Gastric urease remained a little-studied phenomenon until the high urease production of H. pylori and other helicobacters was observed first by Langenberg (23). This and the observations of others led to the wide­ spread use of the currently available rapid urease tests (e.g., the CLOtest 31), a simple diagnostic for detect­ ing H. pylori by showing presence of its urease enzyme in gastric tissue. Knowledge of gastric urease and observations on the urea concentration in gastric juice by Marshall and Langton in 1984 (29) led to the development of the urea breath test, a noninvasive form of detecting gastric urease (14, 30).

BISMUTH SALTS F O R GASTRIC DISEASE The final thread tracking the history of H. pylori is that associated with bismuth use. Bismuth com­ pounds were used in ancient times, along with other heavy metals, as an antiseptic and cosmetic. From the 17th century, bismuth, antimony, and mercury were used in various concoctions, usually to induce purg­ ing. In retrospect, prior to the 20th century, almost all individuals were infected with H. pylori (2), so it was only a matter of time before a patient with severe gastric symptoms underwent sudden remission after being treated with large doses of oral heavy metal salts. As a result, by the 19th century, bismuth com­ pounds were advocated in the form of bismuth subnitrate, subcarbonate, and subcitrate for the treatment of nonspecific gastrointestinal symptoms. Ogle de­ scribed "the fruit acid of bismuth" for "nervous disor­ ders of the bowels" (35). His formulation may have been the precursor to the modern drugs De-Nol and ranitidine bismuth citrate. In the United States, a pat­ ent medicine called Bismosal was devised by the Nor-

22

MARSHALL

wich Company. Bismosal was a suspension of bis­ muth subsalicylate, which, unlike some other salts of bismuth, for example, the subnitrate, was relatively insoluble and had low toxicity. Bismosal was used for treating infantile cholera (probably Campylobacter jejuni infection) and, according to the packet insert, was very useful for patients with gastritis. Subsequent experiments confirmed that bismuth was extremely toxic to helicobacter and Campylobacter species, and also had inhibitory effects on many other gut organ­ isms (8, 2 7 ) . As well as bismuth, 19th-century physi­ cians used other exotic metals from time to time for severe gastric disturbance, even going so far as silver nitrate gastric lavage for the treatment of refractory gastritis. Whereas German antacid formulations contin­ ued to contain some bismuth, usually in the form of bismuth subnitrate and bismuth subcarbonate, until modern times, American formulations of antacids were without bismuth after 1 9 5 0 . This deliberate omission resulted from the work of Ivy, who observed that these salts were quite poor buffers and therefore contributed very little to the antacid effect. Neverthe­ less, the grandson of Bismosal, Pepto-Bismol, gradu­ ally came into wide use as an over-the-counter medi­ cation for the treatment of nonspecific episodes of vomiting and diarrhea in the United States. It is inter­ esting to note that double-blind clinical trials of ulcer relapse in the United States always saw far fewer re­ lapses than similar studies performed in Europe. Since antibiotics and Pepto-Bismol were never restricted in U.S. studies, it is quite possible that many of the ulcer patients in both the active and placebo groups were freely taking doses of Pepto-Bismol during their fol­ low-up period. In the mid-1970s, the Gist-Brocades Company of Holland, which manufactured and sold bismuth subcitrate solution at that time, developed a tablet form that was far more palatable and, there­ fore, could be widely marketed. However, by that time, bismuth had been banned in France because of chronic overdose and heavy metal toxicity in thou­ sands of people in the 1950s. However, in countries that allowed bismuth use, the drug De-Nol demon­ strated a lower ulcer relapse rate than was seen in patients who merely had acid reduction therapy with H2 blockers such as cimetidine. The low relapse rate, or even absence of relapse in one-third of patients taking the bismuth treatment, suggested that the ulcer problem had been totally cured in these persons. War­ ren and Marshall took this as supportive evidence to­ ward the hypothesis that H. pylori had been eradi­ cated in these persons and that the organism was responsible for the ulcer in the first place. Further evi­ dence of this was obtained in publications by Gregory, Moshal, and Spitaels (16) showing the improved heal­ ing of duodenal ulcer borders after treatment with

bismuth versus treatment with H2 blockers. In their illustrations of the electron micrographic appearance of these duodenal ulcer borders, Gregory and col­ leagues showed numerous helicobacters in the pretreatment patients, but not in the posttreatment pa­ tients, again supporting the developing hypothesis. During the 1980s, as the links between H. pylori urease, epidemic gastritis, and peptic ulcer were be­ coming recognized, investigators tried to explain some of the more subtle features of peptic ulcer dis­ ease on the basis of the helicobacter infection. The most important of these may have been that gastritis, by suppressing D-cell function and somatostatin re­ lease in the antral mucosa, could lead to lifelong mild hypergastrinemia in some persons, thus explaining the shift of the normal acid secretion distribution to the right in populations of patients with peptic ulcer dis­ ease (12, 38). The supposed "hereditary" predisposi­ tion of some families for peptic ulcer, previously ex­ plained on the basis of stress and inherited manifestations such as elevated serum pepsinogens (41), could now also be explained by H. pylori, since serum pepsinogens reflected inflammation in the gas­ tric mucosa and reduced once the bacterium had been eradicated. SYNTHESIS: WARREN AND MARSHALL 1979-84 The observations of Warren and Marshall be­ tween 1979 and 1984 allowed these and other investi­ gators to tie together the various threads that had been constructed in the medical literature in the preceding 100 years. Warren had observed patients with spiral organisms on their gastric mucosa since 1979 and had documented the inflammation associated with the bacteria by the time he and Marshall began a con­ certed attempt to study the organisms in patients with various upper gastrointestinal symptoms. After Au­ gust 1981, the team studied patients attending for en­ doscopy and were able to demonstrate the gram-nega­ tive bacteria on Gram stains but could not culture them at that time. They tentatively treated one patient with tetracycline and were able to observe a decrease in the number of neutrophils in the gastric mucosa as well as apparent disappearance of the bacteria. They recognized, however, that anecdotal evidence of the bacteria's role in gastric inflammation was of little value and therefore commenced a study in 100 con­ secutive endoscopy patients to try to culture the bacte­ ria, as well as determine their association with gastri­ tis and/or other clinical syndromes. Initially, they did not focus specifically on the etiology of peptic ulcer disease, although they were aware that gastritis was strongly associated with duodenal and gastric ulcers,

CHAPTER 3 • H. PYLORI AND PEPTIC ULCER DISEASE

as well as with gastric cancer (47). Thus, in the begin­ ning of 1 9 8 2 , Marshall and Warren commenced a study whereby patients attending for endoscopy were biopsied, after signing the appropriate consent and completing a questionnaire detailing dental hygiene, dietary habits, nonsteroidal anti-inflammatory drug and antacid use, etc. Only two biopsies were taken from the antrum: one was for culture using a variety of techniques but mainly microaerobic incubation similar to that used for Campylobacter; the second

biospsy was sent for histological examination by Dr. Warren using hematoxylin and eosin and WarthinStarry silver stains. It was not until patient 35 was biopsied that the organism was grown. That event came about because of a lucky accident, in which the cultures were left in the incubator over the long Easter weekend and thus the plates were not examined until the fourth or fifth day after biopsy. When the waterspray 1 mm transparent colonies were observed, the technologist realized in hindsight that, prior to this day, the research biopsies had been discarded after 48 h when normal gastrointestinal or throat specimens would have been expected to be overgrown with com­ mensal flora and thus would have been useless for any further diagnostic purpose. This rule did not apply to H. pylori cultures, however, because they were from clean gastric biopsy specimens and, in most cases, there were few throat commensals on the nonselective blood agar plates. After that time, H. pylori could be grown using microaerobic techniques on blood or agar plates with relative ease in Australia. The organ­ ism was first cultured outside Australia in September 1983 by McNulty and Skirrow from a gastric ulcer patient in Worchester, England (33). In the initial 100 patients studied by Marshall and Warren, more than 6 5 % of patients were infected with the organism, and almost all of them had gastritis (P < 0.000001). Of particular interest to the investi­ gators, however, was the fact that all 13 patients with duodenal ulcer in that study were found to have the organism, and 18 of 2 2 gastric ulcer patients also had the organism. Ulcer patients without H. pylori tended to be taking nonsteroidal anti-inflammatory drugs. This finding accelerated interest in the organism among gastroenterologists, and the results were con­ firmed in several countries within a year or so (22, 33). Double-blind trials had limited success in the en­ suing 5 years because the eradication rates of the available therapies (bismuth with one antibiotic, for example) were rather low, so that in a large study, only about one-quarter of treated patients actually achieved H. pylori eradication. Nevertheless, Coghlan et al. (6), Rauws and Tytgat (40), and Graham et al. (15) confirmed that H. pylori eradication cured peptic ulcer.

23

QUESTIONS F O R T H E F U T U R E Now that the H. pylori link with peptic ulcer and the role of antibiotic therapy in this disease have been accepted, therapies have advanced to the point that almost all patients can be eradicated of the bacteria with antibiotic combinations that include an acidlowering or bismuth component. Associated diseases such as gastric cancer, and even gastric lymphoma, continue to excite interest, and Koch's postulates have been fulfilled for peptic ulcer and gastric cancer in an animal model (the Mongolian gerbil) (48). Neverthe­ less, controversy still reigns over H. pylori. Is it always a pathogen, or is it sometimes a commensal? Is there a beneficial role for the organism in some persons or populations, as has been suggested by Blaser and oth­ ers (3)? If H. pylori is a harmful pathogen, how harm­ ful? Is it cost-effective to eradicate all H. pylori or just toxin producers? These and other interesting ques­ tions are addressed by other authors in this volume. REFERENCES 1. Bizzozero, G. 1893. Ueber die schlauchformigen drusen des magendarmkanals und die bezienhungen ihres epithels zu dem oberflachenepithel der schleimhaut. Arch. Mikr Anat. 42:82. 2. Blaser, M. J. 1997. The versatility of Helicobacter pylori in the adaptation to the human stomach. /. Physiol. Pharmacol. 48:307-314. 3. Blaser, M. J. 1997. Not all Helicobacter pylori strains are cre­ ated equal: should all be eliminated? Lancet 349:1020-1022. 4. Castiglioni, A. 1947. A History of Medicine, 2nd ed. Alfred A. Knopf, New York, N.Y. 5. Chen, M., A. Lee, and S. Hazell. 1995. Immunisation against gastric helicobacter infection in a mousdHelicobacter felis model. Lancet 339:1120-1121. 6. Coghlan, J. G., D. Gilligan, H. Humphries, D. McKenna, C. Dooley, E. Sweeney, C. Keane, and C. O'Morain. 1987. Campylobacter pylori and recurrence of duodenal ulcers—a 12-month follow-up study. Lancet ii:l 109—1111. 7. Doenges, J. L. 1939. Spirochetes in the gastric glands of macacus rhesus and of man without related diseases. Arch. Pathol. 27:469-477. 8. Ericsson, C. D., H. L. DuPont, and P. C. Johnson. 1986. Nonantibiotic therapy for travelers' diarrhea. Rev. Infect. Dis. 8(Suppl. 2):S202-S206. 9. Figura, N., and G. Orderda. 1996. Reflections on the first description of the presence of Helicobacter species in the stom­ ach of mammals. Helicobacter 1:4-5. 10. Fitzgerald, O., and P. Murphy. 1950. Studies on the physiolog­ ical chemistry and clinical significance of urease and urea with special reference to the stomach. Ir. J. Med. Sci. 292:97-159. 11. Freedberg, A. S., and L. E. Baron. 1940. The presence of spiro­ chetes in human gastric mucosa. Am. J. Dig. Dis. 7:443-445. 12. Gibbons, A. H., S. Legon, M. W. Walker, M. Ghatei, and J. Calam. 1997. The effect of gastrin-releasing peptide on gastrin and somatostatin messenger RNAs in humans infected with Helicobacter pylori. Gastroenterology 112:1940-1947. 13. Gledhill, T., R. J. Leicester, B. Addis, N. Lightfoot,J. Barnard, N. Viney, D. Darkin, and R. H. Hunt. 1985. Epidemic hypochlorhydria. Br. Med. J. Clin. Res. Ed. 290:1383-1386.

24

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14. Graham, D. Y., P. D. Klein, and D. J. J. Evans, et al. 1987. Campylobacter pylori detected noninvasively by the 13C-urea breath test. Lancet i:1174-1177. 15. Graham, D. Y., G. M. Lew, D. G. Evans, D. J. Evans, Jr., and P. D. Klein. 1991. Effect of triple therapy (antibiotics plus bismuth) on duodenal ulcer healing. A randomized controlled trial. Ann. Intern. Med. 115:266-269. 16. Gregory, M. A., M. G. Moshal, and J. M. Spitaels. 1982. The effect of tri-potassium di-citrato bismuthate on the duodenal mucosa during ulceration. An ultrastructural study. S. Afr. Med. J. 62:52-55. 17. Harford, W. V., C. Barnett, E. Lee, G. Perez-Perez, M. J. Blaser, and W. L. Peterson. Acute gastritis with hypochlorhy­ dria: report of 35 cases with long-term follow-up. Gut, in press. 18. Heilmann,K. L., and F. Borchard. 1991. Gastritis due to spiral shaped bacteria other than Helicobacter pylori: clinical, histo­ logical, and ultrastructural findings. Gut 32:137-140. 19. Hirschowitz, B. I., D. H. P. Streeten, J. A. London, and H. M. Pollard. 1956. A steroid induced gastric ulcer. Lancet ii: 1081-1083. 20. Ito, S. 1967. Anatomic structure of the gastric mucosa, p. 705-741. In C. F. Code (ed.), Alimentary Canal. American Physiological Society, Washington, D.C. 21. Krienitz, W. 1906. Ueber das Auftreten von Spirochaeten verschiedener Form im Mageninhalt bei Carcinoma ventriculi. Dtsch. Med. Wochenscbr. 32:872. 22. Lambert, J. R., K. L. Dunn, E. R. Eaves, M. G. Korman, J . Hansky, and K. J. Pinkard. 1985. Pyloric CLO in the human stomach. Med. J. Aust. 143:174. (Letter.) 23. Langenberg, M. L., G. N. J. Tytgat, M. E. I. Schipper, P. J. G. M. Rietra, and H. C. Zanen. 1984. Campylobacter-like organisms in the stomach of patients and healthy individuals. Lancet i:1348. 24. Lee, A., S. L. Hazell, J . O'Rourke, and S. Kouprach. 1988. Isolation of a spiral-shaped bacterium from the cat stomach. Infect. Immun. 56:2843-2850. 25. Lockard, V. G., and R. K. Boler. 1970. Ultrastructure of a spiraled micro-organism in the gastric mucosa of dogs. Am.}. Vet. Res. 31:1453-1462. 26. Luck, J. M., and T. N. Seth. 1924. Gastric urease. Biochem. J. 18:1227-1231. 27. Marshall, B. J., J. A. Armstrong, G. J. Francis, N. T. Nokes, and S. H. Wee. 1987. Antibacterial action of bismuth in rela­ tion to Campylobacter pyloridis colonization and gastritis. Digestion 37(Suppl. 2):16-30. 28. Marshall, B. M., J. A. Armstrong, D. B. McGechie, and R. J. Glancy. 1985. Attempt to fulfil Koch's postulates for pyloric Campylobacter. Med. J. Aust. 142:436-439. 29. Marshall, B. J., and S. R. Langton. 1986. Urea hydrolysis in patients with Campylobacter pyloridis infection. Lancet i: 965-966. 30. Marshall, B. J., and I. Surveyor. 1988. Carbon-14 breath test for the diagnosis of Campylobacter pylori associated gastritis. /. Nucl. Med. 29:11-16. 31. Marshall, B. J., J. R. Warren, G. J. Francis, S. R. Langton, C. S. Goodwin, and E. Blincow. 1987. Rapid urease test in the

32.

33. 34.

35. 36. 37. 38.

39.

40. 41.

42. 43.

44. 45. 46.

47. 48. 49.

management of Campylobacter pyloridis-associated gastritis. Am. J. Gastroenterol. 82:200-210. Matysiak-Budnik, T., F. Brief, M. Heyman, and F. Megraud. 1995. Laboratory-acquired Helicobacter pylori infection. Lan­ cet 346:1489-1490. (Letter.) McNulty, C. A. M., and D. M. Watson. 1984. Spiral bacteria of the gastric antrum. Lancet i:1068-1069. Morris, A. J., M. R. AH, G. I. Nicholson, G. I. Perez Perez, and M. J . Blaser. 1991. Long-term follow-up of voluntary ingestion of Helicobacter pylori. Ann. Intern. Med. 114: 662-663. Ogle, J. W. 1964. Effervescing bismuth water. Br. Med. J. 1: 249-250. Osier, W., and T. McCrae (ed.). 1920. The Principles and Practice of Medicine, 9th ed. Appleton, New York, N.Y. Palmer, E. D. 1954. Investigation of the gastric mucosa spiro­ chetes of the human. Gastroenterology 27:218-220. Peterson, W. L., C. C. Barnett, D. J. Evans, Jr., M. Feldman, T. Carmody, C. Richardson, J. Walsh, and D. Y. Graham. 1993. Acid secretion and serum gastrin in normal subjects and patients with duodenal ulcer: the role of Helicobacter pylori. Am. ] . Gastroenterol. 88:2038-2043. Ramsey, E. J., K. V. Carey, W. L. Peterson, J. J. Jackson, F. K. Murphy, N. W. Read, K. B. Taylor, J. S. Trier, and J. S. Fordtran. 1979. Epidemic gastritis with hypochlorhydria. Gas­ troenterology 76:1449-1457. Rauws, E. A., and G. N. Tytgat. 1990. Cure of duodenal ulcer associated with eradication of Helicobacter pylori. Lancet 335: 1233-1235. Rotter, J. I., J. Q. Sones, I. M. Samloff, C. T. Richardson, J. M. Gursky, J. H. Walsh, and D. L. Rimoin. 1979. Duodenalulcer disease associated with elevated serum pepsinogen I: an inherited autosomal dominant disorder. N. Engl. J. Med. 300: 63-66. Salomon, H. 1896. Ueber das spirillum saugetiermagens und sien verhalten zu den belegzellen (abstract 1). Zentralbl. Bakteriol. 19:433-442. Steer, H. W., and D. G. Colin-Jones. 1975. Mucosal changes in gastric ulceration and their response to carbenoxolone sodium. Gut 16:590-597. Steer, H. W. 1984. Surface morphology of the gastroduodenal mucosa in duodenal ulceration. Gut 25:1203-1210. Sumner, J. B. 1926. The isolation and crystallization of the enzyme urease./. Biol. Chem. 69:435-441. Takata, T., T. Shirotani, M. Okada, M. Kanda, S. Fujimoto, and J. Ono. 1998. Acute hemorrhagic gastropathy with multi­ ple shallow ulcers and duodenitis caused by a laboratory infec­ tion of Helicobacter pylori. Gastrointest. Endosc. 47: 291-294. Warren, J . R., and B. Marshall. 1983. Unidentified curved bacilli on gastric epithelium in active chronic gastritis. Lancet i:1273-1275. Watanabe, T., M. Tada, H. Nagai, S. Sasaki, and M. Nakao. 1998. Helicobacter pylori infection induces gastric cancer in Mongolian gerbils. Gastroenterology 115:642-648. Wiersinga, W. M., and G. N. Tytgat. 1977. Clinical recovery owing to parietal cell failure in a patient with Zollinger-Ellison syndrome. Gastroenterology 73:1413-1417.

II. BACTERIOLOGY

Helicobacter pylori: Physiology and Genetics Edited by H . L. T. Mobley, G. L. Mendz, and S. L. Hazell C 2 0 0 1 ASM Press, Washington, D.C.

Chapter 4

Basic Bacteriology and Culture LIEF PERCIVAL ANDERSEN AND TORKEL WADSTROM

CAMPYLOBACTERIACEAE In 1957 E. O. King (81) described human infec­ tions due to members of the Campylobacteriaceae, with high mortality in patients with septicemia and meningitis caused by Vibrio fetus and "related vib­ rios." Taxonomic studies in the early 1960s (133, 155) introduced a new genus, Campylobacter, that included microaerobic vibrios that did not resemble Vibrio cholerae. Vibrio fetus was renamed as Campy­ lobacter fetus and the "related vibrios" included Campylobacter jejuni and Campylobacter coli. In the 1970s Butzler et al. isolated C. jejuni frequently from fecal cultures of children with diarrhea (23), and Skirrow (136) introduced a selective medium for stool cultures for isolating Campylobacter spp., which was a breakthrough in the research of thermophilic Campylobacter species. The family Campylobacteria­ ceae was proposed by Vandamme et al. in 1991 (154). This family includes the genera Campylobacter, Arcobacter, and Helicobacter as well as Tbiovulum (87), Wollinella (164), "Flexispira" (7), two Bacteroides species (B. gracilis and B. ureolyticus) (119), and the closely related Anaerobiospirillum (22). Taxonomic studies (152, 153, 154) divided the genus Campylo­ bacter into three new genera: (i) Campylobacter in­ cluding B. gracilis ( 1 3 8 , 1 5 2 ) , (ii) Helicobacter includ­ ing "Flexispira," and (iii) Arcobacter, Wollinella, Tbiovulum, and Anaerobiospirillum were reported as separate genera.

T H E GENUS

HELICOBACTER

after corrected to C. pylori (91, 93). New intestinal CLOs were discovered at the same time, and C. pylori was sometimes referred to as gastric CLO (GCLO) and GCLO-1 when another CLO (GCLO-2, C. jejuni subsp. doylei) was isolated from the human stomach (78, 152). It soon became clear that even though C. pylori resembles Campylobacter in many aspects, it differs in important features such as flagellum mor­ phology, fatty acid content, and 16S rRNA sequence (53, 5 4 , 9 2 , 127). C. pylori was transferred to a new genus, Helicobacter, and named Helicobacter pylori in 1989, together with Campylobacter fenelliae and Campylobacter cinaedae (52). During the last decade, the genus Helicobacter has expanded tremendously and new species are regu­ larly included. The majority of these new Helico­ bacter species are found in the stomachs and intestines of different animals (7, 2 1 , 3 5 , 4 0 , 4 6 , 4 8 , 4 9 , 5 0 , 5 1 , 52, 5 8 , 7 1 , 89, 9 1 , 9 3 , 9 6 , 144). Cell Biology of H.

pylori

Factors involved in colonization and adhesion Several virulence factors for gastric colonization, tissue damage, and survival have been identified in H. pylori (Table 1). Flagella, urease, and adhesins are all essential factors for H. pylori to colonize the gastric mucosa. Mutants of H. pylori without flagella or without urease are unable to colonize the gastric mu­ cosa in laboratory animals (41, 4 2 ) . Flagella and motility. The curved morphology of H. pylori and the polar motility caused by flagella in one end cause screw-like movements, which may enable the organism to penetrate the mucin layer. The motility of H. pylori is increased when the viscosity

When a new slow-growing Campylobacter-iike organism (CLO) was cultured by Marshall in 1982, it was classified as Campylobacter pyloridis and shortly

Lief Percival Andersen • Department of Clinical Microbiology 7806, National University Hospital (Rigshospitalet), Tagensvej 20, DK2200, Copenhagen, Denmark. Torkel Wadstrom • Department of Infectious Diseases and Medical Microbiology, University of Lund, Sweden.

27

28

ANDERSEN AND WADSTROM

Table 1. Virulence factors identified in H. pylori Virulence factor Colonizing Flagella Urease Adhesins Tissue damaging Proteolytic enzymes 120-kDa cytotoxin (Gac A) Vacuolating cytotoxin (Vac A)

Effect Active movements through mucin Neutralization of acid Anchoring H. pylori to epithelium Glucosulfatase degrades mucin Related to ulcer and severe gastritis Damage of the epithelium

Urease

Toxic effect on epithelial cells, disrupting cell tight junctions Phospholipase A Digest phospholipids in cell membranes Alcohol dehydrogenase . . . Gastric mucosal injury Survival Intracellular surveillance Prevent killing in phagocytes Superoxide dismutase . . . . Prevent phagocytosis and killing Catalase Prevent phagocytosis and killing Coccoid forms Dormant form Heat shock proteins Urease Sheathing antigen Other Lipopolysaccharide Low biological activity Lewis X/Y blood group homology Autoimmunity

of the media is increased in vitro and transverses a methyl glucose solution 10 times more efficiently than Escherichia coli (61), but the motility is pH dependent and impaired at a pH below 4 (97). Urease, catalase, superoxide dismutase, and alkylhydroperoxidase reductase. Urease is one of the key enzymes in H. pylori pathogenesis. It has a molec­ ular weight of 5 5 0 kDa and consists of three subunits of 26.5 kDa (Ure A), 61 kDa (Ure B), and 13 kDa (Ure C) (84, 100). Urease is necessary for H. pylori to maintain a pH-neutral microenvironment around the bacteria, necessary for survival in the acidic stom­ ach (59, 123). Urease induces self-destruction of H. pylori in vitro in nonacidic media (110). Urease is strongly immunogenic and chemotaxic for phago­ cytes. Superoxide dismutase has been isolated from H. pylori, which breaks down superoxide produced in polymorphonuclear leukocytes and macrophages and thereby prevents the killing of these organisms (143). Catalase protects H. pylori against the damaging ef­ fects of hydrogen peroxide released from phagocytes (60). Urease and catalase may be excreted from H.

pylori to the surrounding environment and may pro­ tect this pathogen from the humoral immune response (59). Outer membrane proteins, phospholipids, glycolipids, and other adhesins. H. pylori adheres to mucin and binds specifically to gastric mucosa epithelial cells both in vivo and in vitro ( 4 5 , 6 4 , 1 5 0 ) . Different adhe­ sion patterns, which are different in children and in adults, have been described (14). Several putative gas­ tric tissue receptor structures have been described for H. pylori such as (i) sialoglycoconjugates in gastric mucins and on epithelial cells, phagocytes, and extra­ cellular matrix (43, 6 9 , 1 5 7 ) , (ii) sulfated glycoconjugates such as heparan sulfate and other glycosaminoglycans (158), and (iii) sulfatides (9, 77, 1 3 0 , 151) and various sialylated and nonsialylated glycolipids (97). Binding of H. pylori to cell surface fucosylated blood group antigens, H I and Lewis part of the blood group O in the ABO system, was first described by Boren and collaborators (18) and was shown to me­ diate adherence to human and rhesus monkey gastric tissue surface cells (19). More recently, Petersson and collaborators (124) identified the H. pylori blood group antigen-binding adhesins, Bab A and Bab B, purified the proteins, and cloned the babA and babB genes. Two closely related basic proteins of 78 kDa were characterized. The sialic acid-specific lectins of 19 and 23 kDa have also been purified and the corre­ sponding genes cloned (T. Boren, personal communi­ cation). These proteins, unlike BabA and -B, do not belong to the family of proteins, mostly named helico­ bacter outer membrane proteins by a nomenclature introduced by T. J . Trust and associates (see chapter 7). Cell surface adhesins recognizing sulfatides were not identified, whereas heparan sulfate binding pro­ teins (HeBPs) were purified and characterized by Utt etal. (151; unpublished). None of the glycolipid-binding surface proteins has been purified and character­ ized yet. Interestingly, no similar putative mucin and cell adhesin has been identified in H. felts or other newly identified species, except for H. mustelae (P. O'Toole and T. Trust, personal communication). In vitro affinity binding studies for the Lewis binding give high-affinity constants (K , - 2 . 5 ; 10 to 11 M) and also reveal high affinity for human mucin-binding glycoconjugates to hemagglutinating sialic acid-spe­ cific adhesins or lectins (SALs) (157). Recent studies by Syder et al. (139) in a transgene mouse model sug­ gest that SALs may become key adhesins in inflamed gastric mucosa. It is noteworthy that these SALs are produced already in the exponential growth phase while the Bab A and B adhesins appear mainly in the stationary phase cells. SALs and HeBPs may be the key receptors on leukocytes and macrophages, and a

CHAPTER 4 • BASIC BACTERIOLOGY AND CULTURE

trigger lectinophagocytosis as for several other micro­ bial pathogens (158). Factors involved in tissue damage and survival factors Enzymes: protease, etc. Conflicting results have been reported concerning proteolytic enzymes in H. pylori. It seems probable that H. pylori glycosulfatase degrades gastric mucin ( 1 3 5 , 1 3 7 ) . H. pylori possesses phospholipase A, which can digest phospholipids of cell membranes (86). Urease has a cytotoxic activity (61, 98). Recently, alcohol dehydrogenase has been described to contribute to gastric mucosal injury (128). Toxins: vacuolating cytotoxin A, lipopolysaccharide. H. pylori contains a toxin, Vac A, which can produce vacuoles in gastric epithelial cells and has been related to peptic ulcer, severe gastritis (25, 3 1 , 32, 1 3 1 , 147), and mucosal integrity (47). Lipopolysaccharide (LPS) in H. pylori has a low biological ac­ tivity as compared to LPS from other gram-negative bacteria (30, 112), which may be explained by the unusual composition of lipid A (95). LPS from H. pylori stimulates phenotypic transcription and func­ tional changes in monocytes (112). Assays using gas­ tric mucosal laminin (integrin) receptor binding to a laminin-coated surface have revealed a significant de­ crease in receptor binding occurring in the presence of H. pylori LPS (126). When the gastric epithelial barrier is weakened by disruption of the mucosal sur­ face cells and the extracellular matrix, LPS is responsi­ ble for a marked increase in epithelial cell apoptosis (126). LPS from H. pylori has recently attracted inter­ est because LPS from most strains mimic Lewis and/ or Lewis blood group antigens (102). This mimicry may play a role in the regulation of the immune re­ sponse and induce autoantibodies against the gastric proton pump. Other putative virulence factors. Several heat shock proteins (Hsp) such as 58.2-kDa Gro-El (Hsp B), 13-kDa Gro-Es (Hsp A), and a 70-kDa Hsp have been identified in H. pylori (37, 4 4 , 106). Hsp are produced by all cells and are involved in stabilizing and probably repairing proteins under harsh condi­ tions that may be important to survive in the stomach. H. pylori transforms into coccoid forms (15, 2 4 , 39) under certain conditions such as nutrient starva­ tion and media containing growth inhibitors (bis­ muth, proton pump inhibitor, or certain antibiotics). These coccoid forms have been reported to survive for several years in river water and have been proposed by

29

some to be an important factor for transmission, by fecal excretion, and for therapy failure. Microscopy and Growth of H. pylori Specimens for culture of H. pylori H. pylori is the microorganism most frequently found in the human gastric mucosa in association with gastric epithelial cells, but other curved bacteria have also been found in the human gastric mucosa: C. jejuni subsp. doylei (GCLO-2) ( 5 5 , 7 8 , 1 4 0 ) , "Heli­ cobacter beilmannii" ("Gastrospirilium hominis," "H. germanium") (34, 6 3 , 6 6 ) , and H. felis (120). In our hospital laboratory, Campylobacter sputorum, Campylobacter upsaliensis, and Selenomonas species have occasionally been cultured from human gastric biopsies (L. P. Andersen, unpublished data). Some of these microorganisms may be difficult to distinguish by routine laboratory methods. Apart from H. pylori, "H. beilmannii" is the most common bacterium in human gastric mucosa, with a prevalence of up to 0 . 5 % in dyspeptic patients in western Europe (66). "H. beilmannii" is usually found in the foveolae asso­ ciated with mild chronic gastritis whereas H. pylori is usually found on the surface epithelium associated with severe gastritis. The contact with epithelial cells is usually more superficial for "H. beilmannii" than for H. pylori (66). Occasionally, "H. beilmannii" and H. pylori are found simultaneously (34). Gastric specimens. H. pylori is most regularly found in the antral part of human gastric mucosa of untreated persons. In persons treated with acid-suppressive drugs (proton pump inhibitors and H antag­ onists) H. pylori may be present in higher numbers in the body of the stomach. H. pylori is more frequently found in gastric antrum than in duodenal biopsies even in persons with duodenitis and duodenal ulcer (about 5 0 % ) . H. pylori can only be cultured from gastric juice in about 1 5 % of persons with H. pylori cultured from gastric antrum and from less than 5 0 % of esophageal biopsies from untreated persons with esophagitis, even though H. pylori can be cultured from gastric antrum (1, 3 ) . Thus, it is always impor­ tant to obtain antral biopsies as well as corpus biop­ sies from persons recently treated with acid-suppressive drugs, whereas duodenal and esophageal biopsies and gastric juice are of less importance for routine diagnostics but may be useful for PCR diagnostics (163) and special research purposes. The number of biopsies necessary to diagnose H. pylori by culture has been estimated in a study where more than 9 5 % of H. pylori was cultured from one antral biopsy. For optimal results at least four biopsies 2

30

ANDERSEN AND WADSTROM

should be cultured (108). It is generally accepted, ac­ cording to the modified Sydney classification of gas­ tritis (36), that at least one biopsy from antrum and two biopsies from corpus should be taken for culture to ensure a sufficient diagnosis. Extragastric specimens. H. pylori has occasion­ ally been cultured from ectopic gastric mucosa in Meckel's diverticulum, esophagus, rectum, urinary bladder, dental plaque, and feces ( 1 2 , 2 8 , 3 8 , 7 9 , 1 2 2 , 1 4 6 , 1 4 8 ) . Recently, H. pylori has also been detected by PCR in specimens from the gallbladder and liver (114). H. pylori has mainly been identified by PCR in dental plaque, liver specimens, and fecal specimens. Biopsies should be taken from sites with gastric meta­ plasia and dental plaque or gingival crest. No system­ atic studies have been carried out to recommend opti­ mal sample sites for extragastric H. pylori infections. Several Helicobacter and Campylobacter species are harbored in the mouth and intestine. Culture-con­ firmed microbiological identification is preferable to ensure the bacteriological diagnosis of isolates from these sites, at least until molecular biological methods have been better evaluated than they are today. Microbiological detection of H.

pylori

Primary microscopy of helicobacter-like organ­ isms. Microscopy of gram-stained smears or imprint of gastric biopsies reveals curved gram-negative rods resembling Helicobacter in 60 to 1 0 0 % of the culturepositive biopsies (1, 3, 7 3 , 1 0 1 , 109, 111). "H. heilmannii" and H. felis are easy to distinguish from H. pylori by microscopy because of the long corkscrew shape of "H. heilmannii" and H. felis (5, 6 3 , 6 6 , 88). In smears, helicobacter-like organisms are usually seen unevenly distributed in clusters or rows along the epithelium cells. The presence of polymorphonuclear leukocytes is not always a dominant feature in smears. Phase-contrast microscopy (125) and other staining methods such as silver stain (118) and acridine orange have been described. The probability of detecting H. pylori by microscopy may be increased by using spe­ cific immunofluorescence or peroxidase conjugated antibodies to H. pylori. The atmosphere for culture of H. pylori. In gen­ eral, primary cultures of H. pylori have less oxygen tolerance than most Campylobacter species, with a growth maximum at 3 to 7% of 0 . H. pylori is usu­ ally grown in jars with gas-generation kits (4, 85, 94) or a standard microaerobic atmosphere, in C O 2 incu­ bators or anaerobic chambers with a microaerobic atmosphere. Most studies with standardized atmo­ 2

spheres for culture of H. pylori have used 2 to 5 % 0 , 5 to 1 0 % (optimal closer to 1 0 % ) C 0 , and 0 to 1 0 % H ( 1 - 3 , 5, 6, 7 3 , 109, 111). H. pylori strains are variable in their growth re­ sponse to different culture conditions, but no system­ atic studies on the different atmospheres and growth systems have been published. In our hands, the atmo­ spheres produced by gas-generation kits were un­ stable for reliable culture of H. pylori when the gas generator was changed every third day (L. P. Ander­ sen, unpublished data). Other laboratories have used this technique successfully by changing the gas gener­ ator every day (F. Megraud, personal communica­ tion). No quantitative differences were found between atmospheres with (jars) and without (chambers) H (L. P. Andersen, unpublished data). Subcultures of H. pylori can rapidly be adapted to grow anaerobically or in a standard C 0 mixture ( 1 8 % 0 , 1 0 % C 0 ) in an incubator even under aerobic conditions (149). 2

2

2

2

2

2

2

Nonselective and selective media for growing H. pylori. H. pylori can grow on different solid media containing blood or blood products (blood or lysed blood agar plates). Most studies have used Brucella agar or Columbia agar as the agar base. An amount of 7 to 1 0 % blood improves the growth of H. pylori as compared with 5 % blood. Horse blood may also improve the growth of H. pylori as compared to sheep blood (33, 83). Supplement of agar with cyclodextrin B can be used for blood-free culture media for H. pylori, but with large differences between different batches of cyclodextrin (116). Egg yolk emulsion agar has also been described as a blood-free medium for growth of H. pylori (161). In a small study, egg yolk emulsion as a liquid medium was compared with four other liquid media described in the literature and it was found superior with regard to growth rate of H. pylori, but it contained too many non-H. pylori pro­ teins to be useful for antigen production (A. K. V. Jensen, unpublished data). Often H. pylori grows poorly or not at all on selective media containing antibiotics. Skirrows and Dents selective media seem to be the best available commercial selective media and have been used in sev­ eral studies (62, 76, 7 8 , 85). There seem to be greater differences between horse and sheep blood agar, in favor of horse blood, than between horse blood agar with and without antibiotics (33, 83). By comparing agar plates containing 5 % horse blood, 1 0 % horse blood, 7% lysed horse blood, 7% lysed horse blood with trimethoprim, and selective Campylobacter plates (modified Skirrows medium) (all from SSI Diagnostica, Hillerod, Denmark), we found that H. py­ lori grew with more and larger colonies on 1 0 % horse blood agar plates and 7% lysed horse blood agar

CHAPTER 4 • BASIC BACTERIOLOGY AND CULTURE

plates than on the other media, but the numbers of H. pylori-positive patients were almost equal with all media (unpublished data). Usually H. pylori grows slowly in liquid media, with formation of a high number of coccoid forms (161, 162). Contaminating microorganisms (staphy­ lococci, yeasts, etc.) usually grow much faster than H. pylori and make liquid media useless for primary culture of biopsies. Because of the risk of contami­ nated samples, a selective medium is usually recom­ mended in addition to the nonselective media for rou­ tine culture. Transportation and handling of biopsies for cul­ ture of H. pylori. Some studies have shown that suffi­ cient culture of H. pylori will be obtained after trans­ portation of the biopsies in a medium such as Stuarts transport medium for up to 2 4 h at low temperature (about 4°C), whereas a higher temperature (about 20°C) decreases the number of positive cultures signif­ icantly (82, 129, 141). Our experience is, however, that there is a more than 9 5 % concordance between culture and histological detection of H. pylori when the biopsies are inoculated on agar plates within 4 h after the biopsies are taken (1, 3). H. pylori was only cultured from about 8 0 % of biopsies with helicobacter-like organisms detected in histological sections in a study with similar culture conditions but a trans­ portation time for biopsies of up to 2 4 h (4). Thus, a decrease in culture rate of about 1 5 % was found when biopsies were transported or stored overnight. A long transportation time decreases the number of H. pylori especially after antibiotic therapy, and if the number of bacteria is low, culture may become false negative. This yield can be improved by prolonged incubation, up to 12 days. The above data are based on culture of unhomogenized biopsies by inoculating the biopsies or the agar plates and subsequently spreading the material step­ wise using the conventional technique. Grinding the biopsies 10 to 15 s before culture has been proposed to increase the number of H. pylori colonies and im­ prove H. pylori isolation. However, this method may not increase the number of positive biopsies (A. Hirschl, personal communication), and in our hands the growth of contaminations improved more than the growth of H. pylori (L. P. Andersen, unpublished data). Identification of H. pylori. H. pylori colonies are small (0.5 to 2 mm), translucent to yellowish colonies on 7% lysed horse blood agar and with translucent to pale grayish colonies of 0.5 to 1 mm in size on blood agar. In very young cultures H. pylori may ap­

31

pear as almost straight rods on microscopy. After 3 to 5 days of incubation the bacteria look pleomorphic, with irregular curved rods, several being U shaped. In old cultures, H. pylori appears as degenerative coc­ coid forms that Gram stain poorly (unpublished data). Because of their small size, H. pylori colonies may be difficult to identify and isolate when there are few colonies and additional contaminating oral microbiota is present. Some contaminating microor­ ganisms may grow as small colonies but differ usually from H. pylori in color. H. pylori is biochemically closely related to Campylobacter, Arcobacter, and Wollinella species but also resembles Bacteroides, Tbiovulum, and Selenomonas species. They are all characterized as being gram-negative rods that are able to grow microaerobically or anaerobically. The rods may be more or less curved, depending on the growth conditions. There are conflicting data in the literature about the nitrite and nitrate reaction of H. pylori. The urease reaction is a key reaction in identifying Helicobacter species, but some Campylobacter lari (UPTC) strains are ure­ ase positive and at least one urease-negative H. pylori strain has been isolated from a patient (F. Megraud, personal communication). Several Helicobacter spe­ cies are gram-negative motile curved rods that are oxi­ dase, catalase, and urease positive, and it may, there­ fore, be necessary to undertake protein profiles or genomic analysis to ensure the correct identification. Detection of H. pylori from extragastric speci­ mens by culture and genome methods. Several new species have been discovered during the past years, mainly isolated from animals. Only H. pylori and "H. beilmannii" have been recognized regularly in the human gastric mucosa. H. pylori has also been de­ tected from several extragastric sites. Successful cul­ tures have mainly been associated with findings of gastric metaplasia in esophagus (20, 90), Meckel's di­ verticulum ( 1 0 , 1 0 4 ) , and rectum (38), whereas in one case H. pylori could not be cultured from the gallblad­ der with gastric metaplasia (8). Occasionally, H. py­ lori has been cultured from dental plaque (28, 156) and fecal samples ( 7 9 , 1 4 8 ) . The culture methods used in these cases were similar to those described earlier in this chapter. Detection of extragastric H. pylori from dental plaque, fecal samples, atherosclerotic plaques, and liver was mainly achieved by genome methods and serology (13, 57, 107, 1 2 1 , 1 3 2 ) . Transformation and survival of H. pylori Factors responsible for the survival of cultured H. pylori. The conversion and morphological change of spiral-shaped H. pylori into coccoid forms are

32

ANDERSEN AND WADSTROM

achieved in various ways: (i) by nutrient deprivation ( 3 9 , 1 6 0 ) , (ii) by exposure to anti-ulcer drugs and anti­ biotics (12, 17, 2 7 , 7 0 ) , (iii) by extended incubation (27, 39, 105), (iv) by pH adjustment (9, 5 0 , 72), and (v) by attachment to the gastric epithelium (134). Changes in the morphology of H. pylori in culture on agar plates over time can be observed: after 3 days spiral forms dominated, after 6 days about half of the bacteria had converted into U-shaped or coccoid forms, and after 10 days only coccoid forms may be found ( 1 1 , 1 7 , 2 4 , 2 6 , 2 7 , 2 9 , 4 1 , 7 5 , 1 0 5 , 1 1 3 , 1 6 0 ) . Morphological changes are induced faster with expo­ sure to detrimental environmental circumstances (68, 160). Transformation of H. pylori from rods to coccoids. The spiral form of H. pylori is a curved rod that is 2 to 4 u,m long and 0.5 to 0.8 |xm wide and possesses one to seven sheathed flagella originating from one pole, with a characteristic polar membrane (29, 7 5 , 113). When spiral forms of H. pylori trans­ form into coccoid forms in vitro, a C or U form is initiated by ingrowth of the periplasm on one site of the bacteria. Both in vitro and in vivo coccoid forms of H. pylori vary in size from 1 to 2 u.m in diameter for organisms with dense cytoplasmic bodies up to 3 to 4 |xm in diameter for organisms with less dense cytoplasm (17, 2 4 , 2 9 , 7 5 , 113). The early stages of coccoid forms have preserved the characteristic polar membrane, flagella, and motility as seen in spiral forms ( 1 1 , 17, 7 5 ) . Three-month-old coccoid forms have a complete cell wall, cell membrane, and cyto­ plasm (56, 1 1 3 ) . Coccoid forms of H. pylori are able to maintain an oxidative metabolism at the same level as spiral forms for several months (56, 113). It remains to be established whether this metabolism of coccoids rep­ resents viability or a nonviable "bag" of enzymes (56). The preservation of metabolic activity is supported by a low but constant ATP level over a period of 1 month and the presence of polyphosphates as a phos­ phorus and energy source that allow a certain endoge­ nous metabolism (16, 17, 113). The protein content only changes a little whereas the lipid content changes considerably after coccoid transformation (145). Coc­ coid forms of H. pylori grown on solid media for 4 weeks lost their urease activity and the metabolic ac­ tivity of some enzymes (leucine arylamidase, naphthol-SA-ip-phosphohydrolase) was reduced, whereas the metabolic activities of other enzymes (alcaline and acid phosphatase) were unchanged (67). The genes coding for urease subunit C and a 26-kDa protein were detected unchanged by PCR even though the urease activity was lost, indicating the vitality of the coccoid forms (67). This is confirmed by the fact that

coccoid forms of H. pylori can be detected by means of acridine orange staining (25). Newly synthesized DNA has been detected by bromodeoxyuridine incor­ porated into 3-month-old coccoid forms of H. pylori (17). Viability and regrowth of H. pylori from coccoid forms. Coccoid forms of H. pylori are usually nonculturable and difficult to study (17, 5 6 , 1 1 3 ) . At­ tempts to document the biological significance and the viability of coccoid forms of H. pylori have been carried out in vitro in cell cultures and in vivo in ani­ mal models. In cell cultures, the initial contact and attachment of H. pylori to human gastric epithelial cells occur rapidly, often within minutes, at the aflagellated end of the bacterium. Transformation into coccoid forms is common after attachment or internalization of the bacterium (134). H. pylori does not multiply intracellularly, and many of the coccoid forms that are found within a few hours after uptake are degenerative forms (134). Coccoid forms of H. pylori adhere to cultured Kato III cells and human gastric carcinoma cells and cause similar morphological changes as spi­ ral forms (25, 134). Actin rearrangement occurs di­ rectly beneath the site of attachment of H. pylori, forming a fine condensed structure concentric to the bacterium. Coccoid H. pylori appears to induce an earlier and stronger cytoskeletal response than spiral forms (134). The coccoid forms of H. pylori adhere to the same components as the spiral forms and also contain cell-surface agglutinins and heparan sulfatebinding proteins (43, 6 5 , 80, 1 0 3 , 117). In mice, coccoid forms of H. pylori colonize the stomach, induce inflammation, and cause humoral immune response similar to that of spiral forms (26, 7 4 , 1 5 9 ) . The inflammatory response might be similar to that of a passive immunization with dead organ­ isms, but H. pylori could be cultured from the stom­ ach of mice 1, 2, and 4 weeks after inoculation with the coccoid forms of H. pylori (26, 159). H. pylori grown in liquid media for 2 0 days formed coccoids that were nonculturable on solid media but could after inoculation in mice be isolated from the murine stomach (27). In gnotobiotic piglets, urease-negative mutants of H. pylori are unable to colonize the gastric mucosa, and nonmotile or weakly motile strains of H. pylori are less virulent and colonize the gastric mu­ cosa to a lesser extent than motile strains (39, 4 1 ) . H. pylori can be found free in the mucin layer or attached to the gastric epithelial cells in the human stomach, where it induces changes in epithelial cell architecture: adhesion pedestals, indentation sites, abutting adhesion, membrane fusions or condensa­ tions, vacuolation, and internalization (14, 6 1 , 6 4 ,

CHAPTER 4 • BASIC BACTERIOLOGY AND CULTURE

115, 1 4 2 ) . Epithelial degeneration is present when more than 2 0 % of the bacteria have formed adhesion sites (64). Mucosal epithelial cells have been found in increased numbers in the vicinity of the attachment of coccoid forms (11). Degeneration is recognized his­ tologically by ragged cytoplasmic margins, a high nucleocytoplasmic ratio, and a heaping up of cells to form syncytium-like masses. In children, coccoid forms of H. pylori have been found to be closely asso­ ciated with damaged mucous cells whereas spiral forms have been found in proximity to unchanged or less damaged cells ( 7 2 ) . Coccoid forms of H. pylori are found more frequently and in larger numbers in the gastric mucosa of patients with gastric cancer than in patients with peptic ulcer disease (29, 1 2 6 ) .

16.

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CHAPTER 4 • BASIC BACTERIOLOGY AND CULTURE

127. Rieggs, S. J., B. E. Dunn, and M. J. Blaser. 1995. Microbiol­ ogy and pathogenesis of Helicobacter pylori, p. 535-563. In M. J. Blaser, P. D. Smith, J. I. Ravdin, R. L. Greenberg, and R. L. Guerrant (ed.), Infection of the Gastrointestinal Tract. Raven Press, New York, N.Y. 128. Roine, R. P., K. S. Salmela,J. Hook-Nikanne, T. U. Kosunen, and M. Salaspuro. 1992. Alcohol dehydrogenase mediated acetaldehyde production by Helicobacter pylori—a possible mechanism behind gastric injury. Life Sci. 51:1333-1337. 129. Roosendaal, R., E. J. Kuipers, A. S. Pefla, and J. de Graaff. 1995. Recovery of Helicobacter pylori from biopsy specimens is not dependent on the transport medium used. /. Clin. Mi­ crobiol. 33:2798-2800. 130. Saitoh, T., H. Natomi, W. L. Zhao, K. Okuzumi, K. Sugano, M. Iwamori, and Y. Nagai. 1991. Identification of glycolipid receptors for Helicobacter pylori by TLC-immunostaining. FEBS Lett. 282:385-387. 131. Schmitt, W., and R. Hass. 1994. Genetic analysis of Helico­ bacter pylori vacuolating cytotoxin: structural similarities with the IgA protease type of exported protein. Mol. Micro­ biol. 12:308-319. 132. Scragg, R. K., A. Fraser, and P. A. Metcalf. 1996. Helico­ bacter pylori seropositivity and cardiovascular risk factors in a multicultural workforce. /. Epidemiol. Community Health 50:578-579. 133. Sebald, M., and M. Veron. 1963. Teneur en bases de l'ADN et classification des vibrions. Ann. Inst. Pasteur (Paris) 105: 897-910. 134. Segal, E., S. Falkow, and L. S. Thompkins. 1996. Helico­ bacter pylori attachment to gastric cells induces cytoskeletal rearrangement and tyrosine phosphorylation of host cell pro­ teins. Proc. Natl. Acad. Sci. USA 93:1259-1264. 135. Sidebotham, R. L., J. J. Batten, Q. N. Karim, J. Spencer, and J. H. Baron. 1991. Breakdown of gastric mucus in presence of Helicobacter pylori. J. Clin. Pathol. 44:52-57. 136. Skirrow, M. B. 1977. Campylobacter enteritidis: a "new" disease. Br. Med. J. 2:9-11. 137. Slomiany, B. L., V. L. N. Murty, J. Piotrowski, Y. H. Liau, P. Sundaram, and A. Slomiany. 1992. Glycosulfatase activity of Helicobacter pylori toward gastric mucin. Biochem. Biophys. Res. Comm. 183:506-513. 138. Smith, T., and M. S. Taylor. 1919. Some morphological and biochemical characters of the spirilla (Vibrio fetus n. sp.) as­ sociated with disease of the fetal membranes in cattle. /. Exp. Med. 30:299-312. 139. Syder, A. J., J. L. Guruge, Q. Li, Y. Hu, C. M. Olcksiewicz, R. G. Lorenz, S. M. Karam, P. G. Falk, and J. I. Gordon. 1999. Helicobacter pylori attaches to NeuAc alpha 2,3Gal beta 1,4 glycoconjugates produced in the stomach of transgenic mice lacking parietal cells. Mol. Cell 3:263274. 140. Solnick,J.V.,J. O'Rourke,A. Lee, B.J. Paster, F. E. Dewhirst, and L. S. Tompkins. 1993. An uncultured gastric spiral or­ ganism is a newly identified Helicobacter in humans. /. Infect. Dis. 168:379-385. 141. Soltesz, V., B. Zeeberg, and T. Wadstrom. 1992. Optimal survival of Helicobacter pylori under various transport condi­ tions. /. Clin. Microbiol. 30:1453-1456. 142. Sorberg, M., M. Nilsson, H. Hanberger, and L. E. Nilsson. 1996. Morphologic conversion of Helicobacter pylori from baccilar to coccoid form. Eur.}. Clin. Microbiol. Infect. Dis. 15:216-219. 143. Spigelhalder, C , B. Gerstenecker, A. Kersten, E. Schiltz, and M. Kist. 1993. Purification of Helicobacter pylori superoxide dismutase and cloning and sequencing of the gene. Infect. Immun. 61:5315-5325. 144. Stanley, J., D. Linton, A. P. Burnens, F. E. Dewhirst, R. J.

145.

146.

147.

148.

149.

150.

151.

152.

153.

154.

155.

156.

157.

158.

159.

160.

37

Owen, A. Porter, S. L. W. On, and M. Costas. 1993. Helico­ bacter canis sp. nov., a new species from dogs: an integrated study of phenotype and genotype. /. Gen. Microbiol. 139: 2495-2504. Steinhilber, W., S. Loeffler, A. Siebler, M. Rektorschek, K. Melchers, and K. P. Schafer. 1996. Lipid composition is changed in coccoids from Helicobacter pylori and Helico­ bacter felis. Gut 39(Suppl. 2):61-62. Talley, N. J., A. J. Cameron, R. G. Shorter, A. R. Zinsmeister, and S. F. Phillips. 1988. Campylobacter pylori and Barrett's esophagus. Mayo Clin. Proc. 63:1176-1180. Telford, J., P. Ghiara, M. Dell'Orco, M. Comanducci, D. Burroni, M. Bugnoli, M. F. Tecce, S. Censini, A. Covacci, Z. Xiang, E. Papini, C. Montecucco, L. Parente, and R. Rappuoli. 1994. Gene structure of the Helicobacter pylori cyto­ toxin and evidence of its key role in gastric disease. /. Exp. Med. 179:1653-1658. Thomas,J. E., G. R. Gibson, M. K. Darboe, A. Dale, and L. T. Weaver. 1992. Isolation of Helicobacter pylori from human faeces. Lancet ii:1194-1195. Tompkins, D. S.,J. Dave, and N. P. Mapstone. 1994. Adapta­ tion of Helicobacter pylori to aerobic growth. Eur. J. Clin. Microbiol. Infect. Dis. 13:409-412. Tzouvelekis, L. S., A. F. Mentis, A. M. Makris, C. Spiliadis, C. Blackwell, and D. M. Weir. 1991. In vitro binding of Heli­ cobacter pylori to human gastric mucin. Infect. Immun. 59: 4252-4254. Utt, M., and T. Wadstrom. 1997. Identification of heparan sulphate binding surface proteins of Helicobacter pylori: inhi­ bition of heparan sulphate binding with sulphated carbohy­ drate polymers. /. Med. Microbiol. 46:541-546. Vandamme, P., M. I. Daneshvar, F. E. Dewhirst, B. J. Paster, K. Kersters, H. Goosens, and C. W. Moss. 1995. Chemotaxonomic analyses of Bacteroides grasilis and Bacteroides ureolyticus and reclassification of B. gracilis as Campylobacter grasilis comb. nov. Int. J. Syst. Bacteriol. 45:145-152. Vandamme, P., and J . De Ley. 1991. Proposal for a new family, Campylobacteriaceae. Int. J. Syst. Bacteriol. 41: 451-455. Vandamme, P., E. Falsen, R. Rossau, B. Hoste, P. Segers, R. Tytgat, and J. DeLey. 1991. Revision of Campylobacter, Helicobacter, and Wollinella taxonomy: emendatum of ge­ netic descriptions and proposals of Arcobacter gen. nov. Int. ] . Syst. Bacteriol. 41:88-103. Veron, M., and R. Chaterlain. 1973. Taxonomic study of the genus Campylobacter Sebald and Veron and designation of the neotype strain for the type species Campylobacter fetus (Smith and Taylor) Sebald and Veron. Int. ] . Syst. Bacteriol. 23:122-134. Von-Recklinghausen, G., T. Weischer, R. Ansorg, and C. Mohr. 1994. No cultural detection of Helicobacter pylori in dental plaque. Zentralbl. Bakteriol. 281:102-106. Wadstrom, T., S. Hirmo, and T. Boren. 1996. Biochemical aspects of Helicobacter pylori colonization of the human gas­ tric mucosa. Aliment. Pharmacol. Ther. 1:17-27. Wadstrom, T., and A. Ljungh. 1999. Glycosaminoglycanbinding microbial proteins in tissue adhesion and invasion: key events in microbial pathogenicity. /. Med. Microbiol. 48: 223-233. Wang, X., E. Sturegard, R. Rupar, H. O. Nilsson, P. A. Aleljung, B. Carlen, R. Willen, and T. Wadstrom. 1997. Infection of BALB/cA mice by spiral and coccoid forms of Helicobacter pylori. J. Med. Microbiol. 46:657-663. West, A. P., M. R. Millar, and D. S. Tompkins. 1990. Survival of Helicobacter pylori in water and saline. /. Clin. Pathol. 43:609.

38

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161. Westblom, T. U., E. Madan, and B. R. Midkiff. 1991. Egg yolk emusion agar, a new medium for the cultivation of Heli­ cobacter pylori. J. Clin. Microbiol. 29:819-821. 162. Westblom, T. U., E. Medan, B. R. Midkiff, V. W. Ackins, and M. A. Subik. 1988. Failure of Campylobacter pylori to grow in commercial blood culture systems./. Clin. Microbiol. 26:1029-1030.

163. Westblom, T. U., S. Phadnis, P. Yang, and S. J. Czinn. 1993. Diagnosis of Helicobacter pylori infection by means of a poly­ merase chain reaction assay for gastric juice aspirates. Clin. Infect. Dis. 16:367-371. 164. Wolin, M. J., E. A. Wolin, and N. J. Jacobs. 1961. Cytochrome-producing anaerobic vibrio, Vibrio succinogenes, sp. n./. Bacteriol. 81:911-917.

Helicobacter pylori: Physiology and Genetics Edited by H. L. T. Mobley, G. L. Mendz, and S. L. Hazell © 2 0 0 1 ASM Press, Washington, D.C.

Chapter 5

Taxonomy of the Helicobacter Genus JAY V . SOLNICK AND PETER VANDAMME

INTRODUCTION

pylori, the first member of the new genus (26).

Background

Helicobacter

Expansion of the genus Helicobacter

The cultivation of a novel bacterium from gastric mucosa in 1982 marked a turning point in our under­ standing of gastrointestinal microbial ecology and dis­ ease. Marshall and Warren (46) described spiral or curved bacilli in histologic sections from 58 of 100 consecutive biopsies of human gastric antral mucosa. Eleven biopsies were culture positive for a gram-nega­ tive, microaerophilic bacterium, whose proper classi­ fication generated considerable interest at the Second International Workshop on Campylobacter Infec­ tions held in Brussels, Belgium, in September 1983 (57). The organism resembled Campylobacter in sev­ eral respects, including curved morphology, growth on rich media under microaerophilic conditions, fail­ ure to ferment glucose, sensitivity to metronidazole, and a G + C content of 3 4 % . It was therefore first referred to as "pyloric Campylobacter" (pylorus, Greek, gatekeeper, or one who looks both ways) and validated as Campylobacter pyloridis in 1985 (1). The specific epithet was revised to Campylobacter pylori in 1987 to conform to the correct Latin genitive of the noun pylorus (45).

The cultivation of H. pylori and the recognition of its clinical significance served to renew interest in bacteria associated with the gastrointestinal and hepa­ tobiliary tracts of humans and other animals, many of which have now been identified as novel Helicobacter species. The genus Helicobacter presently comprises 18 validly named species and two Candidatus species, a designation adopted by the International Committee on Systematic Bacteriology to record the properties of putative procaryotic taxa that are incompletely de­ scribed (52). The purpose of this chapter is to describe the taxonomic characteristics of the Helicobacter genus and to discuss methods used to differentiate among Helicobacter species. We conclude with a sum­ mary of recent recommendations for the identification of novel Helicobacter species (12).

DESCRIPTION OF T H E GENUS HELICOBACTER

Yet almost from its initial cultivation it was sus­ pected that perhaps C. pylori was not a true Campylo­ bacter. Early electron micrographs showed multiple sheathed flagella at one pole of the bacterium, in con­ trast to the single bipolar unsheathed flagellum typical of Campylobacter spp. (28). Major protein bands and fatty acids of C. pylori were also markedly different from those of Campylobacter species (28, 56). Subse­ quent 16S rRNA sequence analysis showed that the distance between C. pylori and the true Campylobact­ ers was sufficient to exclude it from the Campylo­ bacter genus (60), and it was renamed Helicobacter

Cellular Morphology and Ultrastructure Helicobacters are non-spore-forming gram-nega­ tive bacteria. The cellular morphology may be curved, spiral, or fusiform, typically 0.2 to 1.2 |xm in diameter and 1.5 to 10.0 u.m long. The spiral wavelength may vary with the age, the growth conditions, and the spe­ cies identity of the cells. In old cultures or those ex­ posed to air, cells may become coccoid. Periplasmic fibers or an electron-dense glycocalyx or capsule-like layer has been observed on the cel­ lular surface of several species (26, 4 3 , 5 5 , 63). Elec-

Jay V. Solnick • Department of Internal Medicine, Division of Infectious Diseases, and Department of Medical Microbiology and Immunology, University of California, Davis, Davis, CA 95616. Peter Vandamme • Laboratorium voor Microbiologic, Universiteit Gent, Ledeganckstraat 35, B-9000 Gent, Belgium.

39

40

SOLNICK AND VANDAMME

tron-dense granular bodies have been observed in H. pylori (4) and H. rodentium (63). In H. pylori these bodies are known to be aggregates of polyphosphate and may serve as a reserve energy source. Helicobacter cells are motile, with a rapid cork­ screw-like or slower wave-like motion due to flagellar activity. Strains of most species have bundles of multi­ ple sheathed flagella with a polar or bipolar distribu­ tion. Other species have only a single polar or bipolar flagellum (Table 1). However, flagellation can be peritrichous (H. mustelae) or nonsheathed (H. pullorum, H. rodentium, and "H. mesocricetorum") as well. Growth Characteristics In laboratory conditions, strains typically grow under microaerobic conditions at 37°C. No growth is observed in aerobic conditions. H. rodentium strains grow in anaerobic as well as microaerobic conditions (63). Strains of several species do not require atmo­ spheric hydrogen for growth in microaerobic condi­ tions, although there may be a growth-stimulating ef­ fect of atmospheric hydrogen on the growth of such strains. However, as for some Campylobacter species (e.g., Campylobacter hyointestinalis), hydrogen re­ quirements for microaerobic growth of Helicobacter may be strain dependent. For instance, the isolates initially described as belonging to a novel anaerobic species, "Helicobacter westmeadii," were subse­ quently identified as hydrogen-requiring H. cinaedi strains that grow optimally in microaerobic condi­ tions (77). Many laboratories routinely use microaer­ obic conditions including hydrogen for the routine culture of Helicobacter species and the role of this atmospheric component is therefore not well docu­ mented. Helicobacters will grow at 37°C on a variety of rich agar bases supplemented with 5 % whole blood or serum. Many species require fresh media with moist agar surfaces for optimal growth conditions, though this is not usually the case for H. pylori. In such conditions, growth is often not seen as individual colonies but as a thin, sometimes colorful or watery, spreading film. The most fastidious species such as H. bizzozeronii and H. salomonis prefer very moist conditions, and a thin broth layer can be poured on top of the agar surface to stimulate growth. Prolonged incubation up to 1 week may be required. Biochemical Characteristics Helicobacters are chemoorganotrophs and show a respiratory type of metabolism. They are asaccharolytic when sugar catabolism is examined by standard

methods (neither oxidation nor fermentation is ob­ served). Recent studies have, however, indicated that glucose oxidation occurs in at least H. pylori (6, 72) (see chapter 10). The glycolysis-gluconeogenesis path­ way probably comprises the principal means of en­ ergy production as well as the starting point for many biosynthetic pathways. The Entner-Doudoroff path­ way, the pentose phosphate shunt, and the tricarbox­ ylic acid cycle are at least partially present; the glyoxylate shunt is absent (6, 72). Gelatin, starch, casein, and tyrosine are not hydrolyzed. Helicobacters are methyl red and VogesProskauer negative. Oxidase activity is present in all species. Strains of most species produce catalase. Many species produce urease, alkaline phosphatase, or both. There is no production of pigments.

Other Characteristics The moles percent G + C range of the DNA ranges from 30 (H. acinonychis) to 48 (H. canis). One species, H. nemestrinae, was reported to have a DNA base ratio of 2 4 % (5). However, repeat analyses of the H. nemestrinae type strain yielded a reproduci­ ble value of 39 mol% (P. Vandamme, unpublished observations). It would therefore be appropriate to do a comparative analysis of various subcultures of the H. nemestrinae type strain to verify the strain iden­ tity in the different public and private culture collec­ tions. Internal transcribed spacers or intervening se­ quences have been described in the 16S ribosomal RNA genes of H. canis (44), H. bilis (19), "H. typhlonicus" (24), and in an unnamed helicobacter referred to as Helicobacter sp. cotton-top (61). Simi­ lar intervening sequences have also been found in the 23S rRNA gene of H. canis, H. mustelae, and H. muridarum (34). The length of intervening sequences in the 16S rRNA gene ranges from 187 bp to 2 3 5 bp, while those found in the 23S rRNA gene range from 93 bp to 377 bp. The cellular fatty acid components have been studied for a restricted number of Helicobacter spe­ cies. Major cellular fatty acids reported include tetradecanoic acid; most species contain moderate to high levels of hexadecanoic acid and octadecanoic acid (27, 4 1 ) . The isoprenoid quinone content of H. pylori, H. cinaedi, and H. fennelliae has been determined and identifies menaquinone-6 (2-methyl-3-farnesyl-farnesyl-l,4-naphthoquinone) and a second, unidentified quinone as major respiratory quinones (51).

H

•9

- A

o

3

I

1

||

0
GlcNAc-- > V 1 1 Gal n' l Fuc

Gal->GlcNAc--> 1 1 Fuc Fuc

471

85

r

v

I

J

t

Key to structural unit modules

Gal->GlcNAc~> Lewis antigen termini :

'

'

Fuc Le" Gal->GlcNAc-I Fuc

r

Extended oligomeric chains:

Extended Le

Gal->GlcNAc~>

Gal->GlcNAc->

'

I

I

±Fuc Fuc Ley/Lex

Fuc Fuc Ley

fGal->GlcNAc

[-3 DHep-] ->

LacNAc

D

n

Heptan

x

Gal->GlcNAcI

Glc

Gal->GlcNAcI

Gal

Alternative glycosylated LacNAc units Figure 5. Modular structures of the polysaccharide component of some examples of H. pylori LPS. For an explanation of sugar abbreviations, see the legend to Fig. 3.

a(l,2)-fucosyltransferase gene involved in L e synthe­ sis (113), as well as the p(l,4)-galactosyltransferase gene required for synthesis of N-acetyllactosamine in the backbone of Le antigens (46). Also, an enzyme with both a ( l , 3 ) - and a(l,4)-fucosyltransferase activ­ ity involved in both L e and L e synthesis, respec­ tively, has been cloned and sequenced (91). y

x

a

Three genes in the sequenced genomes that are homologs of the a(l,2)-glucosyltransferase gene (rfaj) have been found, but no a(l,2)-linked glucose has been described in H. pylori LPS (14, 4 9 ) . It has been suggested that these genes encode 0(l,4)-galactosyltransferase and/or 3(l,3)-N-acetylglucosaminyltransferase functions required for Lewis antigen syn-

86

MORAN

thesis (14). Also, a homolog of GDP-D-mannose dehydratase from Vibrio cholerae and homologs of galactosyltransferases from Klebsiella pneumoniae have been suggested to be involved in O-chain synthe­ sis (49). Although of low homology, an ortholog of neuA, which codes for CMP-N-acetylneuraminic (sia­ lic) acid synthetase (106), was suggested to be in­ volved in sialyl-Le expression (14), which has been verified chemically to occur in H. pylori LPS (54). Mutants expressing truncated LPS structures have been generated by insertional mutagenesis oigalE and rfbM genes that encode UDP-galactose-4-epimerase and GDP-D-mannose pyrophosphorylase, a key en­ zyme in the synthesis of GDP-fucose, respectively (25, 41). Since galactose is essential for linking the O-chain polysaccharide to the core of H. pylori LPS, construc­ tion of an isogenic H. pylori galE mutant results in expression of R-LPS without an O-chain ( 2 5 , 4 1 ) . As expected, construction of an rfbM mutant results in expression of S-LPS with an O-chain but without fucosylation (25). x

LPS Expression under Acid Stress Survival of H. pylori below pH 4 is dependent on urease activity, whereas urease-independent mech­ anisms, although less well characterized, operate at greater than pH 4 . Even less studied are the mecha­ nisms that play a role in H. pylori growth under con­ stantly acidic conditions that prevail in the mucus layer. Expression of LPS appears to be involved in the ability of H. pylori to withstand acid shock since a wbcj gene, encoding a protein homolog involved in the conversion of GDP-mannose to GDP-fucose for O-chain biosynthesis, was induced under acid shock conditions and during growth at pH 5 (52). More­ over, a wbcj mutant did not survive an acid shock of pH 3.5 and was impaired in its survival of acid shock at pH 4 . Further emphasizing the importance of the O-chain in the bacterial response to acid, detailed chemical studies have shown structural differences be­ tween the O-chain of H. pylori grown in liquid media at pH 7 and pH 5, whereas no differences were ob­ served in the core and lipid A regions of LPS (69). Other changes may also occur in the bacterial cell wall since Bukholm et al. (16) observed that growth of H. pylori on solid media at pH 5.5 resulted in production of colonies with increased amounts of lysophosphatidyl ethanolamine and phosphatidyl serine as well as increased virulence, compared with those at pH 7. However, these changes in lipid composition can be induced independently of acidic conditions (63) and, thus, may simply reflect growth on solid media rather than an acidic environment.

Low Immunological Activity of LPS Immunological and endotoxic properties Based on the structure of H. pylori lipid A, the hypothesis was proposed that H. pylori LPS should have low biological activities (80) and, subsequently, this has been proven. The properties of H. pylori LPS, which are reviewed extensively elsewhere (58, 5 9 , 62, 65), have revealed significantly lower endotoxic and immunological activities when compared with entero­ bacterial LPS as the gold standard. For example, the pyrogenicity and mitogenicity of H. pylori LPS is 1,000-fold lower, and lethal toxicity in mice, 500-fold lower, compared with Salmonella enterica serovar Typhimurium LPS (80). The induction of interleukin1 (IL-1), IL-6, and tumor necrosis factor alpha (TNFa) from activated human mononuclear cells by H. py­ lori LPS is significantly lower than that by E. coli LPS (15, 85). Also, the induction of IL-8 from neutrophils is 1,000-fold lower when induced by H. pylori LPS than by enterobacterial LPS (20), and only low-level secretion of IL-8 from epithelial cell lines occurs (21). Similar observations were made in other biological assays (13, 2 3 , 83, 88, 103), including a 1,000-fold lower ability by H. pylori LPS to induce production of procoagulant activity (PCA), identified as tissue factor, and plasminogen activator inhibitor type 2 (PAI-2) by human mononuclear leukocytes (98).

Fine structure of lipid A Studies using chemically defined synthetic ana­ logs and partial structures of E. coli and Salmonella spp. lipid A have shown that E. coli hexaacyl lipid A optimally expresses the full spectrum of endotoxic activities associated with LPS (34, 9 3 , 94). Slight modifications to this lipid A architecture, e.g., the ad­ dition or removal of chemical groups, or the presence of long or unusual fatty acids, result in a significant reduction in the immunological activities of lipid A, and hence of LPS (57, 93). These findings suggested that the unusual phosphorylation and acylation in H. pylori lipid A (Fig. 2) could, in part, explain the ob­ served lower biological activities (58, 5 9 , 80). In serum, LPS-binding protein (LBP) acts as a catalytic protein to present LPS to the monocyte-macrophage cell surface, where the newly formed LPS-LBP com­ plex interacts with the CD 14 surface receptor, which, independently or in association with a second recep­ tor, leads to the induction of proinflammatory cyto­ kines (57). H. pylori LPS binds more poorly with slower binding kinetics than E. coli LPS to LBP, and also exhibits poorer binding to CD14 (22). Collec­ tively, as LBP binds LPS through its lipid A compo-

CHAPTER 8 • ROLES OF LIPOPOLYSACCHARIDES

nent, these findings are consistent with a proportion­ ately lower ability of H. pylori LPS to activate monocytes and reflect the unusual phosphorylation and fatty acid substitution in this lipid A (61, 71). In addition, modification and isolation of H. py­ lori LPS components with subsequent testing in im­ munological assays have given insights into the molec­ ular basis for the observed lower immunological activities. With this approach, the phosphorylation pattern in H. pylori lipid A has been shown to influ­ ence induction of TNF-ct, and the core oligosaccha­ ride modulates this effect (85). A similar phenomenon was observed with induction of PCA and PAI-2 re­ lease from mononuclear leukocytes (98), and dephosphorylation of H. pylori LPS influences activity in the Limulus amebocyte lysate assay (85). In contrast, dephosphorylation does not alter the priming activity of H. pylori LPS on neutrophils to release toxic oxygen radicals, which suggests the lesser importance of phosphorylation compared with acylation pattern in priming (83). On the other hand, the lack of abolition of suppressor T-cell activity has been attributed to the presence of long-chain fatty acids in H. pylori lipid A (13). Thus, depending on the particular immunologi­ cal activity examined, the phosphorylation or acyla­ tion pattern of H. pylori lipid A assumes importance. Implications for the host-parasite relationship H. pylori LPS, which is an essential component of the bacterial outer membrane, by inducing a low immunological response may prolong H. pylori infec­ tion for longer than that by more aggressive and short­ lived pathogens. Thus, we have suggested that H. py­ lori LPS, and its lipid A component in particular, have evolved their present structure as a consequence of adaptation to the ecological niche in the gastric mu­ cosa (58, 59). In an analogous manner, the long-term human commensal bacterium Bacteroides fragilis, a member of the normal gut microbiota, produces a lipid A bearing some structural similarity to H. pylori lipid A and which possesses low immunological ac­ tivities (114). Since the primary role of H. pylori LPS is to provide a functional macromolecular matrix in the outer membrane through which the bacterium in­ teracts with its environment ( 5 7 , 5 9 , 80), the LPS mol­ ecule has retained this role but has been modified to reduce the immunological response, hence aiding per­ sistence of the bacterium and thereby aiding develop­ ment of a chronic infection. Despite the low immunological activity of H. py­ lori LPS, H. pylori colonization of the human antral mucosa is associated with inflammation. H. pylori can activate mononuclear cells by an LPS-independent, as well as an LPS-dependent mechanism (48),

87

and other bacterial surface molecules can induce an immunological response contributing to pathology (61). Nevertheless, as a consequence of enzymatic degradation of LPS by phagocytes, some LPS and/or lipid A partially modified structures can be excreted by exocytosis. These compounds, retaining some im­ munological activity, could play a role as subliminal, low-grade, persistent stimuli involved in H. pylori pathogenesis during long-term chronic infection con­ tributing to gastric damage, and potentially to extra­ gastric sequelae (62, 65). Consistent with this, H. py­ lori LPS and derivatives can induce PCA and PAI-2 production by human mononuclear leukocytes, influ­ encing coagulation and fibrin formation (98), and can induce nitric oxide synthase in an in vivo animal model, thereby contributing to gastric damage (42, 43). LPS and Lectin-Like Interactions in Laminin Binding Bacterial interactions with extracellular and basement membrane proteins play an important role in the pathogenesis and virulence of a number of infec­ tions (45). H. pylori strains bind a number of extracel­ lular matrix components and, especially, exhibit highaffinity binding to laminin (45, 7 0 , 107, 108). This glycoprotein is not alone a component of the extracel­ lular matrix, but plays an important role in the struc­ ture of the basement membrane. Preliminary studies indicated the involvement of both a lectin-like interac­ tion (108) and LPS (70) in laminin binding by H. py­ lori. The interaction of H. pylori with laminin is com­ plex and that mediated by LPS involves two mecha­ nisms operating in strains that can be divided into hemagglutinating and nonhemagglutinating strains (109). A phosphorylated structure in the core oligo­ saccharide of LPS mediates the interaction of a hemag­ glutinating strain, whereas a conserved nonphosphorylated structure in the core oligosaccharide mediates the interaction of a poorly hemagglutinating strain. However, the amino acid domain on the laminin mol­ ecule for LPS interaction has not, to date, been identi­ fied. Although these interactions are mediated by the core of LPS, as a serological response against this LPS domain occurs in H. pylori-positive patients (86), structures within this domain can be exposed on the bacterial surface (62). Moreover, the serological re­ sponse against the core domain, and hence the avail­ ability of the core structures for interaction with lami­ nin, is more developed in duodenal ulcer patients than gastritis patients (86), which may reflect the role of the LPS-laminin interaction in the development of peptic ulcers (see below). The lectin-like adhesin involved

88

MORAN

in laminin binding is sialic acid-specific, recognizes a(2,3)-sialyllactose, is conserved in H. pylori strains, and has been identified as a 25-kDa protein (110). A serological response against this protein occurs in H. py/on'-infected individuals, confirming that the ad­ hesin is produced in vivo (63). Conceptually, the bind­ ing of H. pylori to laminin illustrates a dual recogni­ tion system, whereby sugars in the core of LPS interact with a peptide sequence of laminin, and a protein on H. pylori (with lectin-like properties) recognizes a stretch of sugars in laminin. It has been proposed that the initial binding of laminin by H. pylori is mediated by LPS and that this is followed by the specific lectin binding (45, 1 1 0 ) . The capacity of H. pylori strains to bind laminin is unlikely to be involved in the initial colonization of gastric mucosal cells, since H. pylori possesses adhesins that putatively recognize receptors in the mucus layer and on the epithelial cell surface ( 5 8 , 6 0 , 7 5 , 7 6 ) . Importantly, H. pylori is observed associating with intercellular junctions, and laminin binding may ex­ plain its association with this microenvironment (59) and have pathological consequences ( 5 9 , 6 2 ) . An integrin, a 67-kDa protein receptor for laminin on gastric epithelial cells, has been isolated and H. pylori LPS has been shown to interfere with its specific interac­ tion with laminin in vitro (101). Therefore, the bind­ ing of H. pylori to laminin mediated by LPS could disrupt epithelial cell-basement membrane interac­ tions, contributing to the disruption of gastric mu­ cosal integrity and the development of gastric leakiness associated with the bacterium. Interestingly, H. pylori exhibits a significant penetration between cells, and infection is associated with a weakening of adhe­ sion of the tight junctions between cells, which could explain the origin of gastric leakiness. Certain cytoprotective anti-ulcer drugs, e.g., nitecapone, sucral­ fate, ebrotidine, and sulglycotide, have been reported to counteract the anti-adhesive effect of LPS on integrin action (see references 5 9 , 1 0 1 ) , but further studies are required to verify this hypothesis, since other solu­ ble factors of H. pylori have been identified as contrib­ uting to gastric leakiness (105). Pepsinogen Stimulation by LPS Elevated pepsinogen, a precursor of mucolytic and barrier-breaking pepsin, is considered a marker for the development and recurrence of duodenal ul­ cers. Young et al. (119), using gastric mucosa from guinea pigs in Ussing chambers, observed a 50-fold stimulation of pepsinogen secretion with H. pylori LPS compared with only a 12-fold increase with E. coli LPS. Microscopic examination confirmed the structural integrity of chief cells, indicating the ab­

sence of a nonspecific toxic effect induced by H. pylori LPS. Importantly, degranulation of zymogen granules in H. pylori LPS-treated tissue suggested a specific mechanism of pepsinogen release. Other investiga­ tions using guinea pig mucosa have confirmed these observations (65, 1 1 8 ) . Although the pepsinogen stimulatory effect was comparatively lower, stimula­ tion of pepsinogen was induced in isolated rabbit gas­ tric glands by cell sonicates of H. pylori (18), but this may reflect the stimulatory effect of a lower concen­ tration of LPS in the tested cell sonicates (59). Using guinea pig mucosa, comparison of the stimulatory ef­ fect of LPS derived from two duodenal ulcer patients and two asymptomatic individuals showed that the former preparations induced 50-fold stimulation, whereas the latter induced levels like those of controls (79). Hence, strain-dependent differences in LPS-induced pepsinogen secretion induction are related to pathogenesis. Nevertheless, studies using isolated human gastric mucosa have, to date, not been under­ taken. Experiments using LPS components, dephosphorylated LPS, and polymyxin B inhibition studies have indicated the involvement of structures in the core oligosaccharide of H. pylori LPS from duodenal ulcer patients in pepsinogen induction (79, 118). As discussed above, the LPS cores of H. pylori strains exhibit an unusual conformation compared with those of other bacterial species (62, 65). Structural investigations of the LPS cores of pepsinogen-inducing and noninducing strains have identified differ­ ences in substitution in the outer core-O-chain do­ main (7). Thus, core structures are present that may explain the activation of pepsinogen by H. pylori LPS. Mimicry of Lewis Blood Group Antigens Lewis antigen expression and phase variation mechanisms Phase variation is the reversible on-and-off switching of surface epitopes, including those of LPS, and can be genetically paralleled by the on-and-off switching of the specific glycosyltransferase genes in­ volved in LPS biosynthesis (97). In H. pylori, L e is not a stable trait and LPS can display a high frequency of phase variation, resulting in the occurrence of sev­ eral LPS variants in one bacterial cell population in vitro (5). Thus, phase variation could contribute to the phenotypic heterogeneity of H. pylori and may explain the isolation from one individual of several highly related isolates that differ in Le expression (116). For synthesis of an O-chain containing L e poly­ mer terminated with a L e unit, H. pylori requires a x

x

y

CHAPTER 8 • ROLES OF LIPOPOLYSACCHARIDES

series of enzymes including a(l,3)- and a(l,2)-fucosyltransferases that link fucose to C-3 of N-acetylglucosamine (GlcNAc) and C-2 of galactose (Gal), re­ spectively; and GlcNAc and Gal transfersases that form the polylactosamine O-chain backbone to which fucose is attached (Fig. 4). Three main groups of var­ iants have been reported in H. pylori LPS: one variant group had loss of a(l,3)-linked fucose resulting in nonsubstituted polylactosamine O-chains, producing an i-antigen chain; another had loss of the polymeric chain resulting in expression of a truncated LPS with monomeric Le ; and a third arose by acquisition of a(l,2)-fucose, which expressed polymeric L e plus terminal Le (5). Nevertheless, these structural deduc­ tions were solely based on serology and require chemi­ cal structural validation. It is well established in LPS of Neisseria spp. and Haemophilus influenzae that on-off switching occurs during replication because of a slip-strand mechanism that changes the length of nucleotide repeats that in­ troduce translational frameshifts, leading to the pro­ duction of inactive truncated gene products (97). Sub­ sequent changes during replication may switch the gene back on by restoring the reading frame and hence production of an active gene product. Interestingly, the a(l,3)- and a(l,2)-fucosyltransferase genes of H. pylori contain poly(C) tracts and the a(l,3)-fucosyltransferase genes contain oligonucleotide repeats at the 3'-end (1, 106). Moreover, the poly(C) tracts of the a(l,3)-fucosyltransferase genes have been demon­ strated to shorten and lengthen randomly in H. pylori laboratory strains, and changes in Le expression de­ duced to be a direct result of reversible frameshifting and inactivation of gene products (3). However, the length of C repeats in a(l,3)-fucosyltransferase genes did not correlate with L e or L e expression in H. pylori clinical isolates (95), indicating that posttranslational events and the availability of sugar intermedi­ ates, in addition to active enzyme, are important in determining the Le phenotype expressed. y

x

y

x

y

89

Because Le and L e that are expressed in the foveolar epithelium are isoforms of L e and Le (Fig. 4), Wirth et al. (117) extended the original hypothesis and re­ ported, using erythrocyte Lewis (a, b) phenotyping, that the relative proportion of bacterial expression of Le and Le corresponded to the host Le(a + ,b —) and L e ( a - , b + ) blood group phenotypes, respectively. However, in contrast, similar studies in Irish and Ca­ nadian patient populations did not find this correla­ tion (31, 3 2 , 104). The discrepancies between these results may be influenced by the characteristics of the study populations. O f considerable importance is that 2 6 % of the study population of Wirth et al. (117) were of the recessive Lewis phenotype, Le(a —,b — ) , which is higher than that usually observed for a Cau­ casian or European population, and reflects the het­ erogeneous origin of their patients. Distribution of this recessive phenotype group of patients to their true secretor or nonsecretor phenotype by salivary testing would influence the outcome of the obtained results. Importantly, in the other studies, no patients of a re­ cessive phenotype were observed (31). Loss of O-chain and Lewis antigen expression by H. pylori strains results in a lack of ability to infect mouse models of colonization (36, 74). Construction of an isogenic H. pylori galE mutant, expressing RLPS without an O-chain, results in loss of ability to colonize mice compared with the Le -expressing pa­ rental strain (62, 74). Similarly, mutation of a gene encoding a (l,4)-galactosyltransferase affecting syn­ thesis of the O-chain backbone resulted in less effi­ cient colonization of the murine stomach (46). These observations may be attributable to camouflage but also to the involvement of bacterial expression L e in adherence to the gastric mucosa (62). In adherence studies to gastric antral mucosa in situ in which an H. pylori galE mutant and another mutant in which rfbM had undergone insertional mutagenesis, yielding an LPS with an O-chain but without fucosylation and hence without L e expression, no tropic binding of either mutant to the mucosa was observed (25). Fur­ thermore, Le -binding polypeptides in the range of 16 to 29 kDa are found in gastric epithelial cells, but their identity is, to date, unknown (17). a

b

x

x

y

y

x

x

x

x

Camouflage, colonization, and bacterial adherence The L e , Le , and related blood group antigens are present in the human gastric mucosa (96). Surface and foveolar epithelia coexpress either L e and L e in Le(a + , b —) individuals (nonsecretors), or L e and Le in L e ( a - , b + ) individuals (secretors), whereas glandular epithelium lacks type 1 antigens (Le and Le ) but expresses L e and L e (type 2 antigens) irres­ pective of the secretor phenotype. Therefore, it has been speculated that bacterial expression of L e and Le antigens identical to those in the gastric mucosa may camouflage H. pylori in its ecological niche, par­ ticularly in the early phases of infection (59, 6 0 , 64). x

y

a

x

b

y

a

b

x

y

x

y

Lewis antigens and the inflammatory response In patient studies, although it has become appar­ ent that bacterial colonization density and the ensuing inflammatory response can be influenced by host expression of ABO and L e blood group determinants (33), bacterial L e expression is associated with peptic ulcer disease (50) and is statistically related to neutro­ phil infiltration (32). This is consistent with the role of L e in adherence, aiding bacterial interaction and a

x

x

90

MORAN

delivery of secreted products to the mucosa (60, 9 2 ) , hence influencing the inflammatory response. Alter­ natively, it has been speculated that recognition and cross-linking of CD 15 by H. py/on-induced anti-Le autoantibodies could directly potentiate polymorph adhesiveness to the endothelium (6, 6 1 , 73), but this remains a question for further investigation. On the other hand, one study showed that H. pylori isolates positive for both L e and Le were pre­ dominantly cagA positive and that a cagA-ablated strain had diminished expression of L e (115). It was concluded that expression of host Lewis antigens by the bacterium could aid the persistence of cagA-positive H. pylori proinflammatory strains. Although small numbers of isolates from different countries were examined, predominantly the isolates were from North America. In contrast, investigations of H. py­ lori isolates from a geographically distinct Irish popu­ lation have shown the lack of an association between cagA status and Lewis antigen expression (32, 50). These differing results reflect the adaptation of H. py­ lori strains with differing attributes to differing human populations (50). x

x

y

x

Lewis antigens and putative autoimmune mechanisms Expression of L e and L e antigens has been im­ plicated in the pathogenesis of atrophic gastritis by the induction of autoreactive antibodies ( 4 , 6 , 6 4 , 7 3 ) , but this pathogenic mechanism has not been unequiv­ ocally established (2). The presence of antigastric au­ toantibodies in H. pylori-infected individuals has been correlated with the degree of gastric infiltration, with the numbers of inflammatory cells, and with glandu­ lar atrophy (27, 82). In mouse immunization studies, H. pylori isolates from patients with severe atrophy, which expressed L e and Le antigens, yielded a strong gastric autoantibody response, whereas iso­ lates from individuals with near-normal mucosa, which often lacked Lewis antigen expression, were less able to induce autoantibodies (82). Moreover, growth in mice of an H. pylori-induced, anti-Le -secreting hybridoma resulted in gastritis-like histopathological changes (6, 81). The oligosaccharide of the B-chain of the gastric proton pump (H , K -ATPase) of parietal cell canaliculi, which is similar in chemical structure to Lewis antigens, has been implicated in pathogenic autoim­ mune responses. Anti-Le H. py/on'-induced autoreac­ tive antibodies react with the human and murine Bchain (6). In addition, anti-Lewis and related antigenparietal cell antibodies were induced in a transgenic mouse model of H. pylori infection in which gastric pathology developed (30). Collectively, these data x

y

x

+

+

+

+

FUTURE OUTLOOK

y

y

+

have been taken to indicate that Lewis antigen mim­ icry plays a role in the induction of autoimmunity in H. pylori-associated disease. Nevertheless, this inter­ pretation remains controversial. Contradictory results have been obtained from experiments attempting to abolish the binding of anticanalicular serum autoantibodies by absorption with H. pylori. In one such series (81) using Lewispositive lysates of H. pylori, a decrease was observed, but no significant reduction was observed in another (26), and anti-H , K -ATPase serum autoantibodies were not absorbed with H. pylori whole cells in an­ other study (47). Furthermore, patient sera reacted with recombinant H , K -ATPase expressed in Xenopus oocytes, which was deduced not to express Lewis antigens (19). Because of these findings, it has been concluded that H. pylori-associated autoimmunity parallels the classical model for induction of organspecific autoimmunity, with a central role for in­ creased autoantigen presentation resulting in loss of tolerance, and anti-Lewis antibodies only reflecting gastric damage ( 2 , 2 6 ) . However, the outer membrane of H. pylori undergoes blebbing, producing vesicles, the membranes of which contain LPS, and the associ­ ated Lewis antigens, which can be demonstrated by immunoelectron microscopy ( 3 7 , 6 2 ) . Thus, the Lewis antigens of H. pylori can be presented to the immune system in this format, rather than on whole bacterial cells. Because the use of whole cells or lysates of H. pylori may not be optimal to remove H. pylori-in­ duced anti-Lewis antibodies and it is unclear as to what type of glycosylation occurs in the Xenopus oocytes, further studies are required to resolve these issues.

+

y

In addition to the biological properties detailed above, a number of other biological activities that are less well characterized have been attributed to H. py­ lori LPS. A shift from high- to low-molecular-weight mucin production coupled with an inhibitory effect on the process of mucus glycosylation and sulfation by H. pylori LPS in segments of rat stomach has been observed (100). Moreover, H. pylori LPS inhibited mucin binding to a 97-kDa protein receptor that was isolated from gastric epithelial cells (90). Both these in vitro phenomena require independent verification and further evaluation since such mechanisms could profoundly influence the nature and integrity of the mucus perimeter in vivo. H. pylori LPS has been re­ ported to inhibit acid secretion in pylorus-ligated con­ scious rats (84), and the LPS has been shown to influence enterochromaffin-like cell secretion and

CHAPTER 8 • ROLES OF LIPOPOLYSACCHARIDES

proliferation that may contribute to the abnormalities in gastric acid secretion associated with H. pylori in­ fection (38). Furthermore, induction of apoptosis in gastric epithelia of rats by H. pylori LPS has been shown to contribute to the gastric mucosal injury (89). Although of potential pathogenic importance, the molecular structures within H. pylori LPS respon­ sible for these properties, and hence the structuralbioactivity relationships in the LPS, need to be identi­ fied before these phenomena can be unequivocally ac­ cepted. Despite the assignment of roles to putative open reading frames as LPS biosynthetic genes in the se­ quenced H. pylori genomes ( 1 , 1 0 6 ) , interpretation should be performed with caution, particularly since the putative functions have not been subjected to mu­ tational and biochemical analyses. For future studies, mutational analysis of the genes should be followed by chemical verification of the LPS structures present. Subsequent testing of the validated mutants in animal models and other relevant biological test systems may provide further useful tools for gaining deeper insights into the role of LPS in H. pylori pathogenesis. Acknowledgments. This work was supported by grants from the Irish Health Research Board and the Millennium Research Fund. The author thanks his many colleagues for their continued support.

7.

8.

9.

10.

11.

12.

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21.

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25.

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Helicobacter pylori: Physiology and Genetics Edited by H. L. T. Mobley, G. L. Mendz, and S. L. Hazell © 2 0 0 1 ASM Press, Washington, D.C.

Chapter 9

Vacuolating Cytotoxin JOHN C . ATHERTON, TIMOTHY L. COVER, EMANUELE PAPINI, AND JOHN L. TELFORD

Around 1988, Leunk and colleagues discovered that supernatants from broth cultures of Helicobacter py­ lori induced massive vacuolar degeneration of various cultured epithelial cell lines (53). Since then, the na­ ture of this toxic activity and its role in H. pyloriinduced disease have been the subject of intensive study by a number of groups throughout the world. In 1992, the protein mediating the effect was purified and named the vacuolating cytotoxin (10). Determi­ nation of the amino-terminal sequence of the protein led, in 1994, to the cloning and sequencing of the toxin gene, which was designated vacA (13, 80, 9 0 , 103). Following the initial characterization of the toxin and its gene, research has focused on VacA structure, the mechanisms underlying VacA's toxic activity, naturally occurring differences among VacA proteins produced by different strains of H. pylori, and the clinical importance of VacA polymorphism. Interest in VacA has been intense, partly because of its potential as a novel tool for exploring aspects of eukaryotic cell biology, but mainly because of its putative role in the pathogenesis of H. pylori-associ­ ated diseases, in particular peptic ulceration and distal gastric adenocarcinoma. The precise role of VacA in these diseases is still under investigation, but VacA may contribute to the capacity of H. pylori to colonize and persist in the human gastric mucosa and may also contribute directly to gastric epithelial damage. Hence, VacA is currently a target for therapeutic in­ tervention and a candidate for inclusion in a vaccine against H. pylori. T H E vacA GENE All H. pylori strains contain a copy of the toxin gene, vacA. The vacA transcript is monocistronic,

with its transcriptional start point located about 119 nucleotides upstream from the ATG start codon (28, 29, 90). Insertional mutagenesis of vacA abrogates the capacity of H. pylori to induce vacuolation in epi­ thelial cells, and ablates several other vacA-induced toxic effects (13, 7 8 , 90). Alleles of vacA from at least 25 different H. pylori strains have been sequenced and range from 3,864 to 3,933 nucleotides in length (2, 13, 4 4 , 6 8 , 80, 9 0 , 103). As discussed later in this chapter, there is considerable genetic diversity among vacA alleles from different strains, and alleles can be categorized into several families. The most extensively studied form of VacA is encoded by type s l / m l vacA alleles, which typically encode VacA proteins associ­ ated with a high level of vacuolating cytotoxin activity (Fig. 1) (2). Other forms of VacA are associated with lower level or absent vacuolating activity. For simplic­ ity, we will initially describe the characteristics of prototypic s l / m l forms of VacA. The VacA Protein Processing and secretion of VacA VacA is predicted to encode a protoxin with a mass of about 140 kDa, but the mature secreted VacA toxin migrates as a band of approximately 90 kDa under denaturing conditions (10, 13, 80, 9 0 , 103). A comparison of the amino-terminal sequence of the mature secreted toxin with that predicted for the protoxin indicates that a 33-amino-acid amino-terminal signal sequence is cleaved during the process of VacA secretion (Fig. 1). Studies using antisera raised against different regions of recombinant VacA show that a polypeptide of about 33 kDa derived from the carboxy-terminal portion of the protoxin remains local­ ized to the bacteria and is not secreted (Fig. 1) (103).

John Atherton • Division of Gastroenterology and Institute of Infections and Immunity, University of Nottingham, Nottingham NG7 2UH, United Kingdom. Timothy Cover • Division of Infectious Diseases, Vanderbilt University, and Veterans Affairs Medical Center, Nashville, TN 37232-2605. Emanuele Papini • Department of Biomedical Science and Human Oncology, Section of General Pathology, University of Bari, 70124 Bari, Italy. John Telford • IRIS, Chiron S.p.A, 53100 Siena, Italy.

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Vacuolating cytotoxin gene, vacA Signal region (type si or s2)

Mid region (type ml or m2)

Exposed loop

Signal peptide

p37 p58 fragment fragment Secreted protein, VacA

Cell-associated protein

Figure 1. Schematic of the vacuolating cytotoxin gene, vacA, and protein, VacA (not to scale). vacA alleles vary between strains and this variation is most marked in the second part of the signal region and the ~800-bp mid region. Alleles can be classified according to signal and mid-region type. The translated polypeptide is a protoxin that is amino- and carboxy-terminally processed during secretion. The mature processed toxin slowly dissociates into —37- and —58kDa fragments during storage.

This carboxy-terminal portion of VacA is predicted to contain amphipathic fi-sheets capable of forming a (3-barrel structure and has a terminal phenylalaninecontaining motif that is present in many outer mem­ brane proteins (90). These features, together with a pair of cysteine residues near the carboxy terminus of the mature secreted protein, are characteristic of a family of secreted bacterial proteins called autotransporters (42). Autotransporters do not require any an­ cillary proteins for export across the bacterial outer membrane. Our current understanding of autotransporter export is based primarily on studies of Neisse­ ria gonorrhoeae IgAl protease. Translocation of IgAl protease across the bacterial cytoplasmic membrane is accomplished via a Sec-mediated process and is ac­ companied by cleavage of an amino-terminal signal peptide. The carboxy-terminal (3-barrel domain then inserts into the outer membrane and is thought to function as a pore through which the rest of the mole­ cule passes. Autoproteolytic cleavage yields the ma­ ture secreted IgAl protease; the carboxy-terminal do­ main remains associated with the outer membrane (81).

plexes, about 30 nm in diameter, which appear to consist of a central ring surrounded by six or seven "petals" (54). Three-dimensional reconstructions of these deep-etch metal replicas have provided a de­ tailed view of the surface of VacA oligomers (Fig. 2) (50). As well as the classical flower-like complexes, VacA can assemble into a different type of complex, termed a "flat form," which consists of six or seven petals without a prominent central ring (11, 54). The petals that comprise the flat form typically radiate from the center of the complex with a distinctive clockwise chirality. Multiple models have been pro­ posed to explain the assembly of VacA into flower­ like complexes and chiral flat forms. In one model, the flower-like forms are suggested to comprise six or seven monomers of about 90 kDa (50, 54). In another model, the flower-like forms are suggested to be dodecamers or tetradecamers of VacA monomers of about 90 kDa, and flat forms are suggested to be hexamers or heptamers (11). When VacA is exposed to acidic or alkaline pH, VacA oligomers dissociate into monomeric components of about 90 kDa, each measuring about 6 by 14 nm (11, 5 9 , 1 1 7 ) . This pH-mediated disassembly is associated with a marked increase in VacA cytotoxic activity (11, 2 1 , 5 8 , 1 1 7 ) . It is cur­ rently thought that water-soluble VacA oligomers possess relatively little cytotoxic activity compared to VacA monomers. Further investigation of VacA structure has been undertaken using atomic force microscopic imaging of purified toxin bound to supported lipid bilayers

Oligomeric structure of VacA Early studies indicated that, although mature VacA monomers are approximately 90 kDa in mass, the toxin exists as a much larger complex or aggregate under nondenaturing conditions (10). Lupetti et al. examined the ultrastructure of purified VacA using deep-etch electron microscopy and demonstrated that the toxin assembles into large flower-shaped com­

Figure 2. Image of the top surface of a soluble VacA oligomer, which has a diameter of approximately 30 nm. This is a threedimensional reconstruction from electron micrographs of quickfreeze deep-etch metal replicas of VacA. (Reprinted from reference 50 with permission.)

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Figure 3. (A) Atomic force microscopy image of VacA two-dimensional crystals on a supported lipid bilayer. (B) Processed image of VacA two-dimensional crystals. The line diagram in the top right corner illustrates the orientation of a single oligomer within the crystal. Individual oligomers within the crystal have an overall diameter of about 28 nm. (Reprinted from reference 15 with permission.)

(Fig. 3) (15). Isolated VacA oligomers imaged by this approach appear similar to the structures imaged by deep-etch electron microscopy. Two-dimensional crystalline arrays of VacA on lipid bilayers consist of an ordered array of hexagonal central rings attached by thin connectors to peripheral domains (Fig. 3). H. pylori mutant strains, constructed with inframe deletions in the portion of vacA encoding the amino-terminal region of the toxin, express truncated VacA proteins that are secreted but fail to oligomerize and lack detectable cytotoxic activity (83, 111). One of these mutant VacA proteins (VacA A 9 1 - 3 3 0 ) has been characterized in detail, and forms water-soluble dimers that have an ultrastructural appearance similar to that of the peripheral petals of VacA oligomers (83). This suggests that the peripheral petals of VacA oligomers correspond to the carboxy-terminal por­ tion of the mature secreted VacA polypeptide, most likely the 58-kDa domain described below. Identification of functional domains in VacA Limited proteolysis yields two VacA domains. During prolonged storage or upon incubation with trypsin (11, 5 4 , 103) the purified ~90-kDa VacA toxin tends to degrade into —37- and ~58-kDa com­ ponents, which are derived from the amino terminus and carboxy terminus of the protein, respectively (Fig. 1). Proteolytic cleavage occurs at a site containing multiple charged amino acids, which are predicted to be surface exposed (103). The 37- and 58-kDa frag­ ments of VacA have been presumed to represent subunits or domains of the holotoxin. Recent mass spec­

trometry data indicate that the two VacA fragments have masses of 33 and 5 5 kDa (66), but in this chapter we will continue to refer to the fragments as 37 and 58 kDa to avoid confusion with the published litera­ ture. To determine whether cleavage of VacA into 37and 58-kDa fragments is required for toxin activity, Burroni et al. constructed an H. pylori mutant in which the region of vacA encoding the 46 amino acids flanking the VacA cleavage site was deleted (8). This mutant VacA was fully active, indicating that cleavage of the exposed loop is not necessary for activity. Inter­ estingly, however, in contrast to the wild-type VacA produced by the parent strain, which preferentially formed seven-sided complexes, the mutant preferen­ tially formed six-sided complexes, indicating that re­ moving the exposed loop had introduced structural constraints. Activity of the amino-terminal portion of VacA in the cell cytosol. The minimal region of VacA re­ quired for vacuolating activity has been defined in experiments where mutant forms of vacA under the control of a eukaryotic promoter have been expressed from plasmids in the cytosol of epithelial cells (17). In these experiments, epithelial cell lines were transfected with plasmid constructs encoding either the full-length ~90-kDa secreted toxin or amino- or carboxy-terminally truncated fragments. These ex­ periments showed that a VacA protein lacking most of the carboxy-terminal 58-kDa domain retained full vacuolating activity (18, 121). However, activity was completely abrogated by removing 10 amino acids from the amino-terminus and partially abrogated by

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removing 6 amino acids ( 1 8 , 1 2 1 ) . Thus, the minimal VacA domain that exhibited full vacuolating activity when expressed intracellularly was a peptide compris­ ing amino acids 1 - 4 2 2 , that is, the 37-kDa domain plus a fragment of the 58-kDa domain (121). Interest­ ingly, coexpression of the 37-kDa fragment, which alone was inactive, with a fragment containing the amino-terminal 165 amino acids of the 58-kDa frag­ ment resulted in full vacuolating activity (121). Hydrophobicity plots reveal a possible explanation for the importance of the VacA amino terminus: part of this region (amino acids 1 - 3 2 ) is the only hydropho­ bic region in VacA long enough to span a membrane. T o investigate this further, an H. pylori vacA partial deletion mutant was constructed that lacked codons for amino acids 6 - 2 7 (114). This mutant did not ex­ hibit any obvious alterations in structure compared to wild-type VacA but lacked cytotoxic activity. Fur­ thermore, alanine scanning mutagenesis revealed that point mutations at proline 9 or glycine 14 completely abolished VacA activity (120). Finally, addition of an amino-terminal hydrophilic extension to VacA (iden­ tical to that found in the naturally occurring nontoxigenic s2 forms of VacA discussed later) abolished toxin activity (51). Together, these observations sup­ port a critical role for the amino-terminal hydropho­ bic region in toxin activity. Localization of a receptor binding region. Sev­ eral lines of evidence indicate that binding of VacA to cells is mediated by amino acid sequences located in the carboxy-terminal portion of the mature protein (corresponding to the 58-kDa domain). First, studies with the purified 58-kDa fragment from a mutant H. pylori strain indicate that this protein binds to HeLa cells with kinetics similar to those of the intact toxin (83). Second, polyclonal antiserum reactive with the 58-kDa domain inhibits the binding of VacA to cells (31). Third, some naturally occurring forms of VacA,

which have markedly divergent amino acid sequences in the 58-kDa domain (called m2 forms, and discussed further below), cause vacuolation in a more restricted range of cultured epithelial cell lines than the m l forms of VacA discussed thus far, one explanation for which would be differences in cell binding (68). Finally, VacA with a type m2 58-kDa domain, which did not cause HeLa cell vacuolation when applied ex­ ternally, caused vacuolation when expressed from a plasmid in the HeLa cell cytoplasm. This implies that m2 VacA is fully active but cannot get to its site of action; one explanation of this would be inability to bind to the cell (17). Experiments using naturally oc­ curring and engineered ml/m2 chimeric proteins (46) suggest that an —40 amino acid region near the amino-terminal end of the 58-kDa domain is neces­ sary for HeLa cell vacuolation and may be involved in HeLa cell binding. Biological Activity of VacA (Fig. 4 ) Cytotoxicity VacA causes epithelial cell vacuolation in vitro, but this does not lead rapidly to cell death. For pri­ mary human gastric epithelial cells exposed to high doses of toxin, cell death has been documented after 2 days (95). In contrast, cell death does not usually occur in immortalized cell lines exposed to the toxin; for example, incubation of AZ-521 gastric epithelial cells with VacA for several hours causes reduced mito­ chondrial ATP production and reduced oxygen con­ sumption but does not result in cell death (48). Whether VacA causes epithelial cell death in vivo in humans is unknown, but in mice oral administration of VacA leads to erosion of the gastric epithelium, presumably involving cellular loss (107). VacA binding The binding and uptake of VacA by cells are not yet clearly understood, and there are many apparent

Figure 4. Cartoon illustrating a possible model for interaction of VacA with the epithelial cell. The inactive, soluble oligomer, made up of p37 and p58 fragments, can be disrupted by acidic or alkaline treatment to release active monomeric VacA. Binding: Both the oligomeric and the monomeric toxin are thought to undergo nonspecific binding (NB) to the plasma membrane (PM), which probably does not result in endocytosis. Only activated toxin is thought to undergo specific binding (SB), which may allow toxin internalization. The recently identified RPTP-p appears to be one specific VacA receptor (R). Membrane interaction (MI): Hydrophobic VacA monomers can insert into the plasma membrane, where they associate to form a pore. The structure of this membrane-associated oligomeric VacA may be different from that of the soluble inactive oligomer. The membrane-associated toxin is an anion (X~) selective channel, which allows changes in ionic flux at the level of the plasma membrane. Endocytosis (E): Membrane-associated VacA is internalized, presumably by receptor-mediated endocytosis, transported, and accumulated in endolysosomes. The toxin receptors are depicted still associated with the toxin, but it is not clear whether the pore-receptor complex becomes dissociated. Vacuolation: In one possible model, anion conduction by endocytosed VacA channels stimulates electrogenic proton pumping activity of the V-ATPase. In the presence of ammonia (or other weak bases) this may lead to ammonium ion accumulation in the endolysosomal compartment. The increased concentration of the osmotically active ammonium ions is believed to lead to water influx, swelling of endolysosomes, and vacuole formation.

CHAPTER 9 • VACUOLATING CYTOTOXIN

Inactive oligomer

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contradictions in this area of research. The prototypic s l / m l form of VacA binds to HeLa cells in a saturable manner when assessed by flow-cytometry analysis (57). In contrast, saturable binding has not been de­ tected with classical ligand binding assays with I labeled VacA (58). Activation of VacA by acid treat­ ment markedly enhances its vacuolating activity but does not significantly increase its binding to HeLa or BHK cells ( 5 7 - 5 9 ) . In contrast, acid activation en­ hances binding of the toxin to the gastric cell line AZ521 (117). Several specific VacA receptors have been proposed. In the AZ-521 system, activated VacA binds to a 250-kDa receptor protein-tyrosine phos­ phatase B (RPTPB), which regulates intracellular tyro­ sine phosphorylation (67, 1 1 7 ) . Several lines of evi­ dence suggest a vital role for RPTPB in binding of VacA to cells and subsequent intoxication. First, treat­ ment of the HL-60 cell line with phorbol 12-myristate 13-acetate (PMA) leads to induction of RPTPB expression, which is accompanied by induction of VacA sensitivity (20). Second, BHK-21 cells, which are insensitive to VacA, can be made sensitive by transfection with expression vectors containing the RPTPB gene. Finally, ablation of RPTPB synthesis by antisense oligonucleotides in PMA-treated HL-60 cells results in a significant decrease in VacA-induced vacuolation. Two other specific VacA receptors have been reported: an unidentified 140-kDa protein in AZ-521 and AGS cells (116) and the epidermal growth factor receptor in HeLa cells (91). Taken to­ gether, these observations suggest the existence of multiple surface-binding sites recognized by both in­ active and activated VacA and also the presence of specific VacA receptors that are variably expressed in different cell lines. 1 2

VacA internalization and intracellular trafficking Both 58-kDa and 37-kDa regions are required for VacA internalization (83). To be internalized, VacA must be preactivated by exposure to acid or alkali (58). Internalization occurs through an energydependent process, the precise nature of which is un­ clear but which may be receptor-mediated endocytosis. After internalization, VacA molecules localize in membrane vesicles (31) and are transported along the endocytic pathway to vacuolar-type (V-) ATPase-positive late endosomes and lysosomes, where they accu­ mulate and persist for days with little evidence of deg­ radation (85, 9 6 ) .

olar membranes contain both late endosomal and ly­ sosomal markers, suggesting that the vacuoles are derived from these compartments (62, 7 5 ) . The for­ mation of VacA-induced vacuoles requires the full ac­ tivity of V-ATPase and the presence of weak bases (12, 14, 7 4 , 85), suggesting that vacuoles are derived from the accumulation of weak bases within acidic compartments followed by water influx and swelling. In addition, two small GTP-binding proteins have been shown to be involved in vacuole biogenesis: the membrane traffic regulator rab7 and the actin-cytoskeleton-associated R a c l ( 4 3 , 7 6 ) . These two proteins are associated with the membrane of VacA-induced vacuoles. Vacuolation is inhibited by the expression of rab7 or R a c l dominant negative mutants and is potentiated by dominant positive mutants. These ob­ servations suggest that vacuole development is regu­ lated by membrane fusion events and by the cytoskele­ ton supporting late endosomal compartments. In HeLa cells, VacA impairs the transport of acidic hy­ drolases to lysosomes, resulting in release of these en­ zymes into the extracellular medium (89). Moreover, the degradative power of HeLa cell lysosomes (89) and of the antigen-processing compartment of B lym­ phocytes (61) is also reduced by VacA. Such early functional alteration of the endocytic pathway, occur­ ring in the absence of vacuolation, is likely to be due to a partial neutralization of acidic compartments (89). Alteration of epithelial permeability Epithelial monolayers of MDCK I, T 8 4 , or epH4 cells on porous filters are not vacuolated by VacA and do not show signs of endolysosomal dysfunction (77). However, following exposure to VacA, transepithelial electrical resistance (TER) decreases, accompanied by an increase in transepithelial flux of low-molecularweight molecules (77). The size selectivity of this in­ creased epithelial permeation, the lack of accompany­ ing vacuolation, and the lack of redistribution of junc­ tional proteins all suggest that VacA modulates the resistance of these model epithelia through a paracellular effect. Only epithelial cell monolayers able to develop a T E R higher than 1,000 to 1,200 a / c m are affected. The use of isogenic mutant strains confirms that the effect is dependent on VacA (78). The natu­ rally occurring m2 type of VacA discussed later re­ duces TER in MDCK cells but does not cause vacuola­ tion in this cell line even when cells are nonconfluent (78), further confirming that vacuolation and in­ creased permeability of monolayers are discrete and independent effects. 2

Cell vacuolation and impairment of endolysosmal function

Ion channel formation

The induction of intracellular vacuoles was the first characterized action of VacA (14, 53). The vacu­

VacA forms ion channels in model lipid bilayers and cell plasma membranes, and this phenomenon

CHAPTER 9

may underlie all the other effects of VacA. Disassem­ bly of the inactive VacA oligomer by acidic conditions allows insertion of the toxin into lipid bilayers (61, 62, 69). Experiments with planar model membranes show that membrane insertion is followed by the for­ mation of voltage-dependent, low-conductance (10 to 30 pS in 2 M KC1), anion-selective channels ( 4 5 , 1 0 4 ) . Patch clamp analysis of HeLa cells demonstrates that VacA forms plasma membrane channels with proper­ ties similar to those observed in model membranes (100). Various anion channel blockers inhibit VacA channels in vitro with different potencies and are able to prevent and partially reverse vacuolation of HeLa cells (100, 105), indicating an essential role of the anion channel in vacuolation. The development of a dominant negative mutant of VacA, able to form mixed oligomers with wild-type VacA, provides addi­ tional evidence suggesting that functional anion chan­ nel formation is required for cell vacuolation (117). It has been proposed that the endocytosed VacA chan­ nel, by allowing anions to permeate into late endosomes, increases the turnover of the electrogenic VATPase, which leads to accumulation of weak bases (when present) and thence to vacuole formation by water influx ( 1 0 2 , 1 0 8 ) . This hypothesis is in keeping with the observation that internalization of surfacebound VacA is necessary for the subsequent develop­ ment of vacuolation (58). In this model, vacuolation can be thought of as a side effect of the massive accu­ mulation of endocytosed VacA channels in endolyso­ somes. VacA epithelial permeabilization of MDCK I cells can be partially prevented and reversed by 5nitro-2-(3-phenylpropylamine) benzoic acid (NPPB), the most effective blocker of VacA channels, implying that epithelial permeabilization, like vacuolation, is secondary to the formation of apical anion channels (100). In Caco-2 cells, VacA induces an increased api­ cal anion secretion, and this also is blocked by NPPB (37), implying that it too is due to VacA anion channel formation. VacA: more than a channel? VacA has some features in common with A/Btype toxins, which have an active, enzymatic subunit (A) and a binding subunit (B). These features include its putative two-domain structure, the observation that binding is associated with the 58-kDa fragment, and the fact that cytosolic expression of the 37-kDa fragment (admittedly with part of the 58-kDa frag­ ment) induces cell vacuolation. In this A/B toxin model, the active (enzymatic) domain of VacA is hy­ pothesized to be exposed to or released into the cell cytosol, where it modifies an unknown protein in­

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volved in the regulation of endosome-endosome fusion. Recently, a 54-kDa VacA-binding protein, called VIP54, has been identified by yeast two-hybrid screening (19). Although VLP54 interacts in vitro with VacA and is associated with the HeLa cell cytoskele­ ton, so far no evidence of an interaction between VacA and VIP54 in epithelial cells has been provided. Whether or not VacA interacts directly with the cyto­ skeleton, there is other evidence that VacA-induced cytoskeletal changes may occur. For example, expo­ sure of rat gastric epithelial cells to VacA results in inhibition of actin stress fiber formation and disrup­ tion of microtubules (70). Such effects of VacA on cytoskeletal structure are potentially important, not only in dissecting the mechanism of vacuolation, but also in understanding the inhibitory effects of VacA on cell proliferation (71, 84). Variation among vacA Alleles in Different H. pylori Strains vacA evolution H. pylori exhibits enormous genetic variation, re­ sulting mainly from extensive recombination between H. pylori strains—the most extensive described for any bacterial species (36, 9 9 ) . For many strain com­ parisons, this has led to complete linkage equilibrium between genes and also (uniquely among bacteria) be­ tween loci within genes (36). Sequence analyses of vacA alleles from different H. pylori strains have shown that there are more pronounced differences be­ tween alleles in some regions of the gene than in others (2, 12); for example, a region near the 5' end of the gene is relatively well conserved between alleles. Within this region, recombination has destroyed vir­ tually all detectable phylogenetic clonal structure and there appears to be linkage equilibrium between most loci (36, 99). The portion of vacA that exhibits maxi­ mum diversity is an ~ 8 0 0 - b p "mid region," which encodes part of the 58-kDa domain of VacA (Fig. 1) (2, 5). In contrast to the region near the 5' end of vacA, sequences from the mid region can be phylogenetically divided into two families of alleles, termed m l and m2 (2, 5). H. pylori strains with type m l vacA alleles and strains with type m2 alleles are each widespread in all human populations examined, ex­ cept in the Japanese, among whom type m2 alleles are rare, as further discussed below (3, 4 4 , 118). Strains with evidence of recombination within the vacA mid region between type m l and m2 alleles ap­ pear rare, with ml/m2 recombinant alleles described only in a few Chinese strains of H. pylori (5, 7 2 , 1 1 2 ) . Strains with evidence of recombination in the mid re­ gion among m l and among m2 alleles are more fre-

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quent, but recombination has not been sufficient to abolish phylogenetic structure even within the m l and m2 types (5). This residual phylogenetic structure has led to several proposed subdivisions of the main m l and m2 types ( 4 1 , 7 2 , 9 8 , 107, 112). Aside from the vacA mid region, various other smaller regions differ markedly between vacA alleles. In particular, the "sig­ nal region," encoding part of the amino-terminal sig­ nal peptide and the amino terminus of the processed toxin, exhibits striking diversity between strains (Fig. 1) (2). Two main allelic families of signal regions are recognized, termed s i and s2, and the si type can be further subdivided into s l a , s i b , and s i c (2, 109). As a result of extensive recombination among vacA alleles, all combinations of signal and mid regions can theoretically arise ( s l a / m l , s l b / m l , s l c / m l , sla/m2, etc.) and most of these have indeed been described (2, 52, 6 3 , 114). Analysis of nucleotide substitution rates has pro­ vided further interesting insights into vacA evolution. Marked differences in synonymous substitution rates between vacA regions are consistent with extensive recombination and subsequent natural selection of ge­ netic elements within vac A (5). The ratio of nonsynonymous to synonymous substitution rates is a mea­ sure of structural constraints on function. This ratio is similar for comparisons among m l mid regions and among m2 mid regions, but higher for comparisons between m l and m2 regions. This is consistent with the hypothesis that m l and m2 regions encode do­ mains with different functions, including perhaps dif­ ferent cell-binding properties, as described below. vacA polymorphism and toxin activity Differences between vacA alleles were first inves­ tigated in an attempt to understand why culture supernatants from many H. pylori strains did not cause cell vacuolation in vitro, despite all strains having a copy of the toxin gene vacA (2, 12). Nonsense muta­ tions in vacA alleles have been detected, especially in Japan, and these account for absence of toxin activity in some H. pylori strains (44). However, most western isolates of H. pylori express full-length VacA proteins (3), and for such strains, vac A genotype is a major determinant of vacuolating activity. VacA proteins with a type s2 signal sequence undergo amino-termi­ nal signal peptide cleavage at a different site to VacA proteins with a type s i signal sequence (3). As a conse­ quence, secreted type s2 VacA has a hydrophilic amino terminus, which has been shown to block toxin activity (51). Type s l / m l VacA proteins are active on a wide variety of epithelial cell lines, whereas at least some tested type sl/m2 VacA proteins, although ac­ tive on primary gastric epithelial cells and RK-13 cells,

are minimally active on HeLa cells (70). This differ­ ence in cell type specificity appears attributable, at least in part, to different cell-binding properties me­ diated by the part of the 58-kDa VacA domain en­ coded by the vac A mid region (70). Further variations among signal and mid regions beyond the sl/s2 and ml/m2 divisions have not yet been demonstrated to affect VacA toxicity and so are currently of mainly epidemiological and phylogenetic interest. Association between cagA and vacuolating activity The close but not absolute association between the presence of the cytotoxin-associated gene, cagA (discussed fully in chapter 31), and vacuolating activ­ ity has been enigmatic. In particular, insertional muta­ genesis of cagA or deletion of other genes in the cag pathogenicity island (Pal) does not alter VacA expres­ sion or activity (1). The discovery of different vac A allelic types has gone some way to explain the link: there is a close genetic association between the pres­ ence of cagA (and other genes in the cag Pal) and the presence of type s i vac A alleles (2). However, given the high level of intraspecies recombination in H. py­ lori (34, 3 6 , 99) and the separation of vacA and the cag Pal on the H. pylori chromosome, the reason for this genetic association is unclear. It seems likely that there is some as yet unidentified selective advantage to strains that is conferred by either possessing or lacking both type s i vac A and the cag Pal. Clinical Importance of Strain Differences in VacA Association of vacuolating cytotoxin activity with human disease Numerous studies have shown that H. pylori strains that express vacuolating cytotoxin activity in vitro are more commonly associated with disease than are noncytotoxic strains. Studies from around the world (other than, as discussed below, Japan) have shown an association between vacuolating activity and peptic ulcer disease ( 2 , 2 7 , 3 5 , 8 2 , 1 0 2 , 1 1 4 , 1 2 2 ) . However, although the association is consistent and significant, it is not absolute. Patients with peptic ul­ ceration frequently have noncytotoxic H. pylori iso­ lates, and patients without ulcers frequently have cy­ totoxic isolates. Multiple factors complicate the interpretation of these studies, some of which are in­ herent in all studies examining associations with pep­ tic ulcer disease. For example, patients with peptic ulcer disease may have ulcers in remission at the time of upper gastrointestinal endoscopy and thus a nor­ mal endoscopic examination. Similarly, patients with ulcers at endoscopy may be taking nonsteroidal anti-

CHAPTER 9 • VACUOLATING CYTOTOXIN

inflammatory drugs, which could be responsible for the ulcers, or alternatively, they may be colonized by both cytotoxic and noncytotoxic strains of H. pylori and have a noncytotoxic strain isolated. A further spe­ cific problem with studies assessing vacuolating activ­ ity is that these studies have used cell lines only sensi­ tive to type m l VacA. However, even taking all these points into consideration, it is clear that colonization with cytotoxic strains does not always result in ulcers. Few studies have examined the association between cytotoxic activity and gastric carcinoma or precancer­ ous changes, such as gastric atrophy, but where looked for, weak associations have been found (22, 30). Relevance of VacA antibodies Human infection by H. pylori induces a systemic and local antibody response, and antibodies to VacA can be detected by enzyme-linked immunosorbent as­ says (ELISAs) or immunoblot assays. Commercial kits have been marketed for this purpose. Several studies have assessed the association between the presence of VacA antibodies and disease and usually no associa­ tion has been found (23, 9 2 , 1 0 1 , 119). However, some studies have shown weak associations, for ex­ ample, between VacA expression and peptic ulcera­ tion (56) or between the level of antibody response and the level of gastric atrophy (94). The lack of strong associations is not surprising, as the vast ma­ jority of strains express a VacA protein, although this may or may not be toxigenic. Recently, purified s l / m l and s2/m2 VacA proteins have been used in ELISAs, in an effort to distinguish between infection with m l and m2 strains (79). Developments of this sort may allow more meaningful serological testing for different forms of VacA in the future. Association of vacA genotypes with disease The discovery of multiple vacA genotypes and the description of simple and accurate PCR-based methodology for distinguishing between them (2, 3, 108) have stimulated many studies of the association between specific vacA genotypes and disease. The PCR-based methodology means that strain culture is not strictly necessary (38, 87), although many work­ ers have continued to use cultured strains for typing studies. Most such studies from outside Asia have shown that vacA s2 strains are less commonly associ­ ated with peptic ulceration or gastric carcinoma than si strains (2, 4, 7, 2 5 , 2 6 , 4 0 , 4 7 , 6 5 , 7 3 , 86, 87, 97, 9 8 , 1 0 7 , 1 0 8 , 1 1 0 , 1 1 3 ) , although studies from Texas have shown no statistically significant association ( 3 4 , 1 1 8 ) . It is still unclear whether vacA s2 strains are

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entirely nonulcerogenic. Because of the close genetic association between the vac A s i signal type and pres­ ence of the cag Pal, it is possible that one is merely a marker for the other, although it is equally possible that both are important for disease. A third potential virulence determinant is the blood group antigenbinding adhesin, BabA. The babA gene, like vacA, is polymorphic, and one allelic type, babAl, is disease associated (32). Use of all three markers (vacA s i , cagA, babAl) is a better predictor of pathogenic po­ tential than use of one or two (32), supporting both the concept that all may be clinically important and the idea that the genetic association between them may be due to selective advantage from all being posi­ tive, or none. Most studies have not shown an association be­ tween vacA mid region type and disease, although two studies have shown an association of type m l strains with gastric adenocarcinoma (47, 107). Intriguingly, nearly all strains from Japan are vac A s i / m l ( 4 4 , 4 9 , 88, 9 3 , 1 1 9 ) , the most toxic type, making it interesting to speculate that toxicity may contribute to the high prevalence of gastric adenocarcinoma in Japan. This homogeneity also means that vacA geno­ type and presence or absence of toxin activity are not potentially useful markers of more pathogenic strains within Japan. The mechanisms underlying H. pylori-'mduced peptic ulceration and gastric adenocarcinoma are un­ clear. Inflammation, direct toxic activity, or both could be important. The level of inflammation in the gastric mucosa is associated with vac A signal type (si > s2) (4, 39). Gastric epithelial damage is also associ­ ated with vacA signal type, but one study has shown a further association with vacA mid region type (ml > m2) (4). The density of H. pylori colonization in vivo may be important in disease pathogenesis, but studies assessing the association of vacA type with H. pylori density have given conflicting results (6, 4 0 , 110). As well as being important in pathogenesis, VacA effects on inflammation and epithelial damage may be important in H. pylori treatment. Antibiotic treatment of H. pylori is more often successful for vacA si strains than for vacA si strains ( 9 , 1 1 0 ) , and one possible explanation for this is that antibiotics are delivered better across inflamed and damaged mu­ cosa. vacA subtypes as an epidemiological tool Subtyping of vacA signal and mid regions has given useful information on the population genetics of H. pylori. Of the s i subtypes of vacA, type s i c is found exclusively in East Asian strains, type s i a is predominant in many northern European countries,

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and type s i b is predominant in the Iberian Peninsula and South America ( 1 0 6 , 1 0 7 ) . This latter association suggests genetic relatedness between strains from Spain, Portugal, and South America. Specific m l and m2 variants have been found in parts of Europe and Asia (41, 7 2 , 9 8 , 1 0 7 , 1 1 2 ) . The main Asian m2 sub­ type appears specific for this region and, as expected, is commonly associated with the s i c signal type (72, 107, 1 1 2 ) . Construction of dendrograms (phyloge­ netic trees) based on vac A mid region sequences gives further information on relatedness between H. pylori strains from different populations. For example, one analysis shows separate clustering of East Asian, eth­ nic European, and East Indian strains, implying initial strain dissemination and then relative isolation within these populations (64).

role for VacA in colonization; wild-type H. pylori strains and VacA null mutants did not differ in their capacity to colonize gnotobiotic piglets (24), and wild-type strains colonized gerbils only slightly more efficiently than did VacA null mutant strains (115). A further suggestion is that expression of VacA in the human gastric mucosa could potentially offer a survival advantage to H. pylori once colonization is established. Several possible mechanisms have been suggested. VacA-induced epithelial injury or altera­ tions in the integrity of tight junctions might decrease the integrity of the mucosa, and thereby favor H. py­ lori growth by promoting efflux of nutrients from the mucosa into the mucous layer. Consistent with this hypothesis, VacA induces increases in the permeabil­ ity of epithelial monolayers in vitro, which results in increased diffusion of critical nutrients such as N i and F e (77, 7 8 ) . The formation of anion-selective channels in the apical membranes of gastric epithelial cells also might facilitate H. pylori growth in the gas­ tric mucosa. For example, the release of H C 0 ~ from cells via VacA channels might help neutralize gastric acidity in the microenvironments where H. pylori is found (100, 104). Finally, by interfering with normal endocytic pathways, VacA may alter the process of antigen presentation, which may be one of the mecha­ nisms by which H. pylori evades host defenses (59). Thus, although considerable attention has been fo­ cused on the capacity of VacA to cause tissue injury, the toxin may additionally or alternatively have other, more subtle, functions. 2 +

Role of VacA In Vivo H. pylori produces a toxin that has damaging effects on epithelial cells, and H. pylori colonization is a strong risk factor for the development of peptic ulceration. These facts have led to the hypothesis that VacA directly damages the gastric and duodenal epi­ thelium in vivo and hence causes ulcers. This hypothe­ sis is supported by the association of the si type of VacA (which causes vacuolation in vitro) with peptic ulceration and the s2 type (which is nonvacuolating in vitro) with the absence of ulcers. Furthermore, oral administration of VacA to mice causes damage to the gastroduodenal epithelium, including superficial ul­ ceration ( 3 3 , 1 0 3 ) . However, evidence against an epi­ thelial damaging role for VacA in vivo comes from experiments in which piglets or gerbils were infected with wild-type toxigenic strains of H. pylori or VacA null mutants; no differences in epithelial damage were observed (24, 115). This consistent lack of VacA-attributable damage in several infected animal models has led to the suggestion that VacA may offer other, more subtle, advantages to H. pylori in vivo. This suggestion seems logical from an evolutionary view­ point; it would seem unlikely that a bacterium should produce a toxin primarily to cause a condition such as peptic ulceration from which it does not derive any clear benefit. However, it is feasible that causing ul­ cers in some individuals is an unfortunate secondary effect of VacA. One alternative possibility to a directly damaging primary role for VacA is that VacA may play a role in enabling H. pylori to colonize the human stomach. Some evidence supports a role for VacA in gastric colonization; for example, immunization of mice with VacA prevented infection when mice were subse­ quently experimentally challenged with H. pylori (55). However, other experiments do not support a

3 +

3

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CHAPTER 9 • VACUOLATING CYTOTOXIN

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CHAPTER 9 • VACUOLATING CYTOTOXIN

67. Padilla, P. I., A. Wada, K. Yahiro, M. Kimura, T. Niidome, H. Aoyagi, A. Kumatori, M. Anami, T. Hayashi, J. Fujisawa, H. Saito, J. Moss, and T. Hirayama. 2000. Morphologic dif­ ferentiation of HL-60 cells is associated with appearance of RPTPbeta and induction of Helicobacter pylori VacA sensi­ tivity. /. Biol. Chem. 275:15200-15206. 68. Pagliaccia, C , M. de Bernard, P. Lupetti, X. Ji, D. Burroni, T. L. Cover, E. Papini, R. Rappuoli, J. L. Telford, and J . M. Reyrat. 1998. The m2 form of the Helicobacter pylori cytotoxin has cell type-specific vacuolating activity. Proc. Natl. Acad. Sci. USA 95:10212-10217. 69. Pagliaccia, C , X. M. Wang, F. Tardy, J. L. Telford, J. M. Ruysschaert, and V. Cabiaux. 2000. Structure and interac­ tion of VacA of Helicobacter pylori with a lipid membrane. Eur.}. Biochem. 267:104-109. 70. Pai, R., T. L. Cover, and A. S. Tarnawski. 1999. Helicobacter pylori vacuolating cytotoxin (VacA) disorganizes the cyto­ skeletal architecture of gastric epithelial cells. Biochem. Biophys. Res. Commun. 262:245-250. 71. Pai, R., E. Sasaki, and A. S. Tarnawski. 2000. Helicobacter pylori vacuolating cytotoxin (VacA) alters cytoskeleton-associated proteins and interferes with re-epithelialization of wounded gastric epithelial monolayers. Cell Biol. Int. 24: 291-301. 72. Pan, Z. J., D. E. Berg, R. W. van der Hulst, W. W. Su, A. Raudonikiene, S. D. Xiao, J. Dankert, G. N. Tytgat, and A. van der Ende. 1998. Prevalence of vacuolating cytotoxin pro­ duction and distribution of distinct vacA alleles in Helico­ bacter pylori from China. /. Infect. Dis. 178:220-226. 73. Pan, Z. J., R. W. van der Hulst, G. N. Tytgat, J. Dankert, and A. van der Ende. 1999. Relation between vacA subtypes, cytotoxin activity, and disease in Helicobacter pylori-intected patients from The Netherlands. Am. ] . Gastroenterol. 94: 1517-1521. 74. Papini, E., M. Bugnoli, M. De Bernard, N. Figura, R. Rappu­ oli, and C. Montecucco. 1993. Bafilomycin Al inhibits Heli­ cobacter pylori-induced vacuolization of HeLa cells. Mol. Mi­ crobiol. 7:323-327. 75. Papini, E., M. de Bernard, E. Milia, M. Bugnoli, M. Zerial, R. Rappuoli, and C. Montecucco. 1994. Cellular vacuoles induced by Helicobacter pylori originate from late endosomal compartments. Proc. Natl. Acad. Sci. USA 91:9720-9724. 76. Papini, E., B. Satin, C. Bucci, M. de Bernard, J. L. Telford, R. Manetti, R. Rappuoli, M. Zerial, and C. Montecucco. 1997. The small GTP binding protein rab7 is essential for cellular vacuolation induced by Helicobacter pylori cyto­ toxin. EMBO J. 16:15-24. 77. Papini, E., B. Satin, N. Norais, M. de Bernard, J. L., Telford, R. Rappuoli, and C. Montecucco. 1998. Selective increase of the permeability of polarized epithelial cell monolayers by Helicobacter pylori vacuolating toxin. /. Clin. Invest. 102: 813-820. 78. Pelicic, V., J. M. Reyrat, L. Sartori, C. Pagliaccia, R. Rappu­ oli, J. L. Telford, C. Montecucco, and E. Papini. 1999. Heli­ cobacter pylori VacA cytotoxin associated with the bacteria increases epithelial permeability independently of its vac­ uolating activity. Microbiology 145:2043-2050. 79. Perez-Perez, G. I., R. M. Peek, Jr., J. C. Atherton, M. J. Blaser, and T. L. Cover. 1999. Detection of anti-VacA antibody re­ sponses in serum and gastric juice samples using type sl/ml and s2/m2 Helicobacter pylori VacA antigens. Clin. Diagn. Lab. Immunol. 6:489-493. 80. Phadais, S. H., D. liver, L. Janzon, S. Normark, and T. U. Westblom. 1994. Pathological significance and molecular characterization of the vacuolating toxin gene of Helicobacter pylori. Infect. Immun. 62:1557-1565.

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81. Pohlner, J., R. Halter, K. Beyreuther, and T. F. Meyer. 1987. Gene structure and extracellular secretion of Neisseria gonor­ rhoeae IgA protease. Nature 325:458-462. 82. Rautelin, H., B. Blomberg, G. Jarnerot, and D. Danielsson. 1994. Nonopsonic activation of neutrophils and cytotoxin production by Helicobacter pylori: ulcerogenic markers. Scand. J. Gastroenterol. 29:128-132. 83. Reyrat, J. M., S. Lanzavecchia, P. Lupetti, M. de Bernard, C. Pagliaccia, V. Pelicic, M. Charrel, C. Ulivieri, N. Norais, X. Ji, V. Cabiaux, E. Papini, R. Rappuoli, and J. L. Telford. 1999. 3D imaging of the 58 kDa cell binding subunit of the Helicobacter pylori cytotoxin. /. Mol. Biol. 290:459-470. 84. Ricci, V., C. Ciacci, R. Zarrilli, P. Sommi, M. K. Tummuru, C. Del Vecchio Blanco, C. B. Bruni, T. L. Cover, M. J. Blaser, and M. Romano. 1996. Effect of Helicobacter pylori on gas­ tric epithelial cell migration and proliferation in vitro: role of VacA and CagA. Infect. Immun. 64:2829-2833. 85. Ricci, V., P. Sommi, R. Fiocca, M. Romano, E. Solcia, and U. Ventura. 1997. Helicobacter pylori vacuolating toxin ac­ cumulates within the endosomal-vacuolar compartment of cultured gastric cells and potentiates the vacuolating activity of ammonia. /. Pathol. 183:453-459. 86. Rudi, J., C. Kolb, M. Maiwald, D. Kuck, A. Sieg, P. R. Galle, and W. Stremmel. 1998. Diversity of Helicobacter pylori vacA and cagA genes and relationship to VacA and CagA protein expression, cytotoxin production, and associated dis­ eases. /. Clin. Microbiol. 36:944-948. 87. Rudi, J., A. Rudy, M. Maiwald, D. Kuck, A. Sieg, and W. Stremmel. 1999. Direct determination of Helicobacter pylori vacA genotypes and cagA gene in gastric biopsies and rela­ tionship to gastrointestinal diseases. Am. } . Gastroenterol. 94:1525-1531. 88. Sadakane, Y., K. Kusaba, Z. Nagasawa, I. Tanabe, S. Kuroki, and J . Tadano. 1999. Prevalence and genetic diversity of cagD, cagE, and vacA in Helicobacter pylori strains isolated from Japanese patients. Scand. J. Gastroenterol. 34:981-986. 89. Satin, B., N. Norais, J. Telford, R. Rappuoli, M. Murgia, C. Montecucco, and E. Papini. 1997. Effect of Helicobacter pylori vacuolating toxin on maturation and extracellular re­ lease of procathepsin D and on epidermal growth factor deg­ radation. /. Biol. Chem. 272:25022-25028. 90. Schmitt, W., and R. Haas. 1994. Genetic analysis of the Heli­ cobacter pylori vacuolating cytotoxin: structural similarities with the IgA protease type of exported protein. Mol. Micro­ biol. 12:307-319. 91. Seto, K., Y. Hayashi-Kuwabara, T. Yoneta, H. Suda, and H. Tamaki. 1998. Vacuolation induced by cytotoxin from Helicobacter pylori is mediated by the EGF receptor in HeLa cells. FEBS Lett. 431:347-350. 92. Shimoyama, T., B. Neelam, S. Fukuda, M. Tanaka, A. Munakata, and J. E. Crabtree. 1999. VacA seropositivity is not associated with the development of gastric cancer in a Japa­ nese population. Eur. J. Gastroenterol. Hepatol. 11: 887-890. 93. Shimoyama, T., T. Yoshimura, T. Mikami, S. Fukuda, J. E. Crabtree, and A. Munakata. 1998. Evaluation of Helico­ bacter pylori vacA genotype in Japanese patients with gastric cancer. /. Clin. Pathol. 51:299-301. 94. Shirasaka, D., N. Aoyama, K. Satonaka, K. Shirakawa, H. Yoshida, T. Sakai, T. Ikemura, Y. Shinoda, M. Sakashita, M. Miyamoto, K. Yahiro, A. Wada, H. Kurazono, T. Hiray­ ama, and M. Kasuga. 2000. Analysis of Helicobacter pylori vacA gene and serum antibodies to VacA in Japan. Dig. Dis. Sci. 45:789-795. 95. Smoot, D. T., J. H. Resau, M. H. Earlington, M. Simpson,

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100.

101.

102.

103.

104.

105.

106.

107.

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and T. L. Cover. 1996. Effects of Helicobacter pylori vac­ uolating cytotoxin on primary cultures of human gastric epi­ thelial cells. Gut 39:795-799. Sommi, P., V. Ricci, R. Fiocca, V. Necchi, M. Romano, J. L. Telford, E. Solcia, and U. Ventura. 1998. Persistence of Helicobacter pylori VacA toxin and vacuolating potential in cultured gastric epithelial cells. Am. J. Physiol. 275: G681-G688. Stephens, J. C , J. A. Stewart, A. M. Folwell, and B. J. Rathbone. 1998. Helicobacter pylori cagA status, vacA genotypes and ulcer disease. Eur. J. Gastroenterol. Hepatol. 10: 381-384. Strobel, S., S. Bereswill, P. Balig, P. Allgaier, H. G. Sonntag, and M. Kist. 1998. Identification and analysis of a new vacA genotype variant of Helicobacter pylori in different patient groups in Germany. /. Clin. Microbiol. 36:1285-1289. Suerbaum, S., J. M. Smith, K. Bapumia, G. Morelli, N. H. Smith, E. Kunstmann, L. Dyrek, and M. Achtman. 1998. Free recombination within Helicobacter pylori. Proc. Natl. Acad. Sci. USA 95:12619-12624. Szabo, L, S. Brutache, F. Tombola, M. Moschioni, B. Satin, J. L. Telford, R. Rappuoli, C. Montecucco, E. Papini, and M. Zoratti. 1999. Formation of anion-selective channels in the cell plasma membrane by the toxin VacA of Helicobacter pylori is required for its biological activity. EMBO } . 18: 5517-5527. Takata, T., S. Fujimoto, K. Anzai, T. Shirotani, M. Okada, Y. Sawae, and J. Ono. 1998. Analysis of the expression of CagA and VacA and the vacuolating activity in 167 isolates from patients with either peptic ulcers or non-ulcer dyspepsia. Am. J. Gastroenterol. 93:30-34. Tee, W., J. R. Lambert, and B. Dwyer. 1995. Cytotoxin pro­ duction by Helicobacter pylori from patients with upper gas­ trointestinal tract diseases./. Clin. Microbiol. 33:1203-1205. Telford, J. L., P. Ghiara, M. Dell'Orco, M. Comanducci, D. Burroni, M. Bugnoli, M. F. Tecce, S. Censini, A. Covacci, Z. Xiang, E. Papini, C. Montecucco, L. Parente, and R. Rap­ puoli. 1994. Gene structure of the Helicobacter pylori cyto­ toxin and evidence of its key role in gastric disease. /. Exp. Med. 179:1653-1658. Tombola, F., C. Carlesso, I. Szabo, M. de Bernard, J. M. Reyrat, J. L. Telford, R. Rappuoli, C. Montecucco, E. Papini, and M. Zoratti. 1999. Helicobacter pylori vacuolating toxin forms anion-selective channels in planar lipid bilayers: possi­ ble implications for the mechanism of cellular vacuolation. Biophys. J. 76:1401-1409. Tombola, F., F. Oregna, S. Brutsche, I. Szabo, G. Del Giudice, R. Rappuoli, C. Montecucco, E. Papini, and M. Zoratti. 1999. Inhibition of the vacuolating and anion channel activi­ ties of the VacA toxin of Helicobacter pylori. FEBS Lett. 460: 221-225. Van Doom, L. J., C. Figueiredo, F. Megraud, S. Pena, P. Midolo, D. M. Queiroz, F. Carneiro, B. Vanderborght, M. D. Pegado, R. Sanna, W. De Boer, P. M. Schneeberger, P. Correa, E. K. Ng, J. Atherton, M. J. Blaser, and W. G. Quint. 1999. Geographic distribution of vacA allelic types of Helico­ bacter pylori. Gastroenterology 116:823-830. van Doom, L. J., C. Figueiredo, R. Sanna, S. Pena, P. Midolo, E. K. Ng, J. C. Atherton, M. J. Blaser, and W. G. Quint. 1998. Expanding allelic diversity of Helicobacter pylori vacA. /. Clin. Microbiol. 36:2597-2603. van Doom, L. J., C. Figueiredo, R. Sanna, A. Plaisier, P. Schneeberger, W. de Boer, and W. Quint. 1998. Clinical rele­ vance of the cagA, vacA, and iceA status of Helicobacter py­ lori. Gastroenterology 115:58-66.

109. van Doom, L. J., Y. Henskens, N. Nouhan, A. Verschuuren, R. Vreede, P. Herbink, G. Ponjee, K. van Krimpen, R. Blankenburg, J. Scherpenisse, and W. Quint. 2000. The efficacy of laboratory diagnosis of Helicobacter pylori infections in gastric biopsy specimens is related to bacterial density and vacA, cagA, and iceA genotypes. /. Clin. Microbiol. 38: 13-17. 110. van Doom, L. J., P. M. Schneeberger, N. Nouhan, A. P. Plai­ sier, W. G. Quint, and W. A. de Boer. 2000. Importance of Helicobacter pylori cagA and vacA status for the efficacy of antibiotic treatment. Gut 46:321-326. 111. Vinion-Dubiel, A. D., M. S. McClain, D. M. Czajkowsky, H. Iwamoto, D. Ye, P. Cao, W. Schraw, G. Szabo, S. R. Blanke, Z. Shao, and T. L. Cover. 1999. A dominant negative mutant of Helicobacter pylori vacuolating toxin (VacA) inhibits VacA-induced cell vacuolation. /. Biol. Chem. 274: 37736-37742. 112. Wang, H. J., C. H. Kuo, A. A. Yeh, P. C. Chang, and W. C. Wang. 1998. Vacuolating toxin production in clinical isolates of Helicobacter pylori with different vacA genotypes. /. In­ fect. Dis. 178:207-212. 113. Warburton, V. J., S. Everett, N. P. Mapstone, A. T. Axon, P. Hawkey, and M. F. Dixon. 1998. Clinical and histological associations of cagA and vacA genotypes in Helicobacter py­ lori gastritis. /. Clin. Pathol. 51:55-61. 114. Weel, J. F., R. W. van der Hulst, Y. Gerrits, P. Roorda, M. Feller, J. Dankert, G. N. Tytgat, and A. van der Ende. 1996. The interrelationship between cytotoxin-associated gene A, vacuolating cytotoxin, and Helicobacter py/on'-related dis­ eases./. Infect. Dis. 173:1171-1175. 115. Wirth, H. P., M. H. Beins, M. Yang, K. T. Tham, and M. J. Blaser. 1998. Experimental infection of Mongolian gerbils with wild-type and mutant Helicobacter pylori strains. Infect. Immun. 66:4856-4866. 116. Yahiro, K., T. Niidome, T. Hatskeyama, H. Aoyagi, H. Kurazono, P. I. Padilla, A. Wada, and T. Hirayama. 1997. Helico­ bacter pylori vacuolating cytotoxin binds to the 140-kDa pro­ tein in human gastric cancer cell lines, AZ-521 and AGS. Biochem. Biophys. Res. Commun. 238:629-632. 117. Yahiro, K., T. Niidome, M. Kimura, T. Hatakeyama, H. Aoyagi, H. Kurazono, K. Imagawa, A. Wada, J. Moss, and T. Hirayama. 1999. Activation of Helicobacter pylori VacA toxin by alkaline or acid conditions increases its binding to a 250-kDa receptor protein-tyrosine phosphatase beta. /. Biol. Chem. 274:36693-36699. 118. Yamaoka, Y., T. Kodama, O. Gutierrez, J. G. Kim, K. Kashima, and D. Graham. 1999. Relationship between Helico­ bacter pylori iceA, cagA, and vacA status and clinical out­ come: studies in four different countries. /. Clin. Microbiol. 37:2274-2279. 119. Yamaoka, Y., T. Kodama, K. Kashima, and D. Y. Graham. 1999. Antibody against Helicobacter pylori CagA and VacA and the risk for gastric cancer./. Clin. Pathol. 52:215-218. 120. Ye, D., and S. R. Blanke. 2000. Mutational analysis of the Helicobacter pylori vacuolating toxin amino terminus: identi­ fication of amino acids essential for cellular vacuolation. In­ fect. Immun. 68:4354-4357. 121. Ye, D., D. C. Willhite, and S. R. Blanke. 1999. Identification of the minimal intracellular vacuolating domain of the Heli­ cobacter pylori vacuolating toxin. /. Biol. Chem. 274: 9277-9282. 122. Zhang, Q. B., I. M. Nakashabendi, M. S. Mokhashi, J. B. Dawodu, C. G. Gemmell, and R. I. Russell. 1996. Association of cytotoxin production and neutrophil activation by strains of Helicobacter pylori isolated from patients with peptic ul­ ceration and chronic gastritis. Gut 38:841-845.

III. ENERGY METABOLISM AND SYNTHETIC PATHWAYS

Helicobacter pylori: Physiology and Genetics Edited by H . L . T. Mobley, G. L . Mendz, and S. L. Hazell © 2 0 0 1 ASM Press, Washington, D.C.

Chapter 10

Microaerobic Physiology: Aerobic Respiration, Anaerobic Respiration, and Carbon Dioxide Metabolism DAVID J . KELLY, NICKY J . HUGHES, AND ROBERT K . POOLE

MODES OF E N E R G Y TRANSDUCTION

lism of H. pylori appears to be primarily that of an aerobic, respiring bacterium with the potential for sig­ nificant ATP yields via oxidative phosphorylation. However, H. pylori is likely to encounter signifi­ cant acidity in the stomach and after colonization, despite urease secretion and N H generation. The components of the proton motive force have been measured in H. pylori ATCC(R) 4 3 5 0 4 (36) as well as other acidophiles; in all cases, the organism main­ tains an intracellular pH (pH;) much closer to neutral­ ity than is the outside milieu (pH ) under acidic envi­ ronmental conditions (e.g., pH, 6.2 at pH 3.0). Thus the ApH component of Ap is large. An unusual feature of these bacteria is that the smaller membrane poten­ tial component (Ai|i) is positive inside, which atten­ uates or partly counterbalances the force against which protons must be extruded for pH homeostasis. The result is a relatively constant proton motive force at external pH values between 3 and 7 (36). However, another study (39) found that, in the absence of urea, the membrane potential collapsed to zero below pH 4. This aspect of H. pylori bioenergetics needs further investigation. The proton motive force drives not only ATP synthesis but also flagellar rotation and solute uptake, which are outside the scope of this chapter. The prime generator of Ap is the respiratory chain(s), whose components are asymmetrically ar­ ranged across the cytoplasmic membrane. Such a dis­ position allows electron transfer events to consume protons from the cytoplasmic compartment (e.g., in oxygen reduction) and release protons into the extracytoplasmic compartment (the periplasm in gramnegative bacteria), e.g., by quinol oxidation. Alterna­ tively, or in addition, protons (or other ions, such as N a ) not directly involved in the redox chemistry may be actively pumped by the respiratory complexes. Re-

Respiration is a series of coupled oxidation and reduc­ tion reactions that result in the transfer of electrons from an appropriate electron donor (such as a reduced coenzyme) to an appropriate acceptor of electrons. In aerobic respiration, electron transfer is to oxygen (more correctly, the dioxygen molecule), which is re­ duced to water with concomitant, coupled ion trans­ location and generation of an electrochemical gra­ dient. In anaerobic respiration, electron transfer is to a molecule other than oxygen or to an ionic species, again coupled to generation of an electrochemical gra­ dient. Whatever the electron acceptor, respiration provides the ability to conserve energy in the form of adenosine triphosphate (ATP) or perform energydemanding processes (such as solute transport or mo­ tility) through generation of a transmembrane proton motive force (Ap) (see reference 44). In terms of en­ ergy conservation, respiration-coupled oxidative phosphorylation via the proton motive force is sub­ stantially more efficient than fermentation, allowing faster growth and attainment of higher yields of biomass per mole of energy substrate used. Conversion of the proton electrochemical gra­ dient across the bacterial cytoplasmic membrane into ATP is accomplished by the ATP synthase. Genes en­ coding components of both the membrane-embedded F proton-channeling complex (a, b, c subunits) and the catalytic, peripheral "headpiece," F ! (a, 6, y, 8, e) have all been identified in Helicobacter pylori strains 26695 and J 9 9 (3, 61). An additional gene is present (HP1137/JHP1065) that is predicted to encode a subunit homologous to the ATPase b', a diverged and duplicated form of the b subunit found in plants and photosynthetic bacteria. Thus, the energetic metabo­

3

D

D

0

+

David J. Kelly and Robert K. Poole • Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, United Kingdom. Nicky J. Hughes • SmithKline Beecham Pharmaceuticals Research and Development Ltd., Anti-infectives Research, 1250 South Collegeville Road, Collegeville, PA 19426.

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KELLY ETAL.

spiratory chains comprise complex, branched ar­ rangements of components, which together result in oxidation of a wide variety of substrates (typically NADH, succinate, malate, lactate, hydrogen). Elec­ tron transfer between the component parts (dehydro­ genases, quinones, cytochromes, and one or more ter­ minal oxidases) is made possible by the presence of hemes, flavins, iron-sulfur ([Fe-S]) clusters, and metal ions such as copper, all of which can exist in oxidized or reduced forms. The most important characteristics of bacterial respiratory chains are (i) their branched nature at both "dehydrogenase" and "reductase" ends, (ii) the use of oxygen or alternative electron ac­ ceptors, (iii) the presence of numerous types of cyto­ chromes and quinones, (iv) the "cross-talk" between pathways optimizing the possibility of each reductant being paired with a wide choice of oxidants, and (v)

the concomitant proton translocation and energy transduction. Simple, linear pathways involving a small num­ ber of dehydrogenases, a quinone, and a single termi­ nal oxidase or reductase are uncommon in bacteria. Nevertheless, biochemical and genome analyses (17, 32) suggest a surprisingly simple organization (Fig. 1) for the respiratory apparatus of H. pylori, in which dehydrogenases (unusually, including NADPHlinked enzymes; see below) pass reducing equivalents to menaquinone only, then through a cytochrome bc\ complex and a soluble cytochrome c to a single oxi­ dase. This conclusion appears contrary to some spec­ troscopic information on the cytochrome comple­ ment; reconciling and integrating results from several approaches will be an important target for future work.

OUTER MEMBRANE

PERIPLASM

INNER MEMBRANE

CYTOPLASM

succinate

fumarate + 2H*

Figure 1. Hypothetical arrangement of the major components of the respiratory chains of H. pylori. Reduced substrates (DH) are oxidized (to D) via membrane-bound or membrane-associated dehydrogenases. Integral membrane oxidoreductases include an NDH-1 complex (Nuo), the electron donor to which is unknown, and hydrogenase (Hya, also termed Hyd). Peripherally associated oxidoreductases include (among several others) malaterquinone oxidoreductase (Mqo). Reducing equivalents from all these substrates reduce the sole quinone, menaquinone-6, in the lipid bilayer of the inner membrane. Menaquinol reduces the trimeric cytochrome bci complex (Pet), which in turn reduces periplasmic cytochrome C553 (c). Cytochrome c is reoxidized by the sole terminal oxidase of the Ceo (or Fix) type, cytochrome ebb' (or cbb ). Cytochrome c may also be reoxidized by hydrogen peroxide in the periplasm through the activity of cytochrome c peroxidase (CCP). Fumarate reductase (Frd) catalyzes electron transfer from menaquinol to fumarate as terminal acceptor. The reactions catalyzed by CcO, CCP, FrdA, Mqo, and Nuo show substrate/product conversions. The other arrows indicate directions of electron transfer. 3

CHAPTER 10 • MICROAEROBIC PHYSIOLOGY

This chapter is concerned primarily with substrate ox­ idations and the use of oxygen as electron acceptor. We also examine the evidence for facultative respira­ tion in H. pylori and consider the extent to which the respiratory pathways of this bacterium can be gleaned from the somewhat limited biochemical evidence presently available, and from comparisons with mito­ chondrial and bacterial paradigms.

AEROBIC E L E C T R O N T R A N S P O R T IN H. PYLORI Substrate Oxidation and Primary Dehydrogenases Initial studies of respiration in H. pylori showed that intact cells were capable of oxidizing D-glucose, formate, DL-lactate, succinate, and pyruvate (4). Al­ though the rates of respiration were low for D-glucose and formate, DL-lactate, succinate, and pyruvate were much more readily oxidized. A more comprehensive study of the kinetics of substrate oxidation (12) showed that cells from unstirred broth cultures oxi­ dized ethanol, fumarate, glucose, D-lactate, pyruvate, and succinate. Low concentrations (25 u,M) of pyru­ vate, D-lactate, and succinate were rapidly oxidized and the respiration rates were relatively high. A much lower affinity for ethanol and fumarate was reported, which suggested that these substrates may not be oxi­ dized at significant rates in vivo. From this study, it was calculated that the total oxygen taken up during lactate and pyruvate oxidation was insufficient for complete oxidation to C 0 and H 0 via the citric acid cycle. Chang et al. (12) were unable to detect oxygen uptake when acetate, glycerol, L-lactate, oxaloacetate, 2-oxobutyrate, and several amino acids in­ cluding aspartate and glutamate were added to H. pylori cells. From the genome sequences of strains 26695 and J 9 9 , it is possible to predict the likely identity of some of the primary dehydrogenases responsible for feeding electrons from some of these substrates into the quinone pool in H. pylori. A putative flavoprotein Dlactate dehydrogenase (Did; HP1222/JHP1143) would account for lactate-dependent respiration, and a glycerol-3-phosphate dehydrogenase (GlpC; HP0666/JHP611) is also present. Homologs of pro­ line dehydrogenase (PutA; HP0056/JHP48), glycolate oxidase (GlcD; HP0509/JHP459), and a D-aminoacid dehydrogenase (DadA; HP0943/JHP8878) pro­ vide additional possibilities for substrate-derived elec­ trons to be donated to the membrane-bound electron transport chain, but this has not been tested experi­ mentally. There is experimental evidence, however, for the presence in H. pylori of a malate:quinone oxi2

2

115

doreductase (Mqo; HP0086/JHP79), which would allow the use of L-malate as an electron donor (31). H. pylori is known to be capable of hydrogen oxidation (34), and possesses a membrane-bound hydrogenase activity which can couple with a variety of artificial and physiological electron acceptors with a positive redox potential (34). From immunoblotting experiments with antisera raised against the Bradyrhizobium japonicum uptake hydrogenase, Maier et al. (34) identified H. pylori hydrogenase polypeptides of about 65 and 26 kDa. These correspond to the prod­ ucts of the hydB (HP0632/JHP575) and hydA (HP0631/JHP574) genes, respectively, which are part of a hydABCD operon also encoding a cytochrome subunit (HydC) and a maturation protein (HydD). Gilbert et al. (22) cloned an hypB homolog, which in B. japonicum encodes a nickel-binding protein in­ volved in hydrogenase biosynthesis. The hypB gene (HP0900/JHP8837) is apparently cotranscribed with two further biosynthesis/assembly proteins encoded by the hypDC genes. Additional proteins needed for hydrogenase assembly are likely to be encoded by the hypA (HP0869/JHP803) gene and hypEF (HP0047, HP0048/JHP40, JHP41) homologs. Taken together, the available data indicate that H. pylori possesses a NiFe type of H -uptake hydro­ genase, which could act as an electron donor to the respiratory chain and thus contribute to energy con­ servation if molecular hydrogen is present. 2

The Role of NAD(P)H in H. pylori Respiration and the Function of Complex I Conventional wisdom holds that the function of NADH in bioenergetics is to act as the major electron donor for oxidative phosphorylation via interaction with a proton-translocating quinone oxidoreductase (complex I or NDH-1), while the role of NADPH is as a source of electrons for biosynthetic reactions. Sev­ eral studies have shown that membrane preparations of H. pylori exhibit low or insignificant rates of NADH oxidation, but that the rates with NADPH as electron donor are much higher (12, 13, 2 9 ) . This unusual situation strongly suggests that NADPH is the physiological electron donor to the respiratory chain in H. pylori, rather than NADH as in the major­ ity of bacteria. The genome sequence of H. pylori shows that it contains a cluster of genes encoding a potential NADH-quinone oxidoreductase of the NDH-1 type. However, an examination of the de­ duced proteins encoded by this gene cluster led Finel (19) to conclude that the complex may not actually oxidize NADH, because of the lack of the NuoF and

116

KELLY ET AL.

NuoE subunits, which in other bacteria are known to contain the binding site for NADH. Also missing is a flavin mononucleotide (FMN) prosthetic group and an FeS cluster, involved in electron transfer to the NuoG subunit. Two nonhomologous open reading frames present in place of these genes (HP1264/1265) encode proteins of unknown function. Significantly, these proteins do not contain an obvious NADPHbinding motif. A distinct possibility is that electrons from NADPH are transferred to H. pylori complex I via an intermediate that interacts with the NuoF and NuoE replacements (19). Alternatively, coupling of NADPH with the respiratory chain may not occur via the NDH-1 homolog at all, but through an alternative quinone reductase, which may not be proton translo­ cating. It should be noted that there is no obvious homolog of an NDH-2 type protein that may fulfill this role. Further evidence that the unusual NDH-1 homolog in H. pylori is not a conventional NAD(P)H quinone oxidoreductase has come from the observa­ tion that NADH oxidation is insensitive to the classi­ cal complex I inhibitor, rotenone (13). Finel (19) sug­ gested that HP1264/1265 could act as a docking site for a protein that delivers electrons directly to the FeS cluster of the NuoG homolog. Although the identity of such a protein is unknown, one possibility is flavodoxin or ferredoxin, reduced by the activities of the pyruvate and 2-oxoglutarate oxidoreductases, which, in H. pylori, are present in place of the usual NADHproducing multi-enzyme dehydrogenase complexes found in conventional aerobes (28, 29) (see chapter 12). Interestingly, the genome sequence of the closely related microaerophile Campylobacter jejuni indi­ cates that it has a similar type of complex I to that found in H. pylori, with the NuoF and two subunits replaced by orthologs of HP1265 and 1264 (49). Whether this type of enzyme is proton-translocating is an important question for understanding the bioenergetics of these bacteria. If NADH is not a respiratory electron donor in H. pylori, what is its role? There may not be a sharp distinction in the catabolic and anabolic functions of the pyridine nucleotides in H. pylori, and it is interest­ ing that there is no evidence for a transhydrogenase in this bacterium. The NADH oxidases/dehydrogenases that account for the observed oxidase activities in cellfree extracts must be due to cytoplasmic enzymes. One possible function of these enzymes could be oxy­ gen scavenging to keep the cytoplasmic oxygen con­ centration low enough for oxygen-sensitive enzymes like POR and O O R to operate. This type of role was originally suggested by the decreased NADH oxidase activities found in metronidazole-resistant mutants of H. pylori (54). However, it would be surprising if such soluble oxidases could match the very high oxygen

affinities and turnover rates that are characteristic of membrane-bound terminal oxidases (52). An alterna­ tive function for the soluble oxidases might be to transfer electrons directly to quinones, whose reduced status is probably important for managing oxidative stress (55, 5 6 ) . The Quinone Pool and the Cytochrome bc Complex

x

Quinone composition of Helicobacter species Bacterial respiratory quinones are small lipid-soluble hydrogen (i.e., electron and proton) carriers that mediate electron transfer between dehydrogenase and reductase or oxidase components of respiratory chains. Bacteria contain two main types of such qui­ nones: benzoquinones, such as ubiquinone (UQ), and naphthoquinones, such as menaquinone (MK) and demethylmenaquinone (54). Within each class, qui­ nones differ in the length of the isoprenoid side chain, but 6 to 10 isoprene units (as in U Q ) are most com­ mon in bacteria. Some bacteria such as Escherichia coli have both UQ and M K ; the latter is predominant under anaerobic conditions while UQ is present under aerobic conditions (54). Quinone reduction to quinol results from substrate oxidation by respiratory dehy­ drogenases, and quinol reoxidation to quinone is cou­ pled to reduction of downstream oxidase compo­ nents. Thus quinones-quinols act as shuttles of reducing power between "upstream" (low redox po­ tential) and "downstream" (high potential) redox proteins. Although quinones are often considered to be highly mobile in the lipid bilayer of the membrane, several protein components of the respiratory chain have specific quinone-docking or -binding sites. Early work with H. pylori identified M K as the major quinone (14, 4 1 ) . Electron impact mass spec­ trometry of lipid extracts of H. pylori NCTC 1 1 6 3 7 demonstrated that MK-6 is the dominant form with traces (about 1 0 % ) of MK-4 (35). Other strains of H. pylori and other species gave similar results (35). No UQ was detected. H. pylori lacks the novel methyl-substituted MK-6 found in several species of Campylobacter (9, 41). Marcelli et al. (35) also dem­ onstrated that there is no alteration in quinone type during growth at a range of oxygen concentrations from 2 to 1 5 % (v/v), but M K content was highest at 5 to 1 0 % O 2 , the optimum for growth. 8

Quinone biosynthesis Despite clear biochemical evidence for MK, genes for the biosynthesis of M K have not been identified with the exception of a ubiE homolog (HP1483/

CHAPTER 10 • MICROAEROBIC PHYSIOLOGY

JHP1376), predicted to encode a methyltransferase that, in E. coli, is required for the synthesis of both UQ and MK. It is conceivable that H. pylori obtains menaquinones or their precursors in vivo from its host, but it seems more likely that the genes for bio­ synthesis have not been identified by sequence inspec­ tion. Paradoxically, however, genes for the biosyn­ thesis of UQ were annotated by Tomb et al. (61). Thus, H. pylori is predicted to have a ubiA gene (HP1360/JHP1278) encoding a protein that is 2 2 % identical to E. coli UbiA (4-hydroxybenzoate polyprenyltransferase), which catalyzes the second commit­ ted step of UQ biosynthesis (30, 55) but is not re­ quired for M K synthesis. Apparent UbiA homologs have been identified in other microorganisms (archaea, gram-positive bacteria) that also lack ubiqui­ nones and/or respiratory pathways (55). A ubiD gene (HP1476/JHP1369), encoding a putative decarboxyl­ ase that forms 2-polyprenylphenol, and a ubiE gene have also been identified (17, 61). No clear homologs for other ring-modifying enzymes have been found. Doig et al. (17) point out that UbiB, UbiF, and UbiH are monooxygenases and suggest that their absence from H. pylori might reflect its microaerobic lifestyle. The polyisoprene diphosphate "tail" of UQ is synthesized in E. coli and probably most other bacte­ ria by the "nonmevalonate" pathway of isoprenoid biosynthesis (30). Two genes, ispA (HP0929/JHP864) and ispB (HP0240/JHP225), are predicted to encode enzymes involved in tail biosynthesis. The gene ispA is required for the first step in isoprene polymerization. Interestingly, ispB is an essential gene in E. coli for aerobic growth, even on fermentable substrates. This strict requirement may come from ispB involvement in the biosynthesis of both UQ and M K biosynthesis. More work is needed to establish the function(s) of these proteins in H. pylori and to locate other genes involved in quinone synthesis. The cytochrome bc\ complex Quinols act as a reservoir of reducing power in the cytoplasmic membrane, and in principle, can be used as a source of electrons by various redox protein complexes, the most important of which, in the con­ text of aerobic respiration in H. pylori, is the cyto­ chrome bc\ complex. This heterotrimeric protein complex, located within the cytoplasmic membrane, contains redox centers involved in the transfer of re­ ducing equivalents from a two-electron donor (quinol) to a one-electron acceptor (ultimately, cyto­ chrome c) and is a major contributor to the generation of a proton motive force across the cytoplasmic mem­ brane. Genes that encode the conserved subunits of the cytochrome bc complex have been identified in x

117

the genome of H. pylori (3, 6 1 ) . Cytochromes b and c\ are present as separate polypeptide subunits, en­ coded by petB (HP1538/JHP1461) and petC (HP1539/JHP1460, respectively, along with an Fe-S protein (the Rieske protein) containing a 2Fe-2S clus­ ter, encoded by petA (HP1540/JHP1459). The cyto­ chrome b subunit binds two heme B moieties. The transfer of electrons through the cytochrome bc\ com­ plex to cytochrome c involves a proton motive Qcycle: for every two electrons that pass through the protein, four protons are extruded to the periplasm. The overall reaction performed by the cytochrome bc\ complex is given in equation 1: QH

2

+ 2H

+ C

+ 2c

o x

-

Q + 4Hp + 2t~c +

rei

(1)

Subscripts c and p represent protons on the cyto­ plasmic and periplasmic side of the membrane and c and c a refer to oxidized and reduced cytochrome c, respectively. Evidence for the operation of the cyto­ chrome bc\ complex in H. pylori has come from the inhibition of lactate respiration in intact cells by the specific cytochrome bc\ complex inhibitors antimycin A and myxothiazol (1) and by the inhibition of succi­ nate-cytochrome c reductase activity in sonicates (13). o x

re

Cytochromes c in H. pylori: Biosynthesis and Role in Electron Transport Cytochromes c are proteins that contain covalently bound heme and function in a range of redox reactions in bacteria. Cytochromes c are often either soluble, periplasmic, or membrane-anchored proteins with a heme-containing region exposed to the per­ iplasm. Biogenesis of c-type cytochromes is complex; the biosynthesis of heme, export of the apo-protein to the periplasm, and covalent linkage of the heme moiety to the apo-protein are all essential steps in this process (60). The products of at least 10 hem genes are required for the synthesis of heme B (protoheme IX) from glycine plus succinyl CoA or glutamate-1semialdehyde (60). Homologs of these ten hem genes are present in the H. pylori genome sequence, con­ firming that H. pylori has the biosynthetic capacity to manufacture protoheme I X (3, 61). Although ho­ mologs of the genes required to generate heme C from protoheme I X are present in H. pylori, the homologs of the genes required for the biosynthesis of hemes O, A, and D are absent, as predicted by the absence of spectral features corresponding to these cytochromes (34, 41). An important step in the heme biosynthetic process involves the oxidation of coproporphyrinogen III to protoporphyrinogen I X . This step can be catalyzed by an oxygen-utilizing enzyme (HemF) or an oxygen-independent enzyme (HemN). In many facultative anaerobes HemF is used during aerobic

118

KELLY ET AL.

growth and hemN is induced for heme biosynthesis under anaerobic growth conditions (60). In H. pylori there is no hemF gene, but two copies of a hemN homolog are present (61). It has been shown that heme insertion into the apo-cytochrome c occurs in the periplasm after Secdependent export of the apo-protein (6). In members of the a, B, and y proteobacteria and plant mitochon­ dria, a complex system appears to be responsible for cytochrome c assembly with at least 12 gene products involved (23, 60). Searching the H. pylori genome re­ vealed that the proteins of this system were absent (48). However, a simpler system with four compo­ nents has been found in some gram-positive organ­ isms, cyanobacteria, and chloroplasts. Homologs of the genes encoding these components are present in H. pylori (23); if functional, these proteins would rep­ resent the first example of such a system in a gramnegative bacterium. Sonicates of H. pylori were shown to have ascorbic acid-oxidizing activity (46), which was pro­ posed to be responsible for the destruction of gastric vitamin C seen in H. pylori-'miected patients. A watersoluble component of the H. pylori sonicate was shown to be responsible for the ascorbate oxidizing activity, and this was tentatively assigned to a low molecular mass ( < 1 4 kDa) cytochrome c (46). Spec­ troscopic studies on H. pylori (34, 3 5 , 42) confirmed the presence of c-type cytochromes in the organism. The purification and N-terminal sequence of a per­ iplasmic, soluble c-type cytochrome designated CycA has been reported (18) and characterized spectroscopically as cytochrome c . Recently, cytochrome C553 has been identified as a potential electron donor to the cb-type cytochrome c oxidase (62). The genome sequences of H. pylori 26695 and J 9 9 contain only one annotated c-type cytochrome gene (eyeA; HP1227/JHP1148), which encodes the protein identi­ fied in these biochemical studies. Interestingly, the cycA gene is transcribed divergently from a homolog of the hemN gene, an important factor in the anaero­ bic biosynthesis of heme. 5 5 3

Searching the whole genome sequence for genes that encode proteins containing the C X X C H motif has identified a second, putative low molecular mass cytochrome c (2). The gene encoding this putative cy­ tochrome (HP0236, here designated cycB) is found as the distal gene of an eight-gene operon containing several genes of apparently unrelated function. Signif­ icantly, however, in addition to the putative cyto­ chrome gene, there are two genes within this operon that are involved in heme biosynthesis, hemA and hemC. Both CycA and CycB contain a single CxxCH motif, indicating that they are monoheme c type cyto­

chromes. The role of CycB in electron transport is unknown, but mutagenesis studies have indicated that both cycA and cycB are essential genes that cannot functionally substitute for each other (2). Terminal Oxidase of H. pylori Functions of terminal oxidases For the complete reduction of oxygen (which re­ quires four electrons), the standard redox potential (E°) of the couple is + 820 mV. Thus, use of oxygen as an electron acceptor is more likely to result in higher ATP yields by oxidative phosphorylation than is, for example, use of fumarate/succinate ( E ' + 33 mV). However, the use of oxygen in aerobic respira­ tion is not without its difficulties. The kinetic inertness of O 2 requires activation by a metal center, which, in aerobic respiratory chains, generally comprises two transition metals, either a heme-heme couple or a heme-copper couple. Oxygen is only moderately solu­ ble; it is generally assumed that dioxygen, being a small uncharged molecule, will diffuse readily across biological membranes and that no significant oxygen concentration gradient exists across respiring bacte­ rial membranes. Even so, oxygen-reducing oxidases have high affinities for the ligand, with K values typi­ cally in the submicromolar range. Only the four-electron reduction of oxygen to water is "safe," since intermediate reduction products are toxic and reactive (see below). In bacteria there are two major groups of respiratory oxidases that re­ duce 0 to water—those that accept electrons from the quinone pool directly (quinol oxidases), and those that accept electrons from cytochrome c (cytochrome c oxidases). The vast majority of the latter group are closely related and fall within the heme-copper oxi­ dase superfamily. Most bacteria possess at least two terminal oxidases, often a quinol oxidase and a cyto­ chrome c oxidase (52), with different catalytic proper­ ties and under tight transcriptional control. H. pylori appears to be unusual among bacteria in that clear evidence for the presence of a quinol oxidase is lacking (3, 3 5 , 61). No spectral signals due to cytochrome d could be detected by Marcelli et al. (35) in reduced minus oxidized difference spectra, although an un­ identified band in CO difference spectra indicates the presence of a hemoprotein that forms a CO adduct at 628 nm. In contrast, Maier et al. (34) showed reduced minus oxidized spectra of strain Leu, a clinical isolate, with clear peaks at 5 9 5 and 6 2 7 nm, indicative of a cytochrome M-type oxidase. 0

m

2

The cytochrome cfo-type cytochrome c oxidase Within the gram-negative bacteria, two major types of cytochrome c oxidase have been identified

CHAPTER 10 • MICROAEROBIC PHYSIOLOGY

that differ greatly in their subunit structure and the redox centers present. The aa -type cytochrome c oxi­ dase is found in a wide range of bacteria and is very closely related to the mitochondrial oxidase. Spectro­ scopic studies (35, 42) have clearly shown that H. pylori is devoid of this type of oxidase. The genome sequences of H. pylori strains 26695 and J 9 9 suggest that a cb-type cytochrome c oxidase is the sole termi­ nal oxidase present in H. pylori. It is encoded by the ccoNOQP operon, and the subunit structure is essen­ tially the same as in all other organisms from which this enzyme has been identified, except that the CcoN subunit from H. pylori is truncated at the N terminus when compared to CcoN subunits from other bacte­ ria. The amino acid sequences of the ccoO and ccoP genes reveal conserved motifs for the binding of heme C (CxxCH). CcoO is capable of binding a single heme C, and CcoP is capable of binding two hemes C. In­ deed, Nagata et al. (42) showed that H. pylori con­ tains a cb-type oxidase. This type of oxidase was origi­ nally identified in the symbiotic, nitrogen-fixing bacteria B. japonicum and Rhizobium meliloti (20) and is characterized by a very high affinity for oxygen (K in the nM range). The H. pylori oxidase has been reported to have a K for oxygen of 0.4 uM (41) or 0.04 (JLM (62), but the values were determined using a relatively insensitive membrane-covered 0 electrode, which probably underestimates the true affinity. The oxidase has been purified (62) and contains three hemes C and two protohemes (one high-spin, one low-spin) as predicted. However, surprisingly, CcoN and CcOO appear to form a protein complex even in the presence of sodium dodecyl sulfate. There is evidence that the enzyme can pump protons, although the H pumping activity by reconstituted proteoliposomes was low (62). 3

m

m

2

+

Directly downstream of the fix/ccoNOQP op­ eron, a second operon, the fix/ccoGHIS operon, has been found in all organisms studied previously. A role for the FixGHIS proteins in the uptake and metabo­ lism of copper required for the assembly of the cb-type cytochrome c oxidase has been proposed. However, a homologous operon is not found downstream of the ccoNOQP operon in H. pylori. Although homologs of fixGI and S have been identified in the genome sequence of H. pylori 26695 (61) and J 9 9 (3), these genes are not linked as they are in all other organisms and no homolog of fixH can be identified. Another unusual feature in H. pylori is that genes encoding transcriptional regulators that have been identified upstream of the fix/ccoNOQP operon in all other bacteria studied are absent. In organisms that fix nitrogen, two types of regulators have been found, an FNR homolog and a two-component sensor-regu­

119

lator system, FixLJ (20). In organisms that do not fix nitrogen, only an FNR homolog is found. Both the FNR homologue and FixLJ are involved in regulating gene expression in response to alterations in the level of oxygen. From the genome sequences available (3, 61) it is apparent that FixLJ and FNR homologs are completely absent in H. pylori. This may be due to the fact that the cb-type cytochrome c oxidase is ex­ pressed constitutively as it is the sole terminal oxidase in this bacterium.

Cytochrome c Peroxidase Hydrogen peroxide can be degraded to H 0 and 0 by the cytoplasmic enzyme, catalase, in a disproportionation reaction. A second, periplasmic, enzyme can also perform this function via a reduction reaction with the requirement for reduced cytochrome c as an electron donor (equation 2 ) . 2

2

H 0 2

+ 2e c d + 2H — 2 H 0 + 2c _

2

+

r e

2

o x

(2)

The enzyme responsible for this activity, cyto­ chrome c peroxidase (CCP), has been isolated from a number of bacteria and is a diheme cytochrome c. A low potential heme forms the peroxidatic site and a high spin heme donates the second electron in the re­ duction process (40). The electrons for this reaction are usually donated from a low molecular mass per­ iplasmic cytochrome c that has been reduced by the cytochrome bc\ complex. It is often the same cyto­ chrome c that is the electron donor to the cytochrome c oxidase (24, 62). If the cytochrome c electron donor to the CCP is reduced by the bc\ complex, then reduc­ tion of H 0 by CCP contributes to electron flow through the electron transport chain and thus to pro­ ton translocation. In the related microaerophile Campylobacter mucosalis, the oxidation of formate leads to generation of periplasmic H 0 , which is re­ duced by CCP (25), using electrons from cytochrome C553 that has been reduced by the bc\ complex. Thus, removal of H 0 from the periplasm leads to proton extrusion (25). Although H. pylori does not appear to possess formate-oxidizing enzymes (10, 6 1 ) , it is reasonable to assume that H 0 will be generated in the periplasmic space (58). Although H. pylori pro­ duces an abundant, highly active catalase, it is essen­ tially a cytoplasmic protein. H. pylori catalase has been found on the cell surface (26), but as no signal sequence for export is present in the catalase amino acid sequence (45), it is likely that this results from catalase freed from lysed cells binding to the outside of intact cells ("altruistic lysis"). 2

2

2

2

2

2

2

2

120

KELLY ET AL.

ANAEROBIC E L E C T R O N TRANSPORT IN H. PYLORI Fumarate Respiration The reduction of fumarate catalyzed by the en­ zyme fumarate reductase can be an important energy transduction pathway in anaerobic bacteria (33). In facultative anaerobes, the enzyme is specifically in­ duced by low oxygen tensions and in E. coli this is mediated by the global regulator Fnr. Mendz and Ha­ zell (37) originally identified a variety of products of fumarate metabolism in H. pylori cells or lysates using nuclear magnetic resonance (NMR) techniques. Under their experimental conditions, the primary product of fumarate metabolism was malate, and this was subsequently converted to pyruvate. Upon fur­ ther incubation, the final products were identified as succinate, acetate, lactate, alanine, and formate. The production of succinate indicated a fumarate re­ ductase activity, providing initial evidence that H. py­ lori may generate ATP via anaerobic respiration. Using C - N M R and shorter incubation times, Chalk et al. (11) showed that the major end product of fu­ marate metabolism under anaerobic incubation con­ ditions was succinate, again indicating an active fu­ marate reductase, and they also noted the transient accumulation of malate. Fumarate is transported into H. pylori cells by a C4-dicarboxylate carrier(s), most likely in antiport with succinate (38) in a similar man­ ner to the Dcu systems of E. coli, homologs of which are present in the H. pylori 26695 and J 9 9 genome sequences. 1 3

The fumarate reductase enzyme from H. pylori is discussed in more detail by Kelly and Hughes in chapter 12 of this book, but the disposition of the FrdABC proteins in the respiratory chain is depicted in Fig. 1. From the viewpoint of electron transport, the function of fumarate respiration in H. pylori is not entirely clear as there is no evidence (as yet) for anaerobic growth supported by fumarate reduction. An frdA mutant exhibited a greatly extended lag phase compared to the wild-type, suggesting an im­ portant physiological role under the usual microaero­ bic culture conditions (21). Fumarate reductase could contribute to energy conservation in H. pylori and may also have alternative functions such as being part of a mechanism for redox balancing (see chapter 12).

does not possess genes for a nitrate reductase. How­ ever, there is a gene (HP0407/JHP974) encoding a potential N- or S-oxide oxidoreductase (17), which, although annotated as a biotin sulfoxide reductase by Tomb et al. (61), is similar to a number of molybdopterin-containing enzymes that function as periplasmic reductases for alternative oxidants such as dimethylsulfoxide or trimethylamine N-oxide.

MICROAEROPHILY VERSUS OXIDATIVE STRESS All aerobic organisms face the potential threat of damage from oxygen per se or one of the products of partial oxygen reduction. Transfer of a single electron to oxygen generates the superoxide radical anion ( 0 ~ ) , a highly reactive species that attacks many key biomolecules. In vivo, superoxide is scavenged by su­ peroxide dismutases that convert (dismutate) two molecules of superoxide to peroxide and water (see below). Transfer of a second electron to oxygen or a single electron to superoxide gives peroxide ( 0 ) , another reactive species that is scavenged in vivo by catalases and hydroperoxidases (a collective term for peroxide-consuming catalases and peroxidases). The high redox potential for H 0 reduction to water ( E ' + 1.349 V) is put to use by peroxidases, e.g., cyto­ chrome c peroxidase as discussed above. One of the most distinctive features of H. pylori is its microaerophilic lifestyle. The organism is generally grown in the laboratory at oxygen concentrations around 6 % , and it is tacitly assumed that this also approximates oxygen tensions in the gastric mucosa. The requirement for microaerobic conditions is gener­ ally met in the laboratory by incubation in jars incor­ porating a gas-generating system like the CampyGen (Oxoid) or CampyPack (Becton Dickinson) systems, also used for growth of Campylobacter species. The requirement for control of oxygen tension frustrates growth of significant cell quantities for biochemical studies. In fermenters, appropriate oxygen tensions may be measured and controlled automatically throughout growth. However, surface gassing can be done with 6 % oxygen, then with air, and then with 9 5 % 0 (16) to achieve yields equivalent to optical density at 6 0 0 nm of 2.5 in 2 4 h. The principle, which can also be exploited in shake flasks, is that high cell densities maintain actual dissolved 0 levels low enough to accommodate growth of the microaerophile. The molecular and physiological bases of microaerophily are very poorly understood. H. pylori has an obligate requirement for 0 , yet the bacterium pos­ sesses several essential, highly oxygen-labile meta2

2 _

2

0

2

2

2

2

Evidence for the Use of Other Anaerobic Electron Acceptors Apart from fumarate, there is no direct experi­ mental evidence that H. pylori is able to use alterna­ tive electron acceptors other than oxygen. H. pylori

2

CHAPTER 10 • MICROAEROBIC PHYSIOLOGY

bolic enzymes typical of anaerobic-type metabolism. Furthermore, in vivo H. pylori must be exposed to reactive products of partial oxygen reduction as part of the mucosal inflammatory response. This implies that H. pylori must possess some form of protection against oxidative stress. Bacterial defenses against ox­ idative stress can be broadly divided into enzymes cat­ alyzing the destruction of oxidants, proteins that function to limit the formation of oxidants, and pro­ teins acting to repair DNA damage caused by oxi­ dants (the last, exemplified by the SOS response, is outside the scope of this chapter). Oxidative stress responses are coordinated by a number of global regu­ latory proteins. Oxidative stress may be exacerbated by high levels of intracellular iron, and the regulation of iron uptake by Fur and the protective sequestration of iron by ferritin in H. pylori are described elsewhere in this book (chapter 17). Both strains of H. pylori for which there is a ge­ nome sequence have both superoxide dismutase and catalase, in agreement with earlier biochemical studies (for references, see reference 17). There are also two peroxidases that presumably serve to detoxify perox­ ide in a catalase-deficient mutant (45). Thus, failure to detoxify reactive oxygen species does not appear to be a plausible reason for microaerophily. Many other possible explanations for microaero­ phily exist. First, oxygen-consuming oxidases or globins might be absent, leading to 0 accumulation, but it is clear that H. pylori has an active, albeit apparently simple, respiratory chain that terminates in an oxidase with a moderately high affinity for 0 (42). It is prob­ able in fact that the affinity, measured thus far with a polarographic electrode, is underestimated. Second, at high 0 tensions, oxidase activity might be inhib­ ited. There is some evidence that the high affinity quinol oxidase in E. coli, cytochrome bd, is subject to such modulation (15). A respiratory chain termi­ nated by a single oxidase, as in H. pylori, might be shut down by such a mechanism. A third explanation may be extreme 0 sensitivity of essential cytoplasmic proteins or enzymes. H. pylori has in fact been shown to possess such enzymes, for example, the oxygenlabile 2-oxoacid oxidoreductases, which appear to be essential enzymes catalyzing crucial steps in central metabolism (28, 2 9 ) . These are discussed in more de­ tail in chapter 12. In H. pylori, alkylhydroperoxide reductase, cata­ lase, and superoxide dismutase have been particularly implicated in the enzymatic destruction of toxic oxi­ dants and are discussed below. 2

2

2

2

Alkylhydroperoxide Reductase Alkylhydroperoxide reductase (Ahp) is involved in the detoxification of reactive hydroperoxides to

121

their corresponding alcohols. In E. coli and Salmo­ nella enterica serovar Typhimurium, Ahp is composed of two subunits encoded by ahpC and ahpF (51, 59). The AhpC subunit, which catalyzes the reduction of peroxides, was first identified in H. pylori as a major 26-kDa antigen (HP1563/JHP1471) (47). Although direct experimental evidence for the importance of AhpC in resistance to oxidative stress in H. pylori is lacking, an ahpC mutation in the closely related C. jejuni results in increased sensitivity to oxidative stress induced by cumene hydroperoxide and by exposure to atmospheric oxygen (5). C. jejuni AhpC is regu­ lated by iron in a Fur-independent fashion, and in­ creased expression is observed under iron-restricted conditions (64). Under conditions of iron limitation, various iron acquisition systems are induced, and it has been suggested that AhpC protects the organism from reactive oxygen species resulting from the tran­ sient increase in intracellular iron (5). It is currently unknown whether AhpC is subject to regulation by iron in H. pylori. The AhpF subunit acts as the elec­ tron donor to AhpC and is an NAD(P)H oxidase in other bacteria (51). Although no homolog of ahpF has been identified in the genome sequence of H. py­ lori (or C. jejuni), cytoplasmic NADH oxidases or other electron transport proteins may function in this role. An additional antioxidant enzyme has been puri­ fied from H. pylori, termed "scavengase p 2 0 " (65), catalyzing thioredoxin-dependent peroxidase activ­ ity. The cognate gene {tpx; HP0390/JHP0991) lies ad­ jacent to the SOD gene but is divergently transcribed, and it is unknown whether the two enzymes are coregulated. As with AhpC, no mutants have been de­ scribed in this gene, and therefore the contribution of this enzyme to oxidative stress survival in H. pylori is currently unclear. Superoxide Dismutase The potentially lethal accumulation of superox­ ide is prevented by the enzyme superoxide dismutase, which catalyzes the reduction of superoxide to hydro­ gen peroxide. H. pylori possesses a surface-associated, iron-containing, superoxide dismutase, encoded by sodB (HP0389/JHP0992) (50, 5 7 ) . Three different isoforms of H. pylori SOD have been described, which are affected in their electrophoretic migration due to differences in charged amino acids (7). Interestingly, mutants could not be generated in sodB, suggesting that this enzyme is essential for survival of H. pylori in vitro. Despite the presence of superoxide dismutase, H. pylori N C T C 1 1 6 3 7 generates considerable superox­ ide as assayed by a chemiluminescent probe or in the

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conventional cytochrome c reduction assay (43). On a cell basis, superoxide generation was about 30-fold greater than for E. coli, and superoxide dismutasespecific activity was less than a third of that in £ . coli. It has been suggested that the endogenously generated superoxide reacts with nitric oxide (NO) to yield the highly toxic and mutagenic peroxynitrite ( O N O O ) , which irreversibly inactivates respiration of H. pylori (43). The source(s) of superoxide anion have not been identified, but it is worth noting that H. pylori appears to lack a flavohemoglobin such as Hmp of E. coli (53), which generates superoxide by one-electron re­ duction of O 2 , which in turn attacks NO in an oxy­ genase reaction to generate nontoxic nitrate ion. Nevertheless, it is plausible that excessive superoxide generation might contribute to oxygen sensitivity. The hypothesis is particularly attractive if superoxide were generated from an enzyme with a low affinity for 0 ; hence, at low (physiological?) 0 tensions, superoxide generation would be constrained, whereas at high (in vitro?) 0 tensions, superoxide generation would be favored. -

2

2

2

Catalases Catalase functions to detoxify hydrogen perox­ ide generated by SOD through its breakdown to water and oxygen. H. pylori expresses high levels of catalase activity, which is located in both the cytosol and per­ iplasmic space (26). The corresponding katA gene has been identified (HP0875/JHP0809), and mutations in this gene result in a complete loss of catalase activity (44). A second copy of a catalase-like protein is pres­ ent in the genome sequences of 26695 and J 9 9 (HP0485/JHP0437), which is most closely related to the catalase-like protein of Synechococcus sp. How­ ever, the function of this gene is currently unknown.

across the gastric mucosa drops. Second, the action of the abundant urease enzyme of H. pylori also re­ sults in the generation of C 0 . 2

Despite this requirement for high concentrations of C O 2 , Hughes et al. (28) found that H. pylori appar­ ently lacks many of the enzymes associated with anaplerotic C O 2 fixation in other bacteria. For example, pyruvate carboxylase, phosphoenol pyruvate carbox­ ylase, and PEP carboxykinase activities were not de­ tected. In contrast, Hoffman et al. (27) reported very low levels of these enzyme activities in some strains of H. pylori, and homologs of these enzymes have not been identified in either of the genome-sequenced strains (see chapter 12 for further discussion). The major biotin-containing enzyme identified in H. py­ lori cell extracts is acetyl-CoA carboxylase (ACC) (8, 28). ACC catalyzes the first committed step of fatty acid biosynthesis, carboxylating acetyl-CoA to gener­ ate malonyl-CoA. Acetyl-CoA carboxylase, in com­ mon with other biotin-containing proteins, requires bicarbonate rather than C O 2 as a substrate. Kinetic analysis has indicated that the purified H. pylori ACC enzyme has a comparatively high K value for bicar­ bonate in comparison to the ACC from E. coli (8). These authors have suggested that this decreased af­ finity for bicarbonate may play a role in the organ­ ism's requirement for C 0 . However, supplementa­ tion of growth medium with bicarbonate cannot replace the requirement for gaseous C O 2 (63). Two homologs of carbonic anhydrase (HP0004 and 1186/ JHP0004 and 1112) have been identified in the ge­ nome sequences. There are no reports on the role of these enzymes in H. pylori, but they may serve to con­ centrate C 0 in the form of bicarbonate inside the cell. m

2

2

T H E C 0 ENIGMA

CONCLUDING REMARKS

H. pylori is generally regarded as a microaerophile, but the bacterium also requires an elevated level of atmospheric C O 2 for optimal growth. In fact, H. pylori is capable of growth in atmospheric levels of 0 , if C O 2 is supplied at greater than 1 0 % v/v (63), and as such may be considered a capnophile. The C O 2 concentrations encountered by H. pylori in vivo are unknown. However, there are several reasons why C 0 concentrations may be relatively high in the niche inhabited by this bacterium. First, bicarbonate is continually excreted from the surface epithelial cells as a buffer to protect these cells from gastric acid. The bicarbonate diffusing across the gastric mucus will be increasingly converted to C O 2 as the pH gradient

The genome sequences of two strains of H. pylori have undoubtedly given impetus to studies of the re­ spiratory metabolism of this microaerophile. Studies of the respiration of its close relative Campylobacter are at a similar stage of development, again as the result of recent genome sequencing. In H. pylori, ge­ nomics has in many cases confirmed biochemical in­ formation but has raised many new questions. Chief among these are the nature of the biosynthetic path­ ways leading to menaquinone, the function of the pre­ dicted anaerobic respiratory pathways, and the rela­ tive roles of NADH and NADPH in delivering reducing equivalents to the respiratory chain. The ap­ parent lack of gene regulators appears consistent with

2

2

2

CHAPTER 10 • MICROAEROBIC PHYSIOLOGY

the specialized niches occupied by the bacterium in vivo and its fastidious behavior in laboratory culture. Acknowledgments. We thank Jess Alderson for help with refer­ ences and Mark Johnson for Fig. 1. We also acknowledge the U.K. Biotechnology and Biological Sciences Research Council, the Well­ come Trust, and Glaxo-Wellcome Ltd. for financial support.

16.

17.

REFERENCES 1. Alderson, J., C. L. Clayton, and D. J. Kelly. 1997. Investiga­ tions into the aerobic respiratory chain of Helicobacter pylori. G«M1(S1):A7. 2. Alderson, J., and D. J. Kelly. Unpublished data. 3. Aim, R. A., L.-S. Lee, D. T. Moir, B. L. King, E. D. Brown, P. C. Doig, D. R. Smith, B. Noonan, B. C. Guild, B. L. de Jonge, G. Carmel, P. J. Tummino, A. Caruso, M. Uria-Nickelson, D. M. Mills, C. Ives, R. Gibson, D. Merberg, S. D. Mills, Q. Jiang, D. E. Taylor, G. F. Vovis, and T. J. Trust. 1999. Genomicsequence comparison of two unrelated isolates of the human gastric pathogen Helicobacter pylori. Nature 397:176-180. 4. Baer, W., H. Koopman, and S. Wagner. 1993. Effects of sub­ stances inhibiting or uncoupling respiratory chain phosphory­ lation of Helicobacter pylori. Zentralbl. Bakteriol. 280: 253-258. 5. Baillon, M. L., A. H. van Vliet,J. M. Ketley, C. Constaninidou, and C. W. Penn. 1999. An iron-regulated alkyl hydroperoxide reductase (AhpC) confers aerotolerance and oxidative stress resistance to the microaerophilic pathogen Campylobacter je­ juni. ] . Bacteriol. 181:4798-4804. 6. Beckman, D. L., D. R. Trawick, and R. G. Kranz. 1992. Bacte­ rial cytochromes c biogenesis. Genes Dev. 6:268-283. 7. Bereswill, S., O. Neuner, S. Strobel, and M. Kist. 2000. Identi­ fication and molecular analysis of superoxide dismutase isoforms in Helicobacter pylori. FEMS Microbiol. Lett. 183: 241-245. 8. Burns, B. P., S. L. Hazell, and G. L. Mendz. 1995. Acetyl-CoA carboxylase activity in Helicobacter pylori and the require­ ment of increased C 0 for growth. Microbiology 141: 3113-3118. 9. Carlone, G. M., and F. A. L. Anet. 1983. Detection of menaquinone-6 and a novel methyl-substituted menaquinone-6 in Campylobacter jejuni and Campylobacter fetus subsp. fetus. J. Gen. Microbiol. 129:3385-3393. 10. Chalk, P. A., A. D. Roberts, and W. M. Blows. 1994. Metabo­ lism of pyruvate and glucose by intact cells of H. pylori studied by C-NMR spectroscopy. Microbiology 140:2085-2902. 11. Chalk, P. A., A. D. Roberts, A. A. Davison, D. J. Kelly, and P. J. White. 1997. The use of NMR to study the metabolism of Helicobacter pylori, p. 69-80. In C. L. Clayton and H. L. T. Mobley (ed.), Methods in Molecular Medicine—Helicobacter pylori Protocols. Humana Press Inc., New York, N.Y. 12. Chang, H. T., S. W. Marcelli, A. A. Davison, P. A. Chalk, R. K. Poole, and R. J. Miles. 1995. Kinetics of substrate oxidation by whole cells and cell membranes of Helicobacter pylori. FEMS Microbiol. Lett. 129:33-38. 13. Chen, M., L. P. Andersen, L. Zhai, and A. Kharazmi. 1999. Characterization of the respiratory chain of Helicobacter py­ lori. FEMS Immunol. Med. Microbiol. 24:169-174. 14. Collins, M. D., M. Costas, and R. J. Owens. 1984. Isoprenoid quinone composition of representatives of the genus Campylo­ bacter. Arch. Microbiol. 137:168-170. 15. D'mello, R., S. Hill, and R. K. Poole. 1996. The cytochrome bd quinol oxidase in Escherichia coli has an extremely high oxygen affinity and two oxygen-binding haems: implications

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Helicobacter pylori: Physiology and Genetics Edited by H. L . T. Mobley, G. L. Mendz, and S. L. Hazell © 2 0 0 1 ASM Press, Washington, D.C.

Chapter 11

Nitrogen Metabolism HILDE D E REUSE AND STfepHANE SKOULOUBRIS

N I T R O G E N SOURCES F O R PYLORI

HELICOBACTER

Bacteria require large amounts of nitrogen for synthesis of all the key constituents of the cell, includ­ ing amino acids, pyrimidines and purines, NAD, and amino sugars (25). Gram-negative organisms, such as Escherichia coli, use only one inorganic nitrogen com­ pound, ammonia, when growing aerobically, whereas they are able to use nitrate or nitrite as a nitrogen source during anaerobic growth. However, this bac­ terium obtains most of its nitrogen from organic ni­ trogen sources, including macromolecular building blocks such as amino acids and nucleotide bases. In bacteria, ammonia plays a unique role in nitrogen as­ similation: it is the only compound that is absolutely required in nitrogen anabolism. Two major sources of nitrogen are available in the gastric environment: amino acids and urea. Amino acids may be directly incorporated into proteins or deaminated or deamidated, with the resulting amino nitrogen incorporated into numerous metabolites by reactions described below. The study of nitrogen me­ tabolism in Helicobacter pylori was initially made possible by the development of defined growth media, which permitted (i) the determination of the minimal amino acid requirements of this bacterium (23, 26) and (ii) the following of the fate of amino acids in whole bacteria (18). The subsequent publication of the complete genomic sequences of two H. pylori strains, 26695 (34) and J 9 9 (1), has confirmed some of the data obtained and supplied additional genetic information. Amino Acids H. pylori is auxotrophic for several amino acids, supporting the idea that its growth in vivo is strictly dependent on the gastric environment. The minimal

amino acid requirements of this bacterium are arginine, histidine, isoleucine, leucine, methionine, phe­ nylalanine, and valine, with some strains also requir­ ing alanine or serine (23, 2 6 ) . Large amounts of amino acids, dipeptides, and polypeptides are present in the gastric juice owing to the activities of enzymes such as pepsin, which break down proteins efficiently (13). Some of these mole­ cules can be taken up into H. pylori cells by transport­ ers. Genomic analyses have identified several genes, orthologs of those encoding amino acid transporters for serine (sdaC, HP0133 on strain 26695), proline (putP, HP0055), D-alanine (dagA, HP0942), and glutamate (gits, HP1506) (1, 4, 3 4 ) . The open reading frames (ORFs) H P 1 1 6 9 - 1 1 7 2 code for a high-affinity ABC transport system that may be specific for glutamine. Consistent with the results of Mendz and Holmes (20, 21) (see below), gene HP1017 encodes an amino acid permease homolog of the arginine transporter RocE. H. pylori also has genes encoding homologs of three other amino acid uptake systems with unknown specificity, including the YckJ amino acid ABC transporter ( H P 0 9 3 9 - 0 9 4 0 ) . Orthologs (HP0298-0302) of the E. coli dpp genes coding for a dipeptide transport system are also found in the H. pylori chromosome, along with genes encoding a pu­ tative oligopeptide ABC transporter homologous to the E. coli Opp system ( H P 0 2 5 0 - 0 2 5 1 and H P 1 2 5 1 - 1 2 5 2 ) . Finally, the H. pylori 7-glutamyltranspeptidase may be involved in amino acid trans­ port, as suggested by analogy with the homolog E. coli protein (2). Amino acid utilization by H. pylori grown in a defined medium has been investigated by nuclear magnetic resonance (NMR) spectroscopy and amino acid analysis (18). It has been shown that H. pylori can survive using amino acids as basic nutrients. Dur­ ing microaerophilic growth in the absence of glucose, the bacterium utilizes arginine, aspartate, asparagine,

Hilde De Reuse and Stephane Skouloubris • Unite de Pathogenie Bacterienne des Muqueuses Institut Pasteur, Paris, France.

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DE REUSE AND SKOULOUBRIS

glutamine, and serine as substrates, converting them to acetate, formate, succinate, and lactate as principal metabolic products. These results indicate that fer­ mentation is an important mode of amino acid utiliza­ tion in H. pylori and suggest that its physiology has features in common with anaerobes. Continuous cul­ tures of H. pylori in a defined medium in the presence of glucose and amino acids showed utilization of ala­ nine, arginine, asparagine, aspartate, glutamine, glutamate, proline, and serine by the cells, because these amino acids were depleted almost completely from the growth medium (32). The initial steps of amino acid degradation have been investigated by N M R (18), and the results sug­ gested the existence in H. pylori of (i) deaminase ac­ tivities corresponding to an aspartase and a biodegradative serine dehydratase, and (ii) deamidase activities corresponding to an asparaginase (Asnase) and a glutaminase (Glnase) (Fig. 1). These enzymes produce ammonia by amino acid hydrolysis. Their existence was confirmed by Stark et al. (32), who demonstrated the presence of two independent deamidases, one spe­ cific for glutamine, producing ammonia and glutamate, and another specific for asparagine, producing ammonia and aspartate, which is then hydrolyzed by an aspartase to give ammonia and fumarate (Fig. 1). These Asnase and Glnase activities have been found in all strains tested, suggesting that they are important to the survival of H. pylori. Finally, serine dehydratase converts serine into ammonia and pyruvate. The genes encoding most of the corresponding enzymes

were identified in the H. pylori genome on the basis of sequence similarities ( 1 , 4 , 1 5 , 3 4 ) : HP0723 or asnB encodes an L-asparaginase II; HP0649 or aspA, an aspartate-ammonia lyase; HP0132 or sdaA, an L-serine deaminase; and HP1398 or aid, an alanine dehy­ drogenase (Fig. 1). In addition, the dad A gene (HP0943) of H. pylori encodes an ortholog of the E. coli D-amino acid dehydrogenase, which acts on all D-amino acids except D-Asp and D-Glu. Fast amino acid deaminations and deamidations in H. pylori gen­ erate significant amounts of ammonium that are in­ corporated into central intermediary nitrogen metab­ olism, as described in "Nitrogen Assimilation," below. Urea as a Nitrogen Source All H. pylori strains produce a highly abundant urease, which accounts for up to 6 % of total bacterial protein ( 1 1 , 2 2 ) . The H. pylori urease has a high affin­ ity for its substrate and catalyzes the hydrolysis of urea to give ammonia/ammonium and carbonic acid/ bicarbonate. These compounds have buffering prop­ erties and are essential to the protection of H. pylori against gastric acidity, neutralizing the bacterial microenvironment ( 8 , 1 6 ) . Urease is a true virulence fac­ tor in H. pylori as mutants deficient in this enzyme are unable to colonize the stomach of gnotobiotic pig­ lets (6), nude mice (35), or Mongolian gerbils (39). Several observations have led to the notion that the urease of H. pylori is involved not only in acid

Figure 1. Nitrogen sources for H. pylori. The reactions are catalyzed by the following enzymes: (1) glutaminase, (2) L-serine deaminase, (3) aspartate-ammonia lyase, (4) L-asparaginase II, (5) urease, (6) formamidase, (7) aliphatic amidase. The products of these deamination and deamidation reactions are also shown.

CHAPTER 11 • NITROGEN METABOLISM

resistance, but also in nitrogen metabolism. First, the urease-deficient mutant was unable to colonize gnotobiotic piglets in which gastric acidity was artificially buffered (7). Second, urease is also found in closely related nongastric Helicobacter species such as H. muridarum, H. hepaticus, and H. bills, which do not need a specialized enzyme for protection against acid­ ity. An important observation was made by Williams et al. (37), who showed that urea nitrogen could be used for the synthesis of amino acids such as glutamate, glutamine, phenylalanine, aspartate, and ala­ nine. Incorporation of the ammonia produced from urea demonstrates that urease plays a central role in H. pylori nitrogen metabolism. The importance of these functions of urease sug­ gests that to survive in its natural environment H. pylori requires urea to be constantly available. Urea concentrations in gastric juice have been estimated to be around 1 mM. However, the amount of urea in the immediate surroundings of the bacterium is un­ known, and its concentration may be significantly lowered by extracellular urease. This requirement for urea, which under some conditions may be limiting, and the slow diffusion of this compound suggest that a urea transport system may be necessary for the or­ ganism. It has been shown (29) that the Urel protein encoded by the urease gene cluster of H. pylori (i) is not necessary for the synthesis of a functional urease, (ii) is essential for the resistance of H. pylori to acidity in the presence of urea, and (iii) is required for the colonization of mice. Urel has a sequence similar to those of the AmiS proteins, which are thought to be involved in the transport of amides, molecules similar in structure to urea (38). It is therefore possible that Urel is involved in urea transport in H. pylori. Evi­ dence for such a role for Urel was provided by the recent study of Weeks et al. (36), who showed, in the heterologous Xenopus oocyte system, that Urel increased the rate of urea diffusion if the pH was below 6. This role of Urel has yet to be demonstrated in H. pylori, but would be one way of ensuring the very rapid entry of urea in acid shock conditions. Mendz et al., in collaboration with our group, found recently that H. pylori also has a high-affinity urea transport system (manuscript in preparation). This system is independent of Urel and functions between pH 3.3 and 7.3. It would facilitate the uptake and accumulation of urea at low extracellular concentra­ tions and at neutral pH, conditions that may be en­ countered by H. pylori in the mucous layer. The potential existence of two urea uptake sys­ tems demonstrates the importance of urea acquisition in H. pylori, whether the bacterium is growing in acidic conditions close to the stomach lumen or at almost neutral pH close to the stomach epithelium.

127

Other Nitrogen Sources H. pylori contains two amidases, enzymes hydrolyzing short-chain aliphatic amides to produce am­ monia and the corresponding organic acid (27, 2 8 ) . This suggests that the bacterium may be capable of obtaining ammonia from substrates other than urea and amino acids. The nature and origin of physiologi­ cal substrates of these enzymes are unknown but, by analogy with other amidase-producing organisms, these enzymes are probably involved in nitrogen me­ tabolism in H. pylori. The properties of the two H. pylori amidases are described below. Thus, nitrogen does not appear to be a limiting substrate for the growth of H. pylori in its natural environment. Nitrogen for H. pylori metabolism would be supplied mostly in the form of ammonia, which may be present in considerable amounts in the cytoplasm under some conditions. The pK of ammo­ nia (NH ) is 9.25, so at physiological pH most of the ammonia is protonated, in the form of the ammonium ion ( N H ) . An excess of poorly diffusible N H in the cell would rapidly become toxic, and, although ammonium may be directly incorporated into H. py­ lori metabolism, in order to maintain its nitrogen bal­ ance, the bacterium also has developed strategies for the elimination of unincorporated ammonium that are discussed below in some detail. a

3

+

+

4

4

N I T R O G E N ASSIMILATION In many bacteria, the preferred nitrogen donor is ammonia. Primary products of assimilated ammo­ nia are glutamine and glutamate, which constitute the central reservoir of nitrogen for many biosynthetic pathways. Glutamate is the principal source of nitro­ gen for the production of N-amine and is involved in transamination reactions at the core of amino acid metabolism. For example, in E. coli the availability of energy and nitrogen determines the participation of the three key enzymes in ammonia assimilation: glutamine synthetase (GS-ase), glutamate synthase (GOGAT-ase), and glutamate dehydrogenase (GDHase) (25) (Fig. 2 ) . GS-ase catalyzes the incorporation of ammonia into glutamate to produce glutamine, at the expense of one ATP molecule. Glutamine plus aketoglutarate can then be converted to glutamate by the action of GOGAT-ase. This is the major pathway at low ammonia concentrations. In contrast, in en­ ergy-limiting conditions, glutamate may also be syn­ thesized directly by a-ketoglutarate amination, cata­ lyzed by GDH-ase. This reaction is reversible: glutamate is therefore synthesized by this enzyme principally when ammonium concentration is high,

128

DE REUSE AND SKOULOUBRIS

GOGAT-ase

E. coli

NADP+

NADPH

Glutamine

Glutamate

Glutamine

ATP

ADP + Pi

GS-ase

NADP*

GDH-ase

H. pylori a-ketoglutarate

—

^

»

^

•

Glutamate

^

^

ATP

•

Glutamine

ADP + Pj

GS-ase Figure 2. Pathways of nitrogen assimilation in E. coli and H. pylori. GOGAT-ase corresponds to glutamate synthase, GDHase to glutamate dehydrogenase, and GS-ase to glutamine synthetase.

whereas GS-ase is still necessary for glutamine synthe­ sis. Owing to the central role of GS-ase in nitrogen assimilation in E. coli, the expression of the corre­ sponding gene and its activity are highly regulated. No gene encoding a putative GOGAT-ase has been identified in the genome sequence of H. pylori, but the genes HP0380 and HP0512 are orthologous to those of E. coli encoding GDH-ase (gdhA) and GSase (glnA), respectively, and the proteins they encode have 5 9 % and 4 7 % identity with the corresponding E. coli homologs. The absence of GOGAT-ase sug­ gests that H. pylori produces only the enzymes re­ quired for nitrogen assimilation at high ammonium concentrations. This is consistent with its potential to generate large amounts of ammonium and demon­ strates that H. pylori is highly adapted to its particular ecological niche. Characteristics of H. pylori Glutamine Synthetase The glnA gene encoding the H. pylori glutamine synthetase has been cloned, expressed in E. coli, and shown to complement an E. coli GS-ase-deficient mu­

tant (10). In H. pylori, it was not possible to introduce mutations into the glnA gene by standard allelic ex­ change techniques, which suggests that GS-ase per­ forms an essential function in this bacterium when growing on rich medium in vitro (10). Thus, GS-ase is crucial for nitrogen assimilation in H. pylori and may be active under all physiological conditions. Fur­ ther evidence for the role of this enzyme is provided by the absence in H. pylori of the regulatory features observed in most of its bacterial homologs. The posttranslational modification of GS-ase by adenylation is generally a key mechanism for turning off the activ­ ity of the enzyme. H. pylori GlnA lacks the highly conserved target tyrosine residue within the adenyla­ tion motif and seems to be, therefore, not regulated in this way. No consensus sequence for o- -dependent transcription has been found upstream from the H. pylori glnA gene, suggesting that this second type of regulation is also absent in H. pylori. In conclusion, the only nitrogen incorporation pathway found in H. pylori may explain the lack of regulation of GS-ase, a key metabolic enzyme. In H. pylori, this enzyme seems to play an important role 54

CHAPTER 11 • NITROGEN METABOLISM

in nitrogen metabolism and is probably essential for processing the large amounts of ammonia produced within the cell. Is There Evidence for the Regulation of Nitrogen Assimilation? Genes have been identified in the genome se­ quence of H. pylori encoding homologs of proteins involved in the regulation of nitrogen metabolism in other bacteria such as E. coli. These genes encode the alternative sigma 54 factor, CT (HP0714) and an NtrC homolog (HP0703). However, the experimental data available for H. pylori seem to connect these fac­ tors with flagellum synthesis, rather than with the reg­ ulation of nitrogen assimilation. First, the genes for which transcription was shown to be dependent on the H. pylori RNA-polymerase-or encode proteins involved in flagellum morphogenesis (31). Second, the NtrC homolog of H. pylori was renamed FlgR owing to its role as a transcriptional activator for some of the rj -regulated flagellum proteins (31). These results suggest that the H. pylori a factor may play a role different from that of its homologs in most other bac­ teria, but possibly similar to that described in Caulobacter crescentus. Indeed, these regulatory mechanisms may enable H. pylori to coordinate fla­ gellum synthesis with changes in its environment and may also respond to changes in metabolic fluxes, such as that of nitrogen. Although caution is necessary when interpreting the absence from a genome of a consensus-binding sequence, or of a gene encoding a homologous pro­ tein, nitrogen assimilation does not seem to be as tightly controlled in H. pylori as in many well-studied organisms. The regulatory networks of H. pylori have been found generally to be poor, and it has been sug­ gested that this is related to its very restricted coloni­ zation niche.

129

monium, which is toxic. These strategies probably in­ volve two main mechanisms. First, the extrusion of ammonium by a specific transport system. No ammo­ nium exporter has yet been described in prokaryotes, but it remains a possibility to be explored. Second, the transformation of ammonium into a more diffusi­ ble and nontoxic compound, such as urea, which is the principal nitrogenous waste product in mammals.

The Urea Cycle

54

54

54

54

UREA CYCLE AND ARGINASE Nitrogen metabolism in H. pylori generates con­ siderable amounts of free ammonia that either could be incorporated into proteins via the GS-ase pathway or released into the external environment by diffusion in its N H form. Extracellularly, it will be able to capture a proton and thereby help buffer the bacterial microenvironment. However, at physiological intra­ cellular pH, most of the ammonia generated is present in protonated form as N H , which does not diffuse freely through membranes. The cell must therefore have developed strategies for eliminating excess am­ 3

+

4

Complete urea cycles have not been described fre­ quently in prokaryotes. The possibility that H. pylori has a urea cycle was investigated by assessing the ac­ tivity of the four enzymes of this cycle: arginase, ana­ bolic ornithine transcarbamoylase (OTC-ase), arginosuccinate synthetase, and arginosuccinase (Fig. 3), employing one- and two-dimensional N M R spectros­ copy and radioactive tracer analysis (19). Arginase activity was demonstrated in the presence of L-arginine by the detection of its catabolic products, orni­ thine and urea (Fig. 3). The H. pylori enzyme has been characterized, and is described below. An OTC-ase was shown to be present by the detection of heatstable products in the presence of ornithine and radio­ labeled carbamoyl phosphate. Arginosuccinate for­ mation and catalysis indicated that the two remaining enzyme activities of the cycle also were present. These experiments were performed with cell suspensions, lysates, and membrane fractions. Arginase and argininosuccinase activities are associated with the membrane fraction. In contrast, OTC-ase and argininosuccinate synthetase seem to be cytoplasmic (19). The end products generated by the urea cycle are urea and fumarate; the latter has been shown to be metabo­ lized to give succinate, acetate, lactate, and alanine in H. pylori (19). Various observations can be made concerning the presence of a complete urea cycle in H. pylori. In the first place, in H. pylori liquid cultures the orni­ thine produced by arginase activity accumulates (18). The rate of accumulation of this product of the first step in the urea cycle seems to be much higher than its rate of utilization in catabolism. Also, the gene encoding arginase (HP1399) has been identified in the H. pylori genome, but no genes encoding orthologs of the other three enzymes of the urea cycle have been identified. This may be due to considerable divergence during the course of evolution. Orthologs of the CarA and CarB subunits of the carbamoyl phosphate syn­ thetase, an enzyme catalyzing the synthesis of carbam­ oyl phosphate, a substrate of OTC-ase (Fig. 3), are encoded by the HP1237 and HP0919 genes of H. py­ lori, respectively.

130

DE REUSE AND SKOULOUBRIS

Fumarate

Arginine Urease

Arginlnosuccinase

Arginase

Argininosuccinate

Urea

•

2NH

+ HCCy

+ 4

Ornithine Carbamoyl- phosphate synthetase

AMP + PPi

Argininosuccinate synthetase

Ornithine transcarbamoylast

Carbamoyl phosphate

ATP

2ADP +

Citrulline Aspartate

Y

^

N

H

^

+

H

C

Q

3 -

2ATP

2 Pj i

Figure 3. Enzymatic reactions of the urea cycle. Urea hydrolysis by urease in H. pylori is indicated, as is the reaction producing carbamoyl phosphate.

Finally, the simultaneous presence of a urea cycle and a highly active urease appears paradoxical and raises the question of how H. pylori avoids an energet­ ically expensive futile cycle. The coexistence of these two functions has, however, been described in other organisms, such as in the cyanobacterium Synechocystis sp. strain PCC6803 (24). The association with the membrane of arginase and argininosuccinase led Mendz and Hazell (19) to propose that metabolic compartmentalization separates biosynthesis (urea cycle) from degradation (urease), allowing the urea produced by arginase close to the cytoplasmic mem­ brane to be exported before it is hydrolyzed by intra­ cellular urease. Properties of Arginase and the Transport of Arginine L-arginine is required for the growth of H. pylori in vitro. Catabolism of arginine to ammonia depends on the presence of active urease (19), indicating that the only pathway of arginine catabolism in H. pylori is that initiated by arginase. The gene encoding arginase (HP1399) has been identified in the genomic sequence of H. pylori and given the name rocF, by analogy with the Bacillus subtilis gene whose product is the RocF protein (9), which is 2 7 % identical to the arginase of H. pylori. Based on sequence data, the H. pylori 37kDa RocF protein is a type II arginase. The properties

of this enzyme were characterized by Mendz et al. (21), who showed it to be highly specific for arginine, with a K of 22 mM. The low-affinity K and the quaternary structure (100 to 3 0 0 kDa) of H. pylori arginase show that the enzyme is of the ureotelic type, like those of organisms that excrete excess nitrogen as urea. The H. pylori arginase is a metalloprotein with a preference for C o cations and, in whole cells, its activity is enhanced by bicarbonate. This regula­ tion led the authors (21) to suggest that arginase may connect nitrogen metabolism and the microaerophilic nature of H. pylori. The centrifugation through oil method has been used to study arginine transport in H. pylori cell sus­ pensions (20). This transport is rapid, temperature dependent, saturable, and highly specific for arginine, as analogs had no effect. This suggests that there are one or more carriers, and the absence of inhibition by L-lysine and L-ornithine indicated that this transporter is different from the LAO (Lys-Arg-Orn) system com­ monly found in prokaryotes. Indeed, an ORF (HP1017) encoding a homolog of the B. subtilis RocE arginine transporter has been identified in the com­ plete genomic sequence of H. pylori. m

m

2 +

Role of Arginase in H. pylori Physiology Insertional mutants of the rocF gene were gener­ ated by allelic exchange in various H. pylori strains

CHAPTER 11 • NITROGEN METABOLISM

(17). These rocF mutants were completely devoid of arginase activity. The activities of the other enzymes involved in ammonia release in H. pylori were deter­ mined in these arginase-deficient strains. Urease, Asnase, Aspase, and Glnase activities were not affected, but all the mutants had lower than normal levels of serine dehydratase activity. It is not clear how argi­ nase regulates this enzyme. Although there is a close metabolic relationship between arginase and urease, these enzymes do not influence each other's activity. The role of arginase in acid protection was inves­ tigated because of the potential of this enzyme to sup­ ply urea, the hydrolysis of which is critical to the resis­ tance of H. pylori to acid. In the absence of urea or arginine, the rocF mutant was 1,000 times more sensi­ tive than the corresponding wild-type strain to acid (pH 2.3). In the presence of urea, no difference in viability was observed between the two strains. In contrast to the wild-type strain, which could be res­ cued from acid exposure by the addition of arginine, no such effect was observed with the arginase-defi­ cient strain. These results suggest that the H. pylori arginase is critical for acid protection in vitro. The role of arginase in vivo was examined using the mouse model of colonization by H. pylori strain SSI (14). The level of colonization of mice by the arginase mu­ tants was similar or slightly lower than that of the wild-type strain indicating that, at least in this model, this enzyme is not essential for colonization (17).

T W O PARALOGOUS AMIDASES Aliphatic amidases are amidohydrolases that cat­ alyze the hydrolysis of the amide bonds of short-chain aliphatic amides to produce ammonia and the corre­ sponding carboxylic acid. Such activity has been re­ ported in a small number of bacteria from environ­ mental habitats. The identification of two amidases in H. pylori was therefore unexpected and seems to be unique among the bacteria of the gastroduodenal tract. Properties of the Amidases The enzymatic characteristics of the two ami­ dases of H. pylori were determined after purification of these enzymes from recombinant E. coli strains overexpressing the corresponding genes (27). The first amidase, AmiE encoded by HP0294, shares as much as 7 5 % identity with its homologs from Pseudomonas aeruginosa, Rhodococcus sp. R 3 1 2 , and Bacillus stearothermophilus (27). The substrate specificity of AmiE is similar to that of the other sequenced ami­ dases (27). In vitro, its affinity was highest with ace-

131

tamide, followed by acrylamide and propionamide. AmiE does not hydrolyze urea or formamide. H. py­ lori AmiE is therefore a typical aliphatic amidase. The publication of the complete genome se­ quences of two H. pylori strains revealed the existence of a second amidase encoded by HP1238 that was 3 4 % identical to the H. pylori AmiE protein and with its homologs in other species. The corresponding puri­ fied AmiF protein presented a unique specificity for the shortest amide, formamide. It did not hydrolyze the substrates of AmiE or urea. Although they differ in substrate specificity, AmiE and AmiF are both sulfhydryl enzymes with a common catalytic mechanism and a highly conserved cysteine residue shown, by site-directed mutagenesis, to be part of the active site (27). The H. pylori AmiE and AmiF proteins are thus typical paralogs. Role of the Amidases The role of these two paralogous proteins was investigated in H. pylori, using mutant strains dis­ rupted in one or both of the amiE and amiF genes (27). The amidases are not essential for growth in vitro because no differences in viabilities or growth rates are observed between the mutants not expressing the proteins and the wild-type strain. The activity and substrate specificity of each amidase were confirmed in H. pylori mutant strains deficient in AmiE or AmiF. Employing the mouse colonization model with the H. pylori SSI strain (14), it was shown that the AmiE amidase is not essential in vivo. However, it is still unknown whether a strain deficient in both amidases would be able to colonize the mouse stomach. As is the case for other amidase-producing organ­ isms, the AmiE aliphatic amidase probably ensures that the bacterium is supplied with nitrogen. Overexpression of the H. pylori amiE gene in E. coli renders the strain able to grow on acetamide as the sole nitro­ gen source (5). The physiological substrate of AmiE remains unknown. It may be produced within the cell or generated by degradation of larger compounds in the gastric juice. It is not clear whether formamide is imported into the cell and used as a nitrogen source or generated as a toxic product by metabolic path­ ways, with AmiF involved in detoxification. The presence of amiE and amiF genes in several H. pylori strains isolated from patients of various eth­ nic origins was investigated by PCR (in collaboration with D. Berg, St. Louis, Mo.). Both genes were found to be present in all the strains tested. The conservation of these genes suggests that the paralogous AmiE and AmiF functions have been conserved during evolu­ tion.

132

DE REUSE AND SKOULOUBRIS

AmiE amidase activity has been shown to be en­ hanced in a urease-deficient H. pylori strain (28). This increase in activity is correlated with an increase in AmiE protein synthesis in the absence of urease, sug­ gesting that amiE transcription may be regulated by intracellular ammonia concentration (27). Finally, AmiF protein synthesis has been found to be increased in a strain deficient in both urease and arginase activi­ ties (27).

CONCLUSIONS Ammonia is the major source of nitrogen in H. pylori, and the metabolism of this bacterium seems to be adapted to an environment in which this com­ pound is rarely limiting. As described in this chapter, ammonia is produced in various ways in H. pylori, and urease is one of the principal enzymes involved in this process. The networks regulating nitrogen as­ similation commonly found in other bacteria seem to be absent in this organism. However, the low level of serine dehydratase activity in arginase-deficient strains (17) and the relationship between urease, argi­ nase, and amidase activities (27) suggest the existence of a global nitrogen- or ammonia-dependent regula­ tion of H. pylori metabolism. An exceptional characteristic of H. pylori nitro­ gen metabolism is the close connection between am­ monia production and the resistance of this organism to acidic conditions. Urease is central in this response, and the availability of urea is critical. H. pylori has redundant mechanisms for supplying intracellular urea: probably Urel, but also a high-affinity urea transport system, and an arginase, which may be im­ portant when the extracellular urea concentration de­ creases to very low levels. The contribution of the other ammonia-producing enzymes to the survival of H. pylori in acidic environments is unclear. Arginase is important for in vitro resistance to acidity but is apparently not essential in vivo, whereas the hydroly­ sis of acetamide by the AmiE amidase does not enable H. pylori to survive to acidity in vitro (Bury and De Reuse, unpublished results). Preliminary results from McGee et al. (17) suggest that the amino acid deami­ nases do not play an important role in protecting H. pylori against acidity. Finally, the large amounts of ammonia generated by H. pylori are probably involved in pathogenesis. The ammonia produced by urease has been shown to be toxic for various gastric cell lines (22, 3 0 ) . It has been suggested that urease activity damages the gas­ tric epithelium by interacting with the immune system and stimulating an oxidative burst in human neutro­ phils (33). The hypochlorous acid generated during

this stage then probably reacts with ammonia to yield the highly toxic monochloramine (3, 12). REFERENCES 1. Aim, R. A., L. S. L. Ling, D. T. Moir, B. L. King, E. D. Brown, P. C. Doig, D. R. Smith, B. Noonan, B. C. Guild, B. L. dejonge, G. Carmel, P. J. Tummino, A. Caruso, M. Uria-Nickelsen, D. M. Mills, C. Ives, R. Gibson, D. Merberg, S. D. Mills, Q. Jiang, D. E. Taylor, G. F. Vovis, and T. J. Trust. 1999. Genomicsequence comparison of two unrelated isolates of the human gastric pathogen Helicobacter pylori. Nature 397:176-180. 2. Chevalier, C , J.-M. Thiberge, R. L. Ferrero, and A. Labigne. 1999. Essential role of Helicobacter pylori -y-glutamyltranspeptidase for the colonization of the gastric mucosa of mice. Mol. Microbiol. 31:1359-1372. 3. Dekigai, H., M. Murakami, and T. Kita. 1995. Mechanism of Helicobacter pylori-associated gastric mucosal injury. Dig. Dis. Sci. 40:1332-1339. 4. Doig, P., B. L. de Jonge, R. A. Aim, E. D. Brown, M. UriaNickelsen, B. Noonan, S. D. Mills, P. Tummino, G. Carmel, B. C. Guild, D. T. Moir, G. F. Vovis, and T. J. Trust. 1999. Helicobacter pylori physiology predicted from genomic com­ parison of two strains. Microbiol. Mol. Biol. Rev. 63:675-707. 5. Doring, V., and P. Marliere. 1998. Reassigning cysteine in the genetic code of Escherichia coli. Genetics 150:543-551. 6. Eaton, K. A., C. L. Brooks, D. R. Morgan, and S. Krakowka. 1991. Essential role of urease in pathogenesis of gastritis in­ duced by Helicobacter pylori in gnotobiotic piglets. Infect. Immun. 59:2470-2475. 7. Eaton, K. A., and S. Krakowka. 1994. Effect of gastric pH on urease-dependent colonization of gnotobiotic piglets by Heli­ cobacter pylori. Infect. Immun. 62:3604-3607. 8. Ferrero, R. L., and A. Lee. 1991. The importance of urease in acid protection for the gastric-colonising bacteria Helicobacter pylori and Helicobacter felis sp. nov. Microb. Ecol. Health Dis. 4:121-134. 9. Gardan, R., G. Rapoport, and M. Debarbouille. 1995. Expres­ sion of the rocDEF operon involved in arginine catabolism in Bacillus subtilis. J. Mol. Biol. 249:843-856. 10. Garner, R. M., J. Fulkerson, Jr., and H. L. Mobley. 1998. Helicobacter pylori glutamine synthetase lacks features associ­ ated with transcriptional and posttranslational regulation. In­ fect. Immun. 66:1839-1847. 11. Hu, L. T., and H. L. Mobley. 1990. Purification and N-terminal analysis of urease from Helicobacter pylori. Infect. Immun. 58:992-998. 12. Kleiner, D., A. Traglauer, and S. Domm. 1998. Does ammonia production by Klebsiella contribute to pathogenesis? Bull. Inst. Pasteur 96:257-265. 13. Komorowska, M., H. Szafran, T. Popiela, and Z. Szafran. 1981. Free amino acids in gastric juice. Acta Physiol. Pol. 32: 559-567. 14. Lee, A., J. O'Rourke, M. Corazon De Ungria, B. Robertson, G. Daskalopoulos, and M. F. Dixon. 1997. A standardized mouse model of Helicobacter pylori infection: introducing the Sydney strain. Gastroenterology 112:1386-1397. 15. Marais, A., G. L. Mendz, S. L. Hazell, and F. Megraud. 1999. Metabolism and genetics of Helicobacter pylori: the genome era. Microbiol. Mol. Biol. Rev. 63:642-674. 16. Marshall, B. J., L. Barret, C. Prakash, R. McCallum, and R. Guerrant. 1990. Urea protects Helicobacter (Campylobacter) pylori from the bactericidal effect of acid. Gastroenterology 99:269-276.

CHAPTER 11 • NITROGEN METABOLISM

17. McGee, D. J., F. J. Radcliff, G. L. Mendz, R. L. Ferrero, and H. L. Mobley. 1999. Helicobacter pylori rocF is required for arginase activity and acid protection in vitro but is not essential for colonization of mice or for urease activity. /. Bacteriol. 181:7314-7322. 18. Mendz, G. L., and S. L. Hazell. 1995. Amino acid utilization by Helicobacter pylori. Int. J. Biochem. Cell. Biol. 27: 1085-1093. 19. Mendz, G. L., and S. L. Hazell. 1996. The urea cycle of Helico­ bacter pylori. Microbiology 142:2959-2967. 20. Mendz, G. L., and E. M. Holmes. 1998. Metabolic fate of arginine, an essential amino acid for Helicobacter pylori, p. 193-196. In A. J. Lastovica, D. G. Newell, and E. E. Lastovica (ed.), Campylobacter, Helicobacter and Related Organisms. Institute of Child Health, Cape Town, South Africa. 21. Mendz, G. L., E. M. Holmes, and R. L. Ferrero. 1998. In situ characterization of Helicobacter pylori arginase. Biochim. Biophys. Acta 1388:465-477. 22. Mobley, H. L., M. D. Island, and R. P. Hausinger. 1995. Mo­ lecular biology of microbial ureases. Microbiol. Rev. 59: 451-480. 23. Nedenskov, P. 1994. Nutritional requirements for growth of Helicobacter pylori. Appl. Environ. Microbiol. 60: 3450-3453. 24. Quintero, M. J., A. M. Muro-Pastor, A. Herrero, and E. Flores. 2000. Arginine catabolism in the cyanobacterium Synechocystis sp. strain PCC6803 involves the urea cycle and arginase pathway. /. Bacteriol. 182:1008-1015. 25. Reitzer, L. J. 1996. Ammonia assimilation and the biosynthesis of glutamine, glutamate, asparate, asparagine, L-alanine and D-alanine, p. 301-407. In F. C. Neidhardt (ed.), Escherichia coli and Salmonella: Cellular and Molecular Biology, 2nd ed. ASM Press, Washington, D.C. 26. Reynolds, D. J., and C. W. Penn. 1994. Characteristics of Heli­ cobacter pylori growth in a defined medium and determination of its amino acid requirements. Microbiology 140:2649-2656. 27. Skouloubris, S., A. Labigne, and H. De Reuse. The AmiE ali­ phatic amidase and AmiF formamidase of Helicobacter pylori: natural evolution of two enzyme paralogs. Mol. Microbiol, in press. 28. Skouloubris, S., A. Labigne, and H. De Reuse. 1997. Identifica­ tion and characterization of an aliphatic amidase in Helico­ bacter pylori. Mol. Microbiol. 25:989-998. 29. Skouloubris, S., J. M. Thiberge, A. Labigne, and H. De Reuse. 1998. The Helicobacter pylori Urel protein is not involved in urease activity but is essential for bacterial survival in vivo. Infect. Immun. 66:4517-4521.

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30. Smoot, D. T., H. L. Mobley, G. R. Chippendale, J. F. Lewison, and J. H. Resau. 1990. Helicobacter pylori urease activity is toxic to human gastric epithelial cells. Infect. Immun. 58: 1992-1994. 31. Spohn, G., and V. Scarlato. 1999. Motility of Helicobacter pylori is coordinately regulated by the transcriptional activator FlgR, an NtrC homolog. /. Bacteriol. 181:593-599. 32. Stark, R. M., M. S. Suleiman, I. J. Hassan, J. Greenman, and M. R. Millar. 1997. Amino acid utilisation and deamination of glutamine and asparagine by Helicobacter pylori. } . Med. Microbiol. 46:793-800. 33. Suzuki, M., S. Miura, M. Suematsu, D. Fukumura, I. Kurose, H. Suzuki, A. Kai, Y. Kudoh, M. Ohashi, and M. Tsuchiya. 1992. Helicobacter py/on'-associated ammonia production en­ hances neutrophil-dependent gastric mucosas cell injury. Am. ] . Physiol. 263:G719-G725. 34. Tomb, J.-F., O. White, A. R. Kerlavage, R. A. Clayton, G. G. Sutton, R. D. Fleischmann, K. A. Ketchum, H. P. Klenk, S. Gill, B. A. Dougherty, K. Nelson, J. Quackenbush, L. Zhou, E. F. Kirkness, S. Peterson, B. Loftus, D. Richardson, R. Dodson, H. G. Khalak, A. Glodek, K. McKenney, L. M. Fitzegerald, N. Lee, M. D. Adams, E. K. Hickey, D. E. Berg, J. D. Gocayne, T. R. Utterback, J. D. Peterson, J. M. Kelley, M. D. Cotton, J. M. Weidman, C. Fuji, C. Bowman, L. Watthey, E. Wallin, W. S. Hayes, M. Borodovsky, P. D. Karp, H. O. Smith, C. M. Fraser, and J. C. Venter. 1997. The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature 388:539-547. 35. Tsuda, M., M. Karita, M. G. Morshed, K. Okita, and T. Nakazawa. 1994. A urease-negative mutant of Helicobacter pylori constructed by allelic exchange lacks the ability to colonize the nude mouse stomach. Infect. Immun. 1994:3586-3589. 36. Weeks, D. L., S. Eskandari, D. R. Scott, and G. Sachs. 2000. A H -gated urea channel: the link between Helicobacter pylori urease and gastric colonization. Science 287:482-485. 37. Williams, C. L., T. Preston, M. Hossack, C. Slater, and K. E. McColl. 1996. Helicobacter pylori utilises urea for amino acid synthesis. FEMS Immunol. Med. Microbiol. 13:87-94. 38. Wilson, S. A., R. J . Williams, L. H. Pearl, and R. E. Drew. 1995. Identification of two new genes in the Pseudomonas aeruginosa amidase operon, encoding an ATPase (AmiB) and a putative integral membrane protein (AmiS). /. Biol. Chem. 270:18818-18824. 39. Wirth, H. P., M. H. Beins, M. Yang, K. T. Tham, and M. J. Blaser. 1998. Experimental infection of Mongolian gerbils with wild-type and mutant Helicobacter pylori strains. Infect. Immun. 66:4856-4866. +

Helicobacter pylori: Physiology and Genetics Edited by H. L. T. Mobley, G. L. Mendz, and S. L. Hazell © 2 0 0 1 ASM Press, Washington, D.C.

Chapter 12

The Citric Acid Cycle and Fatty Acid Biosynthesis DAVID J . KELLY AND NICKY J . HUGHES

must be cautious about "missing" enzymes from ge­ nome analysis alone; actual biochemical experiments can reveal activities for which no gene appears to exist. More sensitive similarity searching may "find" the gene, or nonhomologous gene displacement with unexpected enzymes performing the expected func­ tion may be responsible (10, 2 4 ) . With this back­ ground, the available biochemical and genomic data are employed to assess the nature of the CAC in Heli­ cobacter pylori. Also described in this chapter is fatty acid biosynthesis, linked to the cycle through the utili­ zation of acetyl-CoA as its starting point.

DIVERSITY AND MULTIPLE ROLES OF T H E CITRIC ACID CYCLE LN PROKARYOTES The citric acid cycle (CAC) is arguably the most important central metabolic pathway in living cells. It has long been appreciated that this pathway often serves a dual role in energy conservation and in the biosynthesis of key cellular intermediates for anabolic reactions; for example, 2-oxoglutarate and oxaloacetate for amino acid biosynthesis, and succinyl-coenzyme A (CoA) for heme synthesis in some bacteria. The latter function is particularly important under an­ aerobic conditions, where the usual complete "aero­ bic" CAC is converted into two branches, an oxida­ tive, or C6 branch, from citrate to 2-oxoglutarate, and a reductive, or C4 branch, from oxaloacetate to succinate. This occurs largely as the result of the an­ aerobic repression of the 2-oxoglutarate dehydrogen­ ase multi-enzyme complex and succinate dehydrogen­ ase, and the induction of fumarate reductase. The regulatory mechanisms by which this occurs in some bacteria are well known. In Escherichia coli, for ex­ ample, they involve the oxygen-sensing activities of Fnr and the ArcA/ArcB system (51). However, not all bacteria conform to this pat­ tern. With the advent of genome sequencing it has become apparent that there are many variations on the basic theme of the "textbook" CAC, and a surpris­ ingly large number of bacteria, particularly patho­ gens, are turning out to have incomplete or otherwise unusual CAC, which deviate from the E. coli para­ digm (10, 2 4 ) . Indeed, in analyses of the CAC pre­ dicted from 19 completely sequenced genomes, Huynen et al. (24) concluded that in the majority of species the cycle is incomplete or even absent, reflecting par­ ticular adaptations to the metabolic lifestyle of that particular organism. While this may be the case, one

T H E CAC IN H.

PYLORI

Predictions from the Genome Sequence Table 1 lists the open reading frames (ORFs) in strains 26695 (53) andJ99 (1) encoding products with homology to CAC enzymes. In both strains, genes en­ coding citrate synthase, aconitase, isocitrate dehydro­ genase, fumarase, fumarate reductase, and fumarase are clearly present. The first unusual feature to note is that genes encoding the usual pyruvate dehydrogen­ ase and 2-oxoglutarate dehydrogenase multi-enzyme complexes, found in most aerobic bacteria, are ab­ sent. Instead, H. pylori utilizes 2-oxoacid:acceptor oxidoreductases specific for pyruvate (POR) and 2oxoglutarate (OOR) to carry out the same biochemi­ cal reactions, a clear example of nonhomologous gene displacement. There are no genes encoding homologs of succinate dehydrogenase, succinyl-CoA synthetase (SCS), or NAD-linked malate dehydrogenase (MDH). There is also no evidence for genes encoding glyoxylate shunt enzymes (isocitrate lyase and malate syn­ thase). From simple genomic predictions alone, the conclusion would be that the CAC is incomplete, owing to the absence of SCS and MDH.

David J. Kelly • Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, United Kingdom. Nicky J. Hughes • SmithKline Beecham Pharmaceuticals Research and Development Ltd. Anti-infectives Research, 1250 South Collegeville Road, Collegeville, PA 19426.

135

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KELLY AND HUGHES

Table 1. Genomic and biochemical evidence for CAC enzymes in H. pylorf Homolog in:

Enzyme Pyruvate:acceptor oxidoreductase Citrate synthase Aconitase Isocitrate dehydrogenase 2-oxoglutarate:acceptor oxidoreductase 2-oxoglutarate oxidase Succinate dehydrogenase Fumarate reductase Fumarase Malate dehydrogenase (NAD-linked) Malate:quinone oxidoreductase Malate synthase

26695

J99

1108/1109/1110/1111 (porCDAB) 0026 (gitA) 0779 (acnB) 0027 (icdA) 0588/0589/0590/0591 (oorDABC)

1035/1036/1037/1038 0022 0716 0023 0536/0537/0538/0539

0191/0192/0193 (frdBAC) 1325 (fumC)

0177/0178/0179 1245

0086

0079

Reference(s) 21-23 21,45 21,45 21,45 21-23 45 21 3,21,45 21,45 21, 45 13, 27, 28 21, 45

" Activity was detected for all enzymes listed. However, no genes for 2-oxoglutarate oxidase, succinate dehydrogenase, NAD-malate dehydrogenase or malate synthase have been identified.

Experimental Evidence for CAC Reactions There have been conflicting biochemical data published on the overall activity of the CAC in H. pylori. Several studies of H. pylori metabolism in in­ tact cells have indicated the lack of an active oxidative cycle. The amount of oxygen uptake during respira­ tion of various carbon substrates was considered by Chang et al. (8) to be insufficient for complete oxida­ tion through the CAC. In nuclear magnetic resonance (NMR) studies (7), the accumulation of significant concentrations of acetate from pyruvate metabolism under aerobic incubation conditions strongly suggests a major diversion of the acetyl-CoA resulting from the P O R reaction toward acetate production rather than oxidation through the citric acid cycle. This could occur via phosphotransacetylase and acetate ki­ nase, genes for which have been identified in strains 2 6 6 9 5 (53) and J 9 9 (1), and would yield one ATP per mole of acetyl-CoA. In contrast, the operation of a complete oxidative CAC with a glyoxylate bypass has been inferred from the results of spectrophotometric enzyme assays by Hoffman et al. (21). However, SCS was not assayed in this study, and other workers have been unable to detect this activity ( 1 1 , 4 6 ) , consistent with the ab­ sence of the cognate genes in the genome sequences. It is also likely that the succinate dehydrogenase activity reported by Hoffman et al. (21) is in fact due to fumar­ ate reductase. Using both spectrophotometric assays and N M R spectroscopy, Pitson et al. (46) concluded that H. pylori possessed a noncyclic branched path­ way, as in anaerobes, with fumarate reductase but no succinate dehydrogenase or SCS. They found no evidence for an operational glyoxylate or gammaaminobutyrate shunt, although malate synthase activ­

ity was detected. The differences between these stud­ ies may be due to differences in the methods used to assay the enzymes. A number of experimental prob­ lems exist in assaying many CAC enzymes, and partic­ ular caution is needed where low activities are mea­ sured with assays prone to interference (e.g., isocitrate lyase) or high background rates, as such activities may not be physiologically significant. Pitson et al. (46) have tried to avoid some of the problems with spectro­ photometric assays by using N M R analyses of sub­ strates and products. Nevertheless, any assay with crude cell-free extracts can be misleading owing to additional or unsuspected metabolism of the sub­ strates, cofactors, or products, and it should be em­ phasized that the kinetic properties of an enzyme can only be reliably determined by initial rate measure­ ments with the pure protein, although the in vivo properties of enzymes may be modulated by interac­ tions with other cell components. Unfortunately, only a few H. pylori CAC enzymes have yet been purified. The enzyme activities that have been detected are dis­ cussed in more detail below. Properties of Individual CAC Enzymes Pyruvate:flavodoxin oxidoreductase The oxidative decarboxylation of pyruvate is an important reaction in archaea, bacteria, and eukaryotes alike, generating acetyl-CoA necessary for CAC reactions, fatty acid biosynthesis, and many other re­ actions requiring acyl-CoA. In many aerobic bacteria and also mammalian systems this reaction is catalyzed by the pyruvate dehydrogenase multi-enzyme com­ plex (43). Under anaerobiosis in bacteria capable of mixed acid fermentation, the generation of acetyl-

CHAPTER 12 • THE CITRIC ACID CYCLE AND FATTY ACID BIOSYNTHESIS

Co A from pyruvate is catalyzed by pyruvate: formate lyase (31). Biochemical and genomic data indicate that both of these enzymes are absent in H. pylori, and instead this reaction is catalyzed by an unusual four-subunit pyruvate:flavodoxin oxidoreductase (POR) enzyme (22, 2 3 ) . POR enzymes are generally associated with an anaerobic-type metabolism, and there has been growing interest recently in this class of enzymes as targets for the development of novel anti-anaerobe compounds (48). Indeed, the first crys­ tal structure of a POR enzyme has recently been pub­ lished (9). These enzymes can be broadly grouped into three types: single-subunit POR, found for example in Clostridium sp. (37), Anabeana sp. and Klebsiella sp. (49), Desulfovibrio africanus (45), and some an­ aerobic protozoa (14, 4 8 , 5 5 ) ; two-subunit POR, so far identified only in the aerobic archaeon, Halobacterium halobium (29); and the four-subunit POR com­ monly associated with the members of the archaea and hyperthermophilic eubacteria ( 4 , 5 , 5 2 ) . POR has been purified from H. pylori and shown to belong to this ancestral, four-subunit group of enzymes (22), and was the first example of a four-subunit POR found in a mesophilic eubacterium. Characteristi­ cally, these enzymes catalyze the reduction of lowpotential electron acceptors in vivo that are com­ monly either ferredoxin or flavodoxin proteins. There is evidence that the in vivo electron acceptor of the H. pylori POR enzyme is a flavodoxin (FldA; HP1161/ JHP1088) (22, 2 3 , 26). The molecular masses of the subunits are as follows: PorA, 4 7 kDa; PorB, 36 kDa; PorC, 24 kDa; and PorD, 14 kDa (22). The structural genes for POR are located adjacent to each other in the chromosomes of both strains 26695 and J 9 9 in the gene order porCDAB (HP1108-1111/ J H P 1 0 3 5 - 1 0 3 8 ) . The molecular mass of the native POR was estimated to be 2 4 0 kDa; however, the stoichiometry of the native complex is unknown. The PorB subunit displays the conserved amino acid bind­ ing motif for thiamine pyrophosphate (TPP) in com­ mon with other POR enzymes (23). The PorD subunit is strikingly similar to bacterial ferredoxins, display­ ing the characteristic cysteine-rich Fe-S binding mo­ tifs. Unlike pyruvate dehydrogenases, POR enzymes do not utilize either lipoic acid or flavin adenine dinucleotide (FAD) as cofactors, as the oxidative decar­ boxylation reaction involves a free-radical mechanism (30, 4 4 , 5 0 ) . The H. pylori POR was found to be highly oxygen labile, and the presence of this enzyme, along with the related oxygen-labile O O R enzyme (see below), may play an important role in the microaerophily of H. pylori (23, 28). Insertion-inactivation of the porB gene was unsuccessful, implying that POR is essential for the growth of H. pylori (23). It is not known how the POR-reduced flavodoxin

137

is reoxidized in H. pylori, but in cell-free extracts a POR-dependent reduction of NADP (but not NAD) can be observed, which would be consistent with re­ duced flavodoxin reducing NADP via a hypothetical flavodoxin:NADP oxidoreductase (23, 2 8 ) . Alterna­ tively, reoxidation of FldA may involve other electron acceptors, including the electron transport chain. Finally, there is indirect evidence for in vivo expression of POR, through identification in the sera of H. pylori-infected individuals of the immunogenic PorA subunit (36). Citrate synthase Citrate synthase (EC 4.1.3.7) catalyzes the first step in the oxidative branch of the CAC in which acetyl-CoA and oxaloacetate are condensed to generate citrate and CoA. Citrate synthases in other bacteria are encoded by git A, and a single copy of a git A homolog is present in both H. pylori 2 6 6 9 5 (HP0026) and J 9 9 (JHP0022) strains. The purification of the H. py­ lori citrate synthase enzyme has been described (15). In common with other gram-negative citrate synthase enzymes, the native enzyme exists as a hexamer, with an approximate molecular mass of 3 0 0 to 3 4 0 kDa and a subunit size of 50 kDa. However, regulation of the H. pylori enzyme is more characteristic of citrate synthases from gram-positive bacteria and eukaryotes. For example, unlike citrate synthases from gramnegative bacteria, the H. pylori enzyme is not inhib­ ited by NADH (46). This has been related to the role of the CAC in H. pylori being directed toward biosyn­ thesis rather than energy generation. Also, 2-oxoglu­ tarate, succinyl-CoA, AMP, and ADP, which inhibit other bacterial citrate synthases, do not inhibit the H. pylori enzyme. However, ATP was found to act as a strong competitive inhibitor with respect to acetylCoA (46). Aconitase The next step in the oxidative branch of the cycle is carried out by aconitase (EC 4.2.1.3), which cata­ lyzes the reversible isomerization of citrate to generate isocitrate via ds-aconitate. Unlike E. coli, which con­ tains two differentially regulated aconitase enzymes, AcnA and AcnB (12), only a single copy of an acnB homolog has been identified in the H. pylori genomes (HP0779/JHP0716). Aconitase activity has been de­ tected in the cytosolic fraction of H. pylori cells both by N M R and spectrophotometric assays (46). How­ ever, full activity was only detected if the cytosolic fraction was recombined with the cell membrane frac­ tion, indicating that interaction with the cell envelope is required for optimum activity.

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KELLY AND HUGHES

Isocitrate dehydrogenase Isocitrate dehydrogenase (EC 1.1.1.42) catalyzes the NAD(P)-dependent oxidative decarboxylation of isocitrate to generate 2-oxoglutarate and C 0 . In E. coli this enzyme acts as a critical branch point between the CAC reactions and the glyoxylate bypass during growth on C2 compounds like acetate. Regulation is achieved through reversible phosphorylation, leading to inactivation of this enzyme by the AceK protein, an isocitrate dehydrogenase kinase/phosphatase (33). The gene icd (HP0027/JHP0023), encoding isocitrate dehydrogenase, is located immediately downstream of the citrate synthase gene in H. pylori (1, 52). It is unlikely that the H. pylori isocitrate dehydrogenase enzyme acts as a branch point in isocitrate metabolism for two reasons: H. pylori probably lacks a functional glyoxylate bypass (see discussion below), and no ho­ mologs of aceK have been identified in the genome sequence. Pitson et al. (46) have reported that the H. pylori enzyme is NADP-specific and cannot utilize NAD as the electron acceptor, whereas Hoffman et al. (21) detected low levels of NAD-dependent activity. Sigmoidal kinetics with isocitrate and NADP indicates that H. pylori isocitrate dehydrogenase may be sub­ ject to allosteric regulation, and the activity is slightly stimulated by AMP (45). 2

2-Oxoglutarate metabolism The next step in the oxidative branch of the con­ ventional CAC is the oxidative decarboxylation of 2oxoglutarate in the presence of CoA to generate succinyl-CoA with the release of C 0 . This reaction is commonly catalyzed by the 2-oxoglutarate dehydro­ genase multi-enzyme complex. Both enzymatic data ( 1 1 , 2 1 , 2 3 , 4 6 ) and genome sequence analysis (1, 53) indicate that H. pylori lacks this enzyme complex. Instead, oxidation of 2-oxoglutarate is carried out by an oxygen-labile 2-oxoglutarate:acceptor oxidore­ ductase enzyme (23), which shares a number of bio­ chemical and structural features in common with the POR enzyme described above. The O O R enzyme has been purified from H. pylori (23) and is composed of four subunits, OorA (43 kDa), OorB (33 kDa), OorC (21 kDa), and OorD (10 kDa). In the chromosome, the structural genes for the O O R enzyme are located together in the order oorDABC (23) and correspond to gene numbers H P 0 5 8 8 - 0 5 9 1 and J H P 0 5 3 6 - 0 5 3 9 in H. pylori strains 2 6 6 9 5 and J 9 9 , respectively (1, 53). OorB and OorD display sequence motifs for TPP and Fe-S cluster binding in common with POR and related enzymes. Interestingly, however, the pair-wise amino acid identity of the corresponding POR and O O R polypeptides is relatively low ( 2 5 % or less), and 2

in contrast to POR, the OorA and OorB subunits are more similar to the two-subunit puruvate:ferredoxin oxidoreductase of H. halobium than to the four-subunit enzymes commonly found in extremophiles. This strongly suggests independent evolutionary origins for these oxidoreductases in H. pylori. Mutants in oorA could not be generated, suggesting that O O R is essential for H. pylori (23). Corthesy-Theulaz et al. (11) concluded that succinyl-CoA was not generated via CAC reactions in H. pylori owing to the absence of the 2-oxoglutarate dehydrogenase complex, but it is now clear that O O R effectively substitutes for this enzyme. The product of the O O R reaction is succinyl-CoA, and Hughes et al. (23) showed that the activity of the purified enzyme was strictly 2-oxoglutarate and CoA-dependent with K values of 0.3 and 13 u,M, respectively. However, Pitson et al. (46) studied 2-oxoglutarate oxidation in H. pylori cell extracts using H - N M R spectroscopy and reported an FAD or benzyl viologen-dependent 2-oxoglutarate "oxidase" activity not absolutely de­ pendent on, but strongly stimulated by, the addition of CoA in an apparently allosteric manner. Unusual for an allosteric activator, 2-mM CoA increased the apparent K for 2-oxoglutarate and FAD to nonphysiological values (26 and 28 mM, respectively) despite the V also being raised. The product of this reac­ tion was succinate, whereas succinyl-CoA production could not be detected. These results are difficult to reconcile with the properties of purified O O R re­ ported by Hughes et al. (23) but may be due to the use of cell extracts in the work of Pitson et al. (46). m

J

m

max

Interconversion of succinate and SCS SCS catalyzes the sole reaction of the CAC in which a nucleotide triphosphate is generated from the conversion of succinyl-CoA to succinate and CoA. The enzyme can also operate in the reverse direction to generate succinyl-CoA. There is no biochemical evi­ dence for the presence of SCS activity in H. pylori (11, 4 6 ) , and no homologs of the sucCD genes have been identified in the either of the H. pylori genome sequences. The absence of this enzyme would be con­ sistent with other evidence that the CAC in H. pylori consists of a reductive branch ending in succinate and an oxidative branch ending in succinyl-CoA, with no physiological need to connect the two branches. A succinyl-CoA:acetoacetate CoA-transferase (SCOT) enzyme has been identified by Corthesy-Theulaz et al. (11), which in principle could substitute for SCS (10), although this would be dependent on acetoacetate or acetoacetyl-CoA. The role of this enzyme is unclear and is further considered below.

CHAPTER 12 • THE CITRIC ACID CYCLE AND FATTY ACID BIOSYNTHESIS

Fumarate reductase

strongly indicate that the enzyme func­ tions preferentially in the reductive direction, i.e., in the formation of fumarate from malate (fumarate K , 121 mM; V , 4.1 u,mol m i n mg of protein ; malate K , 7.3 mM; V , 10.8 ixmol m i n mg of p r o t e i n ) . Again, this is consistent with fumarase op­ erating in a reductive CAC arm leading to succinate via fumarate reductase. V~max

In E. coli, the interconversion of fumarate and succinate can be carried out by two enzymes: succi­ nate dehydrogenase, which is expressed under aerobic conditions, or fumarate reductase, which is induced under anaerobiosis (25, 51). In the absence of oxygen, fumarate can be used as an alternative terminal elec­ tron acceptor for the proton-translocating electron transport chain and is important in ATP generation in many anaerobic bacteria (32). H. pylori lacks succi­ nate dehydrogenase (1, 4 6 , 53), whereas the presence of a fumarate reductase has been confirmed by bio­ chemical analysis (3, 3 8 , 46) and sequence data (1, 1 7 , 5 3 ) . H. pylori fumarate reductase is closely related to that of the related anaerobe Wolinella succinogenes and is encoded by three genes, frdCAB (HP0193-0191/JHP179-177), encoding polypep­ tides of 27, 8 1 , and 31 kDa, respectively (17). FrdA and FrdB display the amino acid motifs for FAD and Fe-S binding, respectively, in common with other fu­ marate reductases and also succinate dehydrogenases. The frdC gene encodes a hydrophobic, di-heme cyto­ chrome b, which may serve as a membrane anchor for FrdA and B. Like the fumarate reductase of W. succinogenes, activity was localized in the membrane fraction ( 3 , 1 7 ) . Mutants in frdA have been generated, indicating this enzyme is not essential for H. pylori. However, the mutants demonstrated a prolonged lag phase on standard growth medium under microaero­ bic conditions (17). FrdA is also immunogenic, and was recognized in 5 5 % of serum samples from H. pylori-infected individuals (3). Expression of fumar­ ate reductase activity appears to be constitutive in H. pylori, as unlike E. coli, levels of activity did not mark­ edly change in cells grown under varying 0 concen­ trations (13). Also, although the presence of fumarate reductase provides evidence of anaerobic-type respi­ ration, H. pylori has not been successfully cultured under anaerobic conditions, even in the presence of additional fumarate as a terminal electron acceptor. Mendz et al. (40) found that the H. pylori enzyme was inhibited by three known inhibitors of fumarate reductase, morantel, oxantel, and thiabendazole, al­ though high MICs would preclude their use as chemotherapeutic agents. 2

Fumarase

v a u i e s

m

- 1

-1

m a x

- 1

m

m a x

-1

Malate dehydrogenase (NAD-linked) and malate quinone oxidoreductase Only very low specific activities (less than 10 nmol m i n mg of p r o t e i n ) of NADH-dependent malate dehydrogenase were detected in H. pylori by Hoffman et al. (21), but much higher activities (around 100 nmol m i n mg of p r o t e i n assayed in the reductive direction) were measured by Pitson et al. (46). It is difficult to reconcile these values except as strain or assay differences. No homologs of a typi­ cal NAD-MDH have been identified in either of the H. pylori genome sequences, suggesting the enzyme responsible for the activity is very divergent or other­ wise novel. Davison et al. (13) reported the presence in H. pylori of a loosely membrane-bound dye-linked (NAD-independent) malate dehydrogenase activity, and L-malate-dependent cytochrome reduction. The enzyme responsible was predicted by Kelly (28) to be a flavoprotein malateiquinone reductase (Mqo), which functions as an electron donor to the quinone pool, analogous to the better known D-lactate dehydrogen­ ase (41). Kather et al. (27) have confirmed this and have shown that the HP0086 O R F encodes a protein with Mqo activity that is only distantly related to known Mqo enzymes. Why should H. pylori possess two types of malate-oxidizing enzymes? It is likely that these function differentially; the Mqo is involved in the use of malate as a respiratory chain electron donor, whereas the NAD-MDH could function in the opposite (reductive) direction to synthesize malate as part of the reductive C4 branch of the CAC pathway. - 1

-1

- 1

-1

The Anaplerotic Formation of Oxaloacetate If a branched CAC exists in H. pylori, a source of oxaloacetate is needed to fuel both branches. One of the puzzling features of H. pylori carbon metabo­ lism is the apparent lack of anaplerotic C 0 fixation enzymes that could form oxaloacetate from pyruvate or phosphoenolpyruvate (PEP). PEP carboxykinase, PEP carboxylase, and pyruvate carboxylase could not be detected in strain 1 1 6 3 7 by Hughes et al. (22), although very low activities were reported in other strains by Hoffman et al. (21). In view of the fact that 2

Fumarase (EC 4.2.1.2) catalyzes the reversible generation of malate from fumarate, and homologs of the structural gene for a type II fumarase, futnC, have been identified in both of the H. pylori genome sequences (HP1325/JHP1245). Fumarase activity has been investigated by Pitson et al. (46). The K and m

139

140

KELLY AND HUGHES

there are no candidate genes for any of these enzymes in strains 26695 or J 9 9 (the assertion of Cordwell [10] that HP0370 is a pyruvate carboxylase is incorrect; it is a subunit of acetyl-CoA carboxylase), and no biotinylated protein in cell-free extracts of the expected size for a pyruvate carboxylase ( 6 , 2 2 ) , the mechanism of anaplerotic oxaloacetate formation remains un­ clear. However, for the reductive arm of the CAC, a supply of fumarate for fumarate reductase-associated electron transport may be obtained from extracellular aspartate via aspartase (see below).

Other Enzymes Which May Have a Role in the CAC Aspartase The assimilation of amino acids may play an im­ portant role in the provision of biosynthetic interme­ diates and energy conservation in H. pylori in vivo, as the bacterium is capable of growth in defined media supplemented with only amino acids as the sole car­ bon source (39). H. pylori was found to metabolize aspartate rapidly, and the initial product of aspartate metabolism was identified as fumarae (39), indicating a role for aspartase in fumarate formation. Aspartase is encoded by the asp A gene, and homologs have been identified in both genome sequences (HP0649/ JHP594).

Enzymes of the glyoxylate bypass Malate synthase and isocitrate lyase together cat­ alyze the two reactions of the glyoxylate bypass, which is an anaplerotic mechanism for C4 dicarboxylic acid synthesis that specifically operates during growth on C2 compounds as sole carbon source. Ma­ late synthase catalyzes the conversion of glyoxylate and acetyl-CoA to malate and CoA, and is encoded by aceB in E. coli. Although significant malate synthase activity has been detected in H. pylori (21, 4 6 ) , no homologs of aceB have been identified in the genome sequences, possibly suggesting that the H. pylori en­ zyme may have diverged from other malate synthases. Hoffman et al. (21) have reported very low isocitrate lyase activity in some strains whereas Pitson et al. (46) could not detect this enzyme activity either spectrophotometrically or by H - N M R . As no homologs of isocitrate lyase genes have been identified in the ge­ nome sequence, and the phenylhydrazine assay is prone to interference from many carbonyl-containing compounds, it seems unlikely that a functional glyox­ ylate bypass is operative in H. pylori. 1

Conclusions: H. pylori Has a Branched Incomplete CAC Figure 1 shows the likely arrangement of CAC reactions in H. pylori. The evidence supporting the operation of separate reductive (C4) and oxidative (C6) branches can be summarized as follows: (i) the use of fumarate reductase rather than succinate dehy­ drogenase in the C4 branch; (ii) a fumarase with kinet­ ics strongly favoring fumarate formation; (iii) the ab­ sence of SCS; (iv) lack of allosteric inhibition of citrate synthase by NADH, supporting a role in biosynthesis rather than energy conservation; and (v) N M R and oxygen uptake studies with intact cells that show sig­ nificant acetate excretion from pyruvate and insuffi­ cient oxygen uptake during respiration of several sub­ strates for a complete oxidative CAC to be operational. There are also some unusual features of the CAC reactions in H. pylori: (i) the use of the oxy­ gen-labile POR and O O R in place of the correspond­ ing 2-oxoacid dehydrogenases; (ii) the presence of NAD-linked M D H activity and also malate synthase without obvious genes encoding them; (iii) the malate: quinone oxidoreductase, which should not really be considered part of the CAC but as a mechanism by which electrons from malate can be used for respira­ tion; and (iv) the apparent absence of anaplerotic re­ actions for oxaloacetate synthesis. The function of the C6-branch CAC reactions in H. pylori is clearly biased toward biosynthesis of 2-oxoglutarate and succinyl-CoA, the key enzyme being O O R , whereas the C4 branch is concerned with fumarate respiration to allow redox balancing and energy conservation. The key enzyme here is fumarate reductase.

F A T T Y ACID BIOSYNTHESIS IN H. Fatty Acid Composition of H.

PYLORI

pylori

The composition of fatty acids varies signifi­ cantly between different bacteria and can be a useful chemotaxonomic marker to aid identification. The study of the lipid and fatty acid profiles of eight Heli­ cobacter species has revealed some characteristic fea­ tures of the Helicobacter genus (18). Helicobacter species can be differentiated into two groups on the basis of fatty acid profiles. Group A, which contains mostly gastric colonizers including H. pylori, contains a high percentage of tetradecanoic ( 1 4 : 0 ) fatty acids and 19-carbon cyclopropane (19:0cyc) fatty acids, and a low proportion of octadecanoic (18:1) fatty acids. Haque et al. (18) also reported the presence of unusual cholesteryl glucosides in 11 out of 13 Helico­ bacter species studied to date. These lipids may be a useful chemotaxonomic marker to differentiate Heli-

CHAPTER 12 • THE CITRIC ACID CYCLE AND FATTY ACID BIOSYNTHESIS

141

Figure 1. Citric acid cycle and related reactions in H. pylori. Enzymes are denoted by numbers. 1, pyruvate:flavodoxin oxidoreductase; 2, phosphotransacetylase; 3, acetate kinase; 4, citrate synthase; 5, aconitase; 6, isocitrate dehydrogenase; 7, 2-oxoglutarate:acceptor oxidoreductase; 8, succinyl-CoA:acetoacetate CoA transferase; 9, NAD-linked malate dehydrogenase; 10, fumarase; 11, fumarate reductase; 12, maIate:quinone oxidoreductase; 13, aspartase; 14, malate synthase. The mechanisms for anaplerotic oxaloacetate synthesis are unknown (thin dashed line). Fid, flavodoxin; Fd, ferredoxin. Solid lines indicate core CAC reactions, which have been demonstrated by enzyme assay. The thick dashed line for enzyme 8 indicates uncertainty about its physiological role.

cobacter species from the closely related Campylo­ bacter and Wolinella species, which apparently lack cholesteryl glucosides. Three different cholesteryl glu­ cosides were identified in H. pylori, which together comprise approximately 2 5 % of the total lipid con­ tent of the bacterium (20). These glycolipids were found to contain an alpha-glycosidic linkage, which is unusual for natural glycosides, and, furthermore, one was a newly identified phosphate-linked glyco­ side. In common with other gram-negative bacteria, the predominant phospholipids identified in H. pylori were phosphatidylethanolamine, cardiolipin, and phosphatidylglycerol. Phosphatidylserine was also detected, but at lower abundance (20). Biosynthesis of Fatty Acids Indications from the genome sequence Few studies have examined the synthesis of fatty acids in H. pylori, and most of our understanding has come from analysis of the genome sequences (1, 53). H. pylori utilizes the type II or dissociated fatty acid synthesis pathway, typical of most bacteria and plants, in which discrete proteins catalyze individual

steps in the pathway. Table 2 lists the relevant genes in strains 26695 and J 9 9 , and Figure 2 shows the pre­ dicted pathway for fatty acid biosynthesis in H. py­ lori, largely based on the E. coli model for fatty acid biosynthesis (reviewed in reference 4 7 ) . The first committed step of fatty acid biosyn­ thesis, in which malonyl-CoA is generated from acetyl-CoA, is catalyzed by acetyl-CoA carboxylase (ACC) (see below). Malonyl-CoA reacts with the acylcarrier protein (ACP) to generate malonyl-ACP, which is catalyzed by the fabD gene product, malonylCoA:ACP transacylase. Malonyl-ACP is required not only for initiation of fatty acid biosynthesis, but also for each subsequent round of elongation of the fatty acid chain. ACP is a small, soluble protein that plays a critical role in fatty acid biosynthesis in most bacte­ ria by covalently binding the intermediates of the pathway. The structural gene for ACP (acpP) has been identified in both genome sequences. Interest­ ingly, there is an additional ACP homolog found only in H. pylori strain 26695 (HP0962), which encodes a larger protein, extended at the N terminus. To func­ tion in fatty acid biosynthesis, the apo-ACP protein must first be activated by transfer of the 4'-phosphopantotheine from CoA, and this reaction is predicted

142

KELLY AND HUGHES

Table 2. Summary of genes associated with fatty acid synthesis in H. pylori Gene accA accD accC fabE fabD acpP acpS fabF fabH fabG fabZ fabl plsX plsC

cfa

cdsA pssA

psd

Function Acetyl-CoA carboxylase-carboxytransferase subunit Acetyl-CoA carboxylase-carboxytransferase subunit Acetyl-CoA carboxylase-biotin carboxylase subunit Acetyl-CoA carboxylase-biotin carboxy carrier protein Malonyl-CoA:ACP transacylase Acyl carrier protein Holo-acyl carrier protein synthase (3-ketoacyl-acyl carrier protein synthase II P-ketoacyl-acyl carrier protein synthase III 0-ketoacyl-acyl carrier protein reductase (3R)-hydroxymyristoyl-(acyl carrier protein) dehydratase Enoyl-(acyl-carrier-protein) reductase Fatty acid/phospholipid synthesis protein of unknown function l-acyl-glycerol-3-phosphate acyltransferase Cyclopropane fatty acid synthase CDP-diglyceride synthetase Phosphatidylserine synthase Phosphatidylserine decarboxylase

J99 ORF

26695 ORF

0504 0884 1011 1010 0083 0506 0744 0505 0188 0508 1290 0181 0187 1267 0968 0201 0354 1275

0557 0950 0370 0371 0090 0559 (0962") 0808 0558 0202 0561 1376 0195 0201 1348 0416 0215 1071 1357

" This second copyof an ACP-like gene was identified only in H. pylori strain 26695.

to be catalyzed by holo-ACP synthase, encoded by acpS in H. pylori. To initiate fatty acid synthesis, acetoacetyl-ACP is generated from malonyl-ACP, and in E. coli there are three potential pathways leading to the formation of acetoacetyl-ACP (47). First, malonyl-ACP can undergo condensation with acetyl-CoA to generate acetoacetyl-ACP. In E. coli, this reaction is catalyzed by B-ketoacyl-ACP synthase III (FabH), which also catalyzes an alternate pathway in which acetyl-CoA is converted to acetyl-ACP. Acetyl-ACP is then con­ densed with malonyl-ACP to generate acetoacetylACP, which in E. coli is catalyzed by B-ketoacyl-ACP synthase I (FabB) and II (FabF). FabB can also func­ tion to decarboxylate malonyl-ACP to generate ace­ tyl-ACP, which then undergoes condensation with a second malonyl-ACP molecule. In the case of H. py­ lori, fabF and fabH homologs are present, whereas fabB has not been identified. Further biochemical studies are required to confirm the function, and to ascertain the substrates and side reactions associated with these enzymes in H. pylori. Acetoacetyl-ACP is converted to B-hydroxyacylACP by the 3-ketoacyl-ACP reductase enzyme (FabG), and the product of this reaction is then dehy­ drated. In E. coli, this dehydration reaction can be carried out by two enzymes, FabA and FabZ. How­ ever, only FabA catalyzes the isomerization of trans2 decenoyl-ACP to c/s-3-decenoyl-ACP, the interme­ diate for unsaturated fatty acid synthesis in E. coli. A homolog of fabZ is present in the H. pylori genome sequences, whereas fab A has not been identified. It is currently unknown whether the H. pylori FabZ has

a similar role to E. coli FabA, as the branch point between the synthesis of saturated and unsaturated fatty acids. FadR, which has a dual role in E. coli as a positive activator for transcription of fab A and a repressor of fatty acid degradation, has not been iden­ tified in H. pylori. The final step in the cycle is catalyzed by enoylACP reductase (Fabl) to generate acyl-ACP, which can then enter another round of elongation or, alter­ natively, enter the pathway for membrane phospho­ lipid synthesis. The first step of phospholipid biosyn­ thesis is the transfer of the acyl chain to the 1-position of glycerol-3-phosphate, the scaffold for this synthe­ sis, by glycerol-3-phosphate acyltransferase (PlsB) (19). A plsB homolog has not been identified in the H. pylori genome. However, a homolog of the gene encoding PlsC, which catalyzes the transfer of a sec­ ond acyl group to glycerol-3-phosphate, is present. H. pylori also contains a homolog of plsX, which by mutational analysis plays an as yet poorly defined role in phospholipid biosynthesis in E. coli (34). Genes associated with the production of CDP-diglycerol (cdsA), phosphatidylserine (pssA), andphosphatidylethanolamine (psd) from phosphatidic acid have been identified in H. pylori, and Ge and Taylor (16) have found that insertion-inactivation mutants of pssA could not be generated, indicating this enzyme is es­ sential in H. pylori. A homolog of pgsA encoding the enzyme for production of phosphatidylglycerophosphate is present, whereas the genes encoding the sub­ sequent enzymes of this pathway, leading to the pro­ duction of phosphatidylglycerol and cardiolipin (pgp and els, respectively), have not been identified.

CHAPTER 12 • THE CITRIC ACID CYCLE AND FATTY ACID BIOSYNTHESIS

Phospholipid Biosynthesis

143

CARDIOLIPIN

c/s* PHOSPHATIDYLGLYCEROL P9P

PHOSPHATIDYLETHANOLAMINE

>

'psd

PHOSPHATIDYLGLYCEROL PHOSPHATE pgsA V + GLYCEROL-3-PHOSPHATE"'\ ATE\

PHOSPHATIDYLSERINE

X

//

pss + SERINE +SERI

DI CDP-DIGLYCEROL

pIsBVplsX pIsC GLYCEROL-3-PHOSPHATE — » > LYSOPHOSPHATIDIC ACID — — » . T

t'

cdsA

PHOSPHATIDIC ACID

NAD(P)*

•

fabD

HOOC-CH, MALONYL-COA

NAD(P)H + H+

MALONYL-ACP

C

O

A

accA accC accD

fabE

HOOC-CH,-C

fabH/F

S

ACP

CH -C 3

R-CH=CH-C.

CP

ACETYL-COA ^ S C O A

fabH

P

R-C-CH -C^* 2

ACP

O

II

H,0

CH,-C-CH,-C'' fabZffabA*

ACETOACETYL-ACP

fabG

ACP

Initiation

NADPH + H*

R-CHOH-CH,-C' ACP

NADP*

Elongation Figure 2. Predicted pathways for fatty acid and phospholipid biosynthesis in H. pylori. During the initiation phase of fatty acid biosynthesis, acetyl-CoA is carboxylated to generate malonyl-CoA, which is then converted to malonyl-ACP. MalonylACP is also required for each subsequent round of elongation. Several potential pathways for the formation of acetoacetylACP are described in the text; for simplicity, only the condensation of acetyl-CoA and malonyl-ACP by FabH is illustrated. Acetoacetyl-ACP is then used as a substrate for the elongation reactions encoded by fabG, A, I, and F. It is noteworthy that no fabA homolog has been identified in H. pylori, which in E. coli acts as the branch point for unsaturated fatty acid synthesis. The acyl-ACP generated by FabI may enter another round of elongation through condensation with malonyl-ACP or act as a substrate for phospholipid biosynthesis. A homolog of the glycerol-3-phosphate acyltransferase enzyme, encoded by plsB, which catalyzes the first acylation of glycerol-3-phosphate, has not been identified. The function and ORF numbers of the H. pylori genes shown in this diagram are summarized in Table 2. Genes that have not been identified in the genome sequence are identified with an asterisk.

144

KELLY AND HUGHES

As discussed above, H. pylori contains cyclopro­ pane fatty acids, which in other bacteria are generated through modification of membrane phospholipids. This modification involves methylenation of the phos­ pholipid by S-adenosylmethionine and is predicted to be catalyzed by the product of the H. pylori cfa gene. H. pylori also contains unusual cholesteryl glucosides, which are synthesized though the action of UDP-glucose:sterol glucosyl-transferases in other organisms. No sequence similarity to genes encoding these en­ zymes has been identified in H. pylori.

2 6 6 9 5 (HP1045). 3-oxidation of fatty acids then pro­ ceeds in a cyclical fashion, via four enzyme activities, acyl-CoA dehydrogenase, 2-enoyl-CoA hydratase, 3hydroxyacyl-CoA dehydrogenase, and thiolase. Apart from thiolase, no other genes have been identified for fatty acid degradation. H. pylori possesses lipolytic activity, and phospholipase A (35), A (42), and C (54) activities have been detected in different strains. However, only one gene with homology to Ai phos­ pholipase has been identified in the genome sequences of H. pylori 26695 (HP0449) and J 9 9 (JHP451).

Enzymes of Fatty Acid Biosynthesis

Effects of Fatty Acids on Host Cells

ACC H. pylori ACC has been characterized by Burns et al. (6), and the possible role of this enzyme in the capnophilic phenotype of H. pylori is discussed else­ where (see chapter 10). Genome sequence data (1, 53) indicate that H. pylori ACC is typical of other prokaryotic enzymes, in that it is composed of four individual proteins: biotin carboxylase, biotin carboxyl carrier protein, and two subunits constituting the carboxytransferase. N M R spectroscopy has con­ firmed that the end product of the ACC reaction is malonyl-CoA, and that the reaction is reversible (6).

t

Several studies have indicated that H. pylori fatty acids directly cause damage to the host. Both 1 9 : Ocyc and 1 4 : 0 fatty acids may have an antisecretory action in vivo, as at high concentrations they were found to inhibit H + /K( + ) ATPase activity in parietal cells, and exhibit detergent action at the apical parietal cell membrane (2). Also, H. pylori cholesteryl glucosides may damage gastric epithelial cells in vivo, as they demonstrate hemolytic activity in vitro (20). Acknowledgments. Work on H. pylori in Professor Kelly's labora­ tory has been funded by the U.K. Biotechnology and Biological Sciences Research Council, The Wellcome Trust, and Glaxo-Wellcome.

S C O T and thiolase Several enzymes have been identified in H. pylori that may contribute to the production of acetyl-CoA in the bacterium. Corthesy-Theulaz et al. (11) have identified a SCOT enzyme in H. pylori, which cata­ lyzes the reversible transfer of CoA from succinylCoA to acetoacetate to generate acetoacetyl-CoA. H. pylori SCOT is composed of two subunits of molecu­ lar masses of 2 6 kDa (encoded by HP0691/JHP0637) and 2 4 kDa (HP0692/JHP0636). SCOT may play a role in the metabolism of acetoacetate in H. pylori, forming acetyl-CoA from acetoacetyl-CoA through the action of a thiolase enzyme, encoded by fadA (HP0690/JHP0638), which lies immediately up­ stream of the SCOT {scoAB) genes. However, the pre­ cise physiological role of SCOT in H. pylori has not been clearly defined, and a role in succinate/succinylCoA interconversion has also been suggested ( 1 0 , 1 1 ) . Fatty Acid Degradation There is currently little information about the metabolism of lipids by H. pylori. The initial step in the oxidation of fatty acids is catalyzed by acyl-CoA synthetase (AcoE), and the corresponding gene has been identified only in the genome sequence of strain

2

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CHAPTER 12 • THE CITRIC ACID CYCLE AND FATTY ACID BIOSYNTHESIS

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oxidoreductases which mediate electron transport to NADP. /. Bacteriol. 180:1119-1128. Huynen, M. A., T. Dandekar, and P. Bork. 1999. Variation and evolution of the citric-acid cycle: a genomic perspective. Trends Microbiol. 7:281-291. Ingeldew, W. J., and R. K. Poole. 1984. The respiratory chains of Escherichia coli. Microbiol. Rev. 48:222-271. Kaihovaara, P., J. Hook-Nikanne, M. Uusi-Oukari, T. U. Ko­ sunen, and M. Salaspuro. 1998. Flavodoxin-dependent pyru­ vate oxidation, acetate production and metronidazole reduc­ tion by Helicobacter pylori. } . Antimicrob. Chemother. 41: 171-177. Kather, B., K. Stingl, M. E. van der Rest, K. Altendorf, and D. Molenaar. 2000. Another unusual type of citric-acid cycle enzyme in Helicobacter pylori: the malate:quinone oxidore­ ductase. /. Bacteriol. 182:3204-3209. Kelly, D. J . 1998. The physiology and metabolism of the human gastric pathogen Helicobacter pylori. Adv. Microb. Physiol. 40:137-189. Kerscher, L., and D. Oesterhelt. 1981. Purification and proper­ ties of two 2-oxoacid:ferredoxin oxidoreductases from Halobacterium halobium. Eur. J. Biochem. 116:587-594. Kerscher, L., and D. Oesterhelt. 1981. The catalytic mecha­ nism of 2-oxoacid:ferredoxin oxidoreductases from Halobacterium halobium. Eur. J. Biochem. 116:595-600. Knappe, J., and G. Sawers. 1990. A radical-chemical route to acetyl-CoA: the anaerobically induced pyruvate formate-lyase system of Escherichia coli. FEMS Microbiol. Rev. 6:383-398. Kroger, A., V. Geisler, E. Lemma, F. Theis, and R. Lenger. 1992. Bacterial fumarate respiration. Arch. Microbiol. 158: 311-314. LaPorte, D. C , and T. Chung. 1985. A single gene codes for the kinase and phosphatase which regulates isocitrate dehydro­ genase. /. Biol. Chem. 260:15291-15297. Larson, T. J., D. N. Ludtke, and R. M. Bell. 1984. sra-Glycerol3-phosphate auxotrophy of plsB strains of E. coli: evidence that a second mutation, plsX, is required. /. Bacteriol. 160: 711-717. Lichtenberger, L. M., S. L. Hazell, J. J. Ramero, and D. Y. Graham. 1990. Helicobacter pylori hydrolysis of artificial lipid monolayers: insight into a potential mechanism of mucosal injury. Gastroenterology 98:A78. McAtee, C. P., K. E. Fry, and D. E. Berg. 1998. Identification of potential diagnostic and vaccine candidates of Helicobacter pylori by "proteome" technologies. Helicobacter 3:163-169. Meinecke, B., J. Bertram, and G. Gottschalk. 1989. Purifica­ tion and characterization of the pyruvate-ferredoxin oxidore­ ductase from Clostridium acetobutylicum. Arch. Microbiol. 152:244-250. Mendz, G. L., and S. L. Hazell. 1993. Fumarate catabolism in Helicobacter pylori. Biochem. Mol. Biol. Int. 31:325-332. Mendz, G. L., and S. L. Hazell. 1995. Aminoacid utilization by Helicobacter pylori. Int. J. Biochem. Cell. Biol. 27: 1085-1093. Mendz, G. L., S. L. Hazell, and S. Srinivasan. 1995. Fumarate reductase: a target for therapeutic intervention against Helico­ bacter pylori. Arch. Biochem. Biophys. 321:153-159. Narindrasorasak, S., A. H. Goldie, and B. D. Sanwal. 1979. Characteristics and regulation of a phospholipid-activated ma­ late oxidase from Escherichia coli. J. Biol. Chem. 254: 1540-1545. Ottlecz, A., J. J. Romero, S. L. Hazell, D. Y. Graham, and L. M. Lichtenberger. 1993. Phospholipase activity of Helicobacter pylori and its inhibition by bismuth salts. Biochem. Biophys. Res. Commun. 38:2071-2080. Patel, M. S., and T. E. Roche. 1990. Molecular biology and

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Helicobacter pylori: Physiology and Genetics Edited by H. L. T . Mobley, G. L. Mendz, and S. L. Hazell © 2 0 0 1 ASM Press, Washington, D.C.

Chapter 13

Nucleotide Metabolism GEORGE L . MENDZ

De Novo Pyrimidine Nucleotide Synthesis

Nucleotides have a central role in the physiology of organisms as building blocks of nucleic acids, storage of chemical energy, carriers of activated metabolites for biosynthesis, structural moieties of coenzymes, and metabolic regulators. A complete understanding of the nucleotide metabolism of Helicobacter pylori is of fundamental interest to microbiology and also will help in the development of new anti-H. pylori therapies. Owing to the complex and varied interac­ tions of nucleotides present in normal functions, cells have an essential need to maintain tightly regulated pools of these compounds, which will serve to keep nucleotide balance and avoid wasting resources on end products not required by the organism. Pyrimidines and purines are essential for the syn­ thesis of nucleoside triphosphates, which are precur­ sors of nucleic acids. Nucleoside polyphosphates are formed by successive phosphorylations of their mono­ phosphate counterparts. 5-Phospho-a-D-ribosyl-l-pyrophosphate (PRPP) is synthesized from ATP and ribose 5-phosphate by the action of phosphoribosyl pyrophosphate synthetase. This enzyme is encoded by gene HP0742 (prsA) in H. pylori. The ribose 5-phosphate moiety of nucleotides is derived from PRPP in de novo synthesis and in some salvage pathways. Ribonucleoside monophosphates are precursors of deoxyribonucleoside monophosphates and may be synthesized de novo from simple precursors or formed via salvage pathways.

The pathway responsible for the de novo synthe­ sis of UTP and CTP, two precursors of RNA, com­ prises nine enzymes (Fig. 1). The ability of H. pylori to grow in a defined medium without preformed py­ rimidines was studied by successive passage experi­ ments, and the results indicate that the bacterium can grow and replicate by relying exclusively on the de novo biosynthesis of pyrimidine nucleotides (19, 31). The enzyme activities of the de novo pyrimidine syn­ thesis pathway of H. pylori have been identified in situ, and putative genes coding for the corresponding enzymes have been found in its genome (3, 33). Carbamoyl phosphate synthetase Carbamoyl phosphate is a substrate for the first reaction of the pathway and is formed by a synthetase that catalyzes the reaction from glutamine or ammo­ nium, bicarbonate, and ATP. Carbamoyl phosphate forms a metabolic branchpoint for the arginine and the de novo pyrimidine pathways. The incorporation of radioactive carbon atoms from bicarbonate into nucleic acids suggests the presence of a de novo pyrim­ idine nucleotide synthesis pathway in H. pylori (17), although it is possible that this incorporation may take place also via de novo purine biosynthesis. Anal­ ysis of the H. pylori DNA sequence identified the genes HP1237 and HP0919, sharing similarities with the pyrAa gene from Salmonella enterica serovar Cholerasuis and the pyrAb gene from Bacillus caldolyticus (3, 3 3 ) , which encode carbamoyl phosphate synthetases; these findings support the interpretation of the experimental data.

PYRIMIDINE RIBONUCLEOTIDES

Pyrimidine ribonucleotide synthesis in H. pylori was investigated by examining the incorporation of pyrimidine ring precursors and preformed pyrimi­ dines and the activities of enzymes involved in their biosynthetic pathways (17).

Aspartate carbamoyl transferase In the first reaction committed solely to pyrimi­ dine biosynthesis, carbamoyl phosphate is converted

George L. Mendz • School of Biochemistry and Molecular Genetics, The University of New South Wales, Sydney NSW 2052, Australia.

147

148

MENDZ

Glutamine or ammonium

Bicarbonate ^ >

Aspartate

• Carbomoylphosphate ^ »

PyA

• Carbomoylaspartate

pyrB

• Dihydroorotic acid pyr

c pyrD

Orotate

pyrE

pyrG CTP

ndk UTP GMP

-•GDP

Succinyl-AMP

- > AMP

-*• ADP"

GTP

IMP

purB

adk

- > ATP

ndk

Figure 3. De novo synthesis of ATP and GTP. The enzymes encoded by the different genes are guaB, IMP dehydrogenase; guaA, GMP synthase; gmk, GMP kinase; ndk, nucleoside diphosphokinase; purA, adenylosuccinate synthetase; purB, adenylo­ succinate lyase; and adk, adenylate kinase. The nucleotides are IMP, inosine monophosphate; XMP, xanthosine monophos­ phate; GMP, guanosine monophosphate; GDP, guanosine diphosphate; GTP, guanosine triphosphate; AMP, adenosine mono­ phosphate; ADP, adenosine diphosphate; and ATP, adenosine triphosphate. The asterisk denotes a gene not identified in the genome or whose corresponding enzyme activity has not been observed.

154

MENDZ

AdR

GdR

HxdR

deoD*

A

Succinyl-AMP ;

G

Hx

GMP «— XMP :

t

t

ATP

GTP

guaA

Figure 4. Salvage and interconversion of purines. The enzymes en­ coded by the different genes are deoD, purine nucleoside phosphorylase; apt, adenine phosphoribosyl transferase; purB, adenylosuc­ cinate lyase; purA, adenylosuccinate synthetase; guaC, GMP reductase; gpt, guanine phosphoribosyl transferase; guaB, IMP de­ hydrogenase; and guaA, GMP synthase. The purine bases are A, adenine; G, guanine; and Hx, hypoxanthine. Ribonucleosides and deoxyribonucleosides are identified by R and dR, respectively. Nu­ cleotide monophosphates are identified by MP with XMP as xanthosine monophosphate. Nucleotide triphosphates are identified by TP. The asterisk represents a gene whose enzyme activity has not been detected.

APRTase and GPRTase (18), and no orthologous gene for a specific HPRTase was found in the H. py­ lori genome. In E. coli and S. enterica serovar Typhimurium, the genes gpt and hpt encode GPRTase and HPRTase, respectively. However, in H. influenzae, hypoxanthine, xanthine, and guanine are substrates for XGPRTase to synthesize IMP, X M P , and GMP, respectively; and in B. subtilis, the same transferase uses guanine or hypoxanthine (23). Thus, it is possible that the enzyme encoded by the gpt gene in H. pylori is able to utilize both guanine and hypoxanthine. In­ terestingly, there is a relationship between the uptake of adenine, guanine, and hypoxanthine and the activi­ ties of the corresponding phosphoribosyltransferases in H. pylori, suggesting that uptake mechanisms are under the same metabolic controls as the salvage bio­ synthesis pathways, similar to what occurs in £ . coli, S. enterica serovar Typhimurium, and B. subtilis. The significant amounts of purine bases incorporated by H. pylori and the relatively high activities measured

for the transferases indicate that the bacterium can salvage purines efficiently via this pathway. A route for the synthesis of IMP or GMP from inosine or guanosine, respectively, involves the phos­ phorylation of the corresponding nucleoside. In £ . coli and S. enterica serovar Typhimurium, the enzyme catalyzing this step is guanosine kinase encoded by gsk. The absence of guanosine kinase activity (18), and of a gene coding for it (3, 33), indicates that this route is not present in H. pylori. In E. coli and S. enterica serovar Typhimurium, a major pathway for the salvage of purine nucleosides is their degradation to the corresponding base by a purine-nucleoside phosphorylase encoded by deoD, followed by the conversion to a nucleotide monophosphate by the ap­ propriate phosphoribosyltransferases (40) (Fig. 4 ) . No purine-nucleoside phosphorylase activity was de­ tected in H. pylori (18), although gene HP1178 or­ thologous to deoD was identified in its genome (3, 33). This discrepancy may be explained by the rela­ tively high adenine and guanine nucleosidase activi­ ties observed in H. pylori cell extracts, which hy­ drolyze the nucleosides to ribose and the corresponding base and could substitute for purinenucleoside phosphorylase in the hydrolysis of the nu­ cleosides. Nucleosidase activities may have masked phosphorylase activities, since the method employed to assess the presence of the latter enzyme measures its activity at the same time as those of the nucleosi­ dases (18). Alternatively, deoD may not be expressed under the bacterial growth conditions employed in that study. In E. coli and S. enterica serovar Typhimu­ rium, the synthesis of purine-nucleoside phosphoryl­ ase is induced by purine nucleosides in the growth medium with concomitant suppression of the contri­ butions of the de novo pathway to the purine nucleo­ tide pool (40). It is of interest that considerable purine nucleo­ side phosphotransferase activities were measured in H. pylori cell extracts (18). These enzymes phosphorylate adenosine or guanosine to AMP or GMP, re­ spectively, and may constitute an alternative nucleo­ side salvage pathway not found in E. coli, S. enterica serovar Typhimurium, or B. subtilis. No genes encod­ ing these enzymes have been identified in the H. pylori genome (3, 3 3 ) . The significant activities of adenosine nucleosi­ dase and adenine phosphoribosyltransferases, the lower activity of adenosine phosphotransferase, and the lack of adenosine kinase activity suggest that the principal route for adenosine utilization in H. pylori is via the salvage of the purine ring after hydrolysis of adenosine to adenine, and phosphorylation by ade­ nine phosphoribosyltransferases, although some pro­ duction of AMP would also occur by direct phosphor-

CHAPTER 13 • NUCLEOTIDE METABOLISM

is directed to the ribonucleoside triphosphate (rNTP) pools. Nonetheless, the relatively small fraction that is directed to synthesizing deoxyribonucleoside tri­ phosphates (dNTPs) is of fundamental importance for the life of the cell since dNTPs are employed almost exclusively in the biosynthesis of DNA. In most or­ ganisms, the first step specific for deoxyribonucleotide synthesis is catalyzed by ribonucleoside diphos­ phate reductase (rNDPase), which converts all four ribonucleoside diphosphates to the corresponding Tdeoxyribonucleoside diphosphates. In H. pylori, the genes HP0680 and HP0364 have similarities with the nrdA gene from E. coli encoding the a subunit of this enzyme and the nrdB gene from Synechocystis sp., which encodes the B subunit, respectively. The biosynthesis of thymidine triphosphate (dTTP) occurs partly from the dUDP produced by rNDPase and partly from deoxycytidine nucleotides. In E. coli, dTTP synthesis requires four additional steps catalyzed by dUTPase (dUTP —• dUMP) encoded by dut, thymidylate synthase (dUMP —• dTMP) en­ coded by thyA, dTMP kinase (dTMP — dTDP) en­ coded by tmk, and nucleoside diphosphokinase (dTDP — dTTP) encoded by ndk (Fig. 5). In H. pylori DNA, the genes HP0865, HP1474, and HP0198 are orthologous to dut, tmk, and ndk, respectively; but no gene orthologous to thyA has been identified (3, 33). The dTTP salvage pathway described in E. coli requires the presence of uridine phosphorylase or thy­ midine phosphorylase, and thymidine kinase, en­ coded by udp, deoA, and tdk genes, respectively (21).

ylation by adenosine phosphotransferase. Similar mechanisms operate for the utilization of guanosine (18). Thus, the catabolism of ribonucleosides and deoxyribonucleosides in H. pylori appears to follow similar pathways as in E. coli and S. enterica serovar Typhimurium. In these bacteria the nucleosides are not phosphorylated directly but are first degraded to free bases that react with PRPP to yield the corresponding nucleoside monophosphate derivative (40). The high activities observed for adenylate kinase and its ability to phosphorylate AMP using ATP, GTP, or ITP suggest the presence in H. pylori of an effective way of contributing to the balance of intra­ cellular purine nucleotide pools. The absence of de­ tectable hydrolysis of AMP by H. pylori lysates indi­ cates very low activities of AMP hydrolase and AMP nucleosidase, if at all present. The synthesis of coen­ zymes containing the adenylate moiety is an impor­ tant metabolic role of purine nucleotides; thus, trans­ fer of phosphate groups from GTP or ITP to AMP in H. pylori may be a mechanism used by the bacterium to keep the necessary supply of flavin nucleotides, nic­ otinamide nucleotides, and coenzyme A.

BIOSYNTHESIS OF DEOXYRIBONUCLEOTIDES The ratio of RNA to DNA in cells is between 5:1 and 1 0 : 1 , indicating that most of the carbon that flows through the nucleotide biosynthesis pathways

ndk

dATP

nrd NDP

dut dNDP"

155

dCTP —

thyA* "> dUMP

dTMP

| dcd dUMP

tmk

dTDP

ndk 1

dTTP

Figure 5. Biosynthesis of deoxyribonucleotides. The enzymes encoded by the different genes are nrd, ribonucleoside diphos­ phate reductase; ndk, nucleoside diphosphokinase; dcd, dCTP deaminase; dut, deoxyuridine triphosphatase; thyA, thymidylate synthase; tmk, thymidylate kinase; and ndk, nucleoside diphosphokinase. In the nucleotides N can be A, C, and U; dNDP, deoxynucleoside diphosphate; dATP, deoxyadenosine triphosphate; dCTP, deoxycytidine triphosphate; dUTP, deoxyuridine tiphosphate; dUMP, deoxyuridine monophosphate; dTMP, deoxythymidine monophosphate; dTDP, deoxythymidine diphos­ phate; and dTTP, deoxythymidine triphosphate. The asterisk denotes a gene that has not been identified in the genome.

156

MENDZ

No genes sharing similarity with these genes were found in H. pylori (3, 3 3 ) . Alternatively, dTTP may be provided by the action of exodeoxyribonuclease on DNA releasing nucleotide 5'-monophosphates, which can be converted to triphosphates as described above. The gene HP1526 is similar to the lexA gene encoding this enzyme in B. subtilis. The salvage route for pyrimidine deoxynucleotide synthesis in H. pylori was investigated by measur­ ing the incorporation of deoxycytidine and thymidine and the activities of the corresponding kinases (17). It is of interest to note that although the bacterium does not appear to take up either deoxynucleoside in any significant amounts, and the genes encoding thymidine phosphorylase (deoA) and thymidine ki­ nase (tdk) have not been identified in the genome (3, 33), the latter enzyme activity is detected in lysates (17). The role of this activity in H. pylori is unclear, and it is possible that the observed thymidine kinase activity reflects a relaxed specificity of a deoxypurine nucleoside kinase. H. pylori salvages deoxyadenosine but at much lower rates than adenosine, and any incorporation of deoxyguanosine is below the detection levels of the radioactive tracer experimental techniques employed (18). Nucleosidases hydrolyze deoxynucleosides to deoxyribose and the corresponding base, and nucleo­ side phosphotransferases produce deoxynucleoside monophosphates by phosphorylation of deoxy­ nucleosides. Significant deoxyadenosine nucleosidase and phosphotransferase activities, as well as deoxygu­ anosine phosphotransferase activity, but no deoxygu­ anosine nucleosidase activity are measured in H. py­ lori (18). The incorporation of deoxyadenosine by H. pylori, together with the presence of deoxyadenosine phosphotransferase, suggests that phosphorylation of the deoxynucleoside is a pathway for the synthesis of adenosine deoxynucleotides (18). However, the oper­ ation of this salvage mechanism is limited by the pres­ ence of an equally active deoxyadenosine nucleosi­ dase, which would partly reroute the purine base moiety via some of the other biosynthetic pathways. The lack of incorporation of deoxyguanosine and the absence of deoxyguanosine nucleosidase activity sug­ gest that the synthesis of guanosine deoxynucleotides follows different pathways, notwithstanding the deoxyguanosine phosphotransferase activity ob­ served. It may be possible that some of the deoxyri­ bose produced by the action of deoxyadenosine nucleosidase is recycled into the synthesis of guanine deoxyribonucleotides (18). CONCLUSIONS There are only a few studies on the nucleotide metabolism of H. pylori, but together with the infor­

mation derived from analyses of the genome of the bacterium, they have provided a wealth of informa­ tion about the pathways of biosynthesis and degra­ dation of pyrimidine and purine nucleotides, and showed that nucleotide biosynthetic enzymes are po­ tential targets for antimicrobials designed against this organism. H. pylori synthesizes pyrimidine nucleotides de novo and this pathway is essential for the growth and survival of the bacterium. This finding is in agreement with its limited capacity to salvage pyrimidines and the absence from its genome of key enzymes of pyrimi­ dine salvage pathways. H. pylori can survive and pro­ liferate by synthesizing de novo purine nucleotides, but it has yet to be established whether this pathway is essential for the bacterium. The organism incorpo­ rates preformed purines, and pathways for purine sal­ vage are clearly identifiable in its genome. Although these salvage pathways act as energy-saving devices by utilizing the preformed purine ring and the ribose moiety of purine nucleosides, it has yet to be eluci­ dated whether H. pylori can survive by having re­ course only to purine salvage; and there is some evi­ dence that these pathways may not be sufficient to support the integrity and proliferation of the bac­ terium. Studies have demonstrated that targeting the in­ hibition of the de novo pyrimidine pathway presents valuable opportunities for the development of antimi­ crobial agents selective for H. pylori. The potential of the pathways of purine synthesis as therapeutic tar­ gets for antibacterials specific to H. pylori has yet to be explored, and to achieve this it is necessary to ob­ tain a better understanding of the metabolism of pu­ rine nucleotides in the bacterium.

REFERENCES 1. Adair, L. B., and M. E.Jones. 1972. Purification and character­ istics of aspartate transcarbamylase from Pseudomonas flu­ orescens. J. Biol. Chem. 247:2308-2315. 2. Ahonkhai, I., M. Kamekura, and D. J. Kushner. 1989. Effects of salts on the aspartate transcarbamylase of a halophilic bac­ terium, Vibrio costicola. Biochem. Cell Biol. 67:666-669. 3. Aim, R. A., L. S. Ling, D. T. Moir, B. L. King, E. D. Brown, P. C. Doig, D. R. Smith, B. Noonan, B. C. Guild, B. L. dejonge, G. Carmel, P. J. Tummino, A. Caruso, M. Uria-Nickelsen, D. M. Mills, C. Ives, R. Gibson, D. Merberg, S. D. Mills, Q. Jiang, D. E. Taylor, G. F. Vovis, and T. J. Trust. 1999. Genomicsequence comparison of two unrelated isolates of the hu­ man gastric pathogen Helicobacter pylori. Nature 397:176180. 4. Bergh, S. T., and D. R. Evans. 1993. Subunit structure of class A aspartate transcarbamylase from Pseudomonas fluorescens. Proc. Natl. Acad. Sci. USA 90:9818-9822. 5. Bethell, M. R., and M. E. Jones. 1969. Molecular size and

CHAPTER 13 • NUCLEOTIDE METABOLISM

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13.

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20.

21.

22.

feedback-regulation of bacterial aspartate transcarbamylase. Arch. Biochem. Biophys. 134:352-365. Burns, B. P., S. L. Hazell, and G. L. Mendz. 1997. In situ properties of aspartate carbamoyltransferase activity in Heli­ cobacter pylori. Arch. Biochem. Biophys. 347:119-125. Burns, B. P., S. L. Hazell, and G. L. Mendz. 1998. A novel mechanism for resistance to the antimetabolite N-phosphonoacetyl-L-aspartate by Helicobacter pylori. J. Bacteriol. 180: 5574-5579. Burns, B. P., S. L. Hazell, G. L. Mendz, T. Kolesnikow, D. Tillett, and B. A. Neilan. 2000. The Helicobacter pylori pyrB gene encoding aspartate carbamoyltransferase is essential for survival. Arch. Biochem. Biophys. 380:78-84. Chang, T.-Y., and M. E.Jones. 1974. Aspartate transcarbamy­ lase from Streptococcus faecalis. Purification, properties, and nature of an allosteric activator site. Biochemistry 13: 629-638. Chu, C , and T. P. West. 1990. Pyrimidine biosynthetic path­ way of Pseudomonas fluorescens. J. Gen. Microbiol. 136: 875-880. Copeland, R. A., J. Marcinkeviciene, T. S. Haque, L. M. Kopcho, W. Jiang, K. Wang, L. D. Ecret, C. Sizemore, K. A. Amsler, L. Foster, S. Tadesse, A. P. Combs, A. M. Stern, G. L. Trainor, A. Slee, M. J. Rogers, and F. Hobbs. 2000. Helicobacter pyloriselective antibacterials based on inhibition of pyrimidine bio­ synthesis. /. Biol. Chem. 275:33373-33378. Grem, J. L., S. A. King, P. J. O'Dwyer, and B. Leyland-Jones. 1988. Biochemistry and clinical activity of N-(phosphonacetyl)-L-aspartate: a review. Cancer Res. 48:4441-4454. Guy, H. I., and D. R. Evans. 1994. Cloning and expression of the mammalian multifunctional protein CAD in Escherichia coli. Characterization of the recombinant protein and a dele­ tion mutant lacking the major interdomain linker. /. Biol. Chem. 269:23808-23816. Jyssum, S., and K. Jyssum. 1979. Metabolism of pyrimidine bases and nucleosides in Neisseria meningiditis. J. Bacteriol. 138:320-323. Kaneko, T., A. Tanako, S. Sato, H. Kotani, T. Sazuka, N. Miyajima, M. Sugiura, and S. Tabata. 1997. Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC 6803.1. Sequence features in 1 Mb region from map position 64% to 92% of the genome. DNA Res. 2: 153-166. Kenny, M. J., D. McPhail, and M. Shepherdson. 1996. A reap­ praisal of the diversity and class distribution of aspartate transcarbamylases in gram-negative bacteria. Microbiology 142: 1873-1879. Mendz, G. L., B. M. Jimenez, S. L. Hazell, A. M. Gero, and W. J. O'Sullivan. 1994. De novo synthesis of pyrimidine nucleotides by Helicobacter pylori. J. Appl. Bacteriol. 77: 1-8. Mendz, G. L., B. M. Jimenez, S. L. Hazell, A. M. Gero, and W.J. O'Sullivan. 1994. Salvage synthesis of purine nucleotides by Helicobacter pylori. J. Appl. Bacteriol. 77:674-681. Mendz, G. L., A. J. Shepley, and S. L. Hazell. 1996. Survival of Helicobacter pylori by de novo synthesis of pyrimidine nu­ cleotides. Gut Suppl. 39:A73. Mendz, G. L., A. J. Shepley, S. L. Hazell, and M. A. Smith. 1997. Purine metabolism and the microaeropohily of Helico­ bacter pylori. Arch. Microbiol. 168:448-456. Neuhard, J., and R. A. Kelln. 1996. Biosynthesis and conver­ sion of pyrimidines, p. 580-599. In F. C. Neidhart (ed.), Esche­ richia coli and Salmonella: Cellular and Molecular Biology, 2nd ed. American Society for Microbiology, Washington, D.C. Neumann, J., and M. E.Jones. 1964. End-product inhibition of

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aspartate transcarbamylase in various species. Arch. Biochem. Biophys. 104:438-447. Nygaard, P. 1993. Purine and pyrimide salvage pathways, p. 359-378. In A. L. Sonenshein (ed.), Bacillus subtilis and Other Gram-Positive Bacteria: Biochemistry, Physiology and Molec­ ular Genetics. American Society for Microbiology, Washing­ ton, D.C. O'Donovan, G. A., and J. Neuhard. (1970) Pyrimidine metab­ olism in microorganisms. Bacteriol. Rev. 34:278-343. Pragobpol, S., A. M. Gero, C. S. Lee, and W. J. O'Sullivan. 1984. Orotate phosphorybosyltransferase and orotidylate de­ carboxylase from Crithidia luciliae. Subcellular location of the enzymes and evidence for substrate channeling. Arch. Bio­ chem. Biophys. 230:285-293. Purcarea, C , G. Erauso, D. Prieur, and G. Herve. 1994. Aspar­ tate transcarbamylase from the deep-sea hyperthermophile archeon Pyrococcus abyssi: genetic organisation, structure, and expression in Escherichia coli. Microbiology 140: 1967-1975. Quinn, C. L., B. T. Stephenson, and R. L. Switzer. 1991. Func­ tional organisation and nucleotide sequence of the Bacillus subtilis pyrimidine biosynthetic operon. /. Biol. Chem. 266: 9113-9127. Reynolds, D. J., and C.W. Penn. 1994. Characteristics of Heli­ cobacter pylori growth in a defined medium and determination of its amino acid requirements. Microbiology 140:26492656. Roland, K. L., F. E. Powell, and C. L. Turnbough. 1985. Role of translation and attenuation in the control of pyrBI expres­ sion in Escherichia coli. ] . Bacteriol. 163:991-999. Roth, B. 1983. Selective inhibitors of bacterial dihydrofolate reductase: structure-activity relationships, p. 107-127. In G. H. Hitchings (ed.), Inhibition of Folate Metabolism in Chemo­ therapy. Springer-Verlag, Berlin, Germany. Shepley, A. J., G. L. Mendz, and S. L. Hazell. 1995. The essen­ tial role of de novo pyrimidine nucleotide synthesis in Helico­ bacter pylori, abstr. P-58. In Proc. 7th FAOBMB Congress. Australian Society for Biochemistry and Molecular Biology, Sydney, Australia. Swyryd, E. A., S. S. Seaver, and G. R. Stark. 1974. N-(phosphonacetyl)-L-aspartate, a potent transition state analog inhibitor of aspartate transcarbamylase, blocks proliferation of mam­ malian cells in culture. /. Biol. Chem. 249:6945-6950. Tomb, J. F., O. White, A. R. Kerlavage, R. A. Clayton, G. G. Sutton, R. D. Fleishmann, K. A. Ketchum, H. P. Kienk, S. Gill, B. A. Dougherty, K. Nelson, J. Quackenbuch, L. Zhou, E. F. Kirkness, S. Peterson, B. Loftus, D. Richardson, R. Dodson, H. G. Khalk, A. Glodek, K. McKenney, L. M. Fitzgerald, M. Lee, M. D. Adams, E. K. Hickey, D. E. Berg, I. D. Gocayne, C. Fujii, C. Bowman, L. Watthey, E. Wallin, W. S. Hayes, M. Borodovsky, P. D. Karp, H. O. Smith, C. M. Fraser, and J. C. Venter. 1997. The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature 388:539-547. Traut, T. W., and M. E. Jones. 1977. Inhibitors of orotate phosphoribosyl-transferase and orotidine-5'-decarboxylase from mouse Ehrlich ascites cells: a procedure for analyzing the inhibition of a multi-enzyme complex. Biochem. Pharmacol. 26:2291-2296. Turner, R. J., Y. Lu, and R. L. Switzer. 1994. Regulation of the Bacillus subtilis pyrimidine biosynthetic (pyr) gene cluster by an autogenous transcriptional attenuation mechanism. /. Bacteriol. 176:3708-3722. Umezu, K., T. Amaya, A. Yoshimoto, and K. Tomita. 1971. Purification and properties of orotidine-5'-phosphate pyrophosphorylase and orotidine-5'-phosphate decarboxylase from baker's yeast. /. Biochem. (Tokyo) 70:249-262.

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37. Wheeler, P. R. 1990. Recent research into the physiology of Mycobacterium leprae. Adv. Microb. Physiol. 31:71-124. 38. Wild, J. R., and W. L. Belser. 1977. Pyrimidine biosynthesis in Serratia marcescens: a possible role for nonsequential enzyme interactions in mimicking coordinate gene expression. Bio­ chem. Genet. 15:157-172. 39. Wild, J. R., and W. L. Belser. 1977. Pyrimidine biosynthesis

in Serratia marcescens: polypeptide interactions of three nonse­ quential enzymes. Biochem. Genet. 15:173-180. 40. Zalkin, H., and P. Nygaard. 1996. Biosynthesis of purine nucleotides, p. 561-579. In F. C. Neidhart (ed.), Escherichia coli and Salmonella: Cellular and Molecular Biology, 2nd ed. American Society for Microbiology, Washington, D.C.

Helicobacter pylori: Physiology and Genetics Edited by H . L . T. Mobley, G. L. Mendz, and S. L . Hazell © 2 0 0 1 ASM Press, Washington, D.C.

Chapter 14

Biosynthetic Pathways Related to Cell Structure and Function PARTHA KRISHNAMURTHY, SUHAS H . PHADNIS, CINDY R . DELONEY, RAOUL S. ROSENTHAL, AND BRUCE E . DUNN

present as a nonreducing 1,6 anhydro sugar. Together the B l —* 4 linked amino sugars and the amide-linked short peptide constitute a monomer and form the basic muropeptide repeating unit of the murein, the specific PG of bacterial cell walls. Between different bacteria the glycan strands exhibit only few structural variations such as O-acetylation or de-N-acetylation of either amino sugar, but the structural diversity of the peptide moiety is substantial, particularly the interpeptide bridges that cross-link peptides from differ­ ent glycan strands (62, 6 4 , 6 5 ) . A unique feature of the peptide backbone of all PG is the alternating se­ quence of optical isomers (L-D-L-D amino acids). Adja­ cent PG strands are generally cross-linked to each other through the peptide side chains by transpeptidation between the carboxyl group of the D-alanine in position 4 of one peptide and the free amino group of di-aminopimelic acid (DAP) in the adjacent strand. These peptide cross-linking bonds between amino acids located on adjacent glycan chains lead to the formation of cross-linked dimers (peptide cross-link­ ing between two monomers on adjacent glycan strands), trimers, and tetramers. In Escherichia coli, a significant percentage of the peptide cross-links do not involve D-alanine-DAP residues, but involve DAPDAP residues of neighboring chains; the relative num­ ber of both types of cross-links depends on the growth phase of the bacterium ( 2 9 - 3 1 ) . Analysis of the muropeptide structures in the PG of E. coli by pulse-chase labeling experiments depict changes in the muropeptide composition as the bacte­ ria reach the stationary phase (4, 10, 3 0 , 6 1 ) . These muropeptide changes include a decrease in pentapeptide side chains, increase in tripeptide side chains, increase in the extent of cross-linking, and decrease

In vivo Helicobacter pylori has a tightly spiraled shape, but in vitro it grows as curved rods, which, after prolonged incubation, evolve into metabolically active but nonculturable coccoid forms (2, 6, 8, 5 7 ) . The spiral morphology of H. pylori appears to confer an advantage to the bacterium in the viscous gastric mucus, since its spiral forms move more effectively in media with high viscosity than more conventional rod-shaped organisms (40). Motility is an essential virulence factor in H. pylori, as nonmotile variants do not colonize the gastric surface in the gnotobiotic piglet animal model (18). Further, the phenomenon of nonculturable but viable coccoid forms of H. pylori may explain the paradox of H. pylori spread via the fecal-oral route in the absence of culturable bacteria from fecal specimens (2, 8). The peptidoglycan of H. pylori, which forms the backbone of the cell wall, probably plays a significant role in maintaining the spiral morphology and in the morphological transfor­ mation of H. pylori from spiral to coccoid forms.

BACTERIAL PEPTIDOGLYCANS Peptidoglycans are the stress-bearing structures of bacteria that maintain the integrity of the cell wall and the shape of the bacterium (28, 4 2 ) . The typical bacterial peptidoglycan (PG) is a heteropolymer of glycan strands made up of the amino sugars N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) linked by B l -+ 4 glycosidic bonds (9, 2 8 , 73). Attached to the carboxyl group of each muramic acid by an amide linkage is the short peptide, L-alanylD-glutamyl-L-meso-diaminopimelyl-D-alanyl-D-alanine (12, 66). The MurNAc at the end of each strand is

Partha Krishnamurthy • Department of Pathology, Medical College of Wisconsin, Milwaukee, WI 53226. Suhas H. Phadnis and Bruce E. Dunn • Department of Pathology, Medical College of Wisconsin, Milwaukee, WI 53226, and Pathology and Laboratory Medicine Service, Department of Veterans Affairs Medical Center, Milwaukee, WI 53295-1000. Cindy R. DeLoney • Division of Biomedical Sciences, University of California, Riverside, CA 92521. Raoul S. Rosenthal • Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN 46202.

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in the average chain length of the glycan strands. Not only are the covalent bonds cleaved during the transi­ tion to the stationary stage, but there is also release of muropeptides from the PG matrix (36, 3 7 ) .

GicNAc

MurNAC 1

L-Ala |

D-Glu |

H. pylori

Peptidoglycan

H. pylori murein has been isolated and character­ ized by Costa et al. (13) and Krishnamurthy et al. (51). High-performance liquid chromatograms of PG fragments obtained by muramidase digestion of H. pylori murein show a relatively small number of peaks, indicating a simple PG structure. The disaccharide backbone of H. pylori PG consists of GlcNAc B l —• 4 linked to MurNAc acid. Adjacent glycan strands are cross-linked via traditional DAP-D-alanine pep­ tide cross bridges. Unlike many gram-negative bacte­ ria, the murein of H. pylori possessed no detectable DAP-DAP cross-links. Monomers (64.8%) and dimers (35.1%) constitute virtually all of the muropep­ tides of H. pylori, and its PG lacks detectable muro­ peptide trimers and tetramers (Table 1). The major muropeptide monomer is pentapeptide. The ratio of nonreducing 1,6-anhydromuramic acid residues at the end of the glycan chains to reducing muramic acid residues is 1:11, suggesting a very low glycan chain length (Table 1). The only amino acids detected by tandem mass spectroscopic analysis are Ala, Glu, Gly, and DAP, linked to the muramic acid moiety in the order L-alanine, D-glutamic acid, DAP, D-alanine, and D-alanine (13, 5 1 ) . H. pylori shows significant variation in murein composition between early log phase (24 h) and late log phase/stationary phase (96 h) cultures (51), sug­ gesting alteration in PG composition as the bacteria change from a helical to a coccoid morphology (13)

Table 1. Structure of peptidoglycan isolated from H.

Strain

NCTC 11637 HP 84-183 HP SPl NCTC 11637 MC 4100

Stage of growth

Early log phase Early log phase Early log phase Late log phase

pylori

Tripeptide

3.0 2.5 0.5 19.0 3.0

8.0 8.2 21.9 3.0 18.6

- AnMurNAc i 1 L-Ala |

D-Ala | D-Ala 11 DAP

D-Glu |

D-Ala DAP DAP 1 1 1 1 1 j • D-Ala DAP D-Ala 1 1 1 D-Glu D-Glu D-Ala D-Ala 1 1 L-Ala L-Ala 1 1 1 GlcNAc - GlcNAc MurNAc MurNAc Figure 1. General composition and age changes in H. pylori PG. As H. pylori cells age from early to late log phase, the composition of their PG changes. The shaded region represents the amino acids that are removed from the PG in late log phase, essentially resulting in a PG with increased dipeptides.

(Fig. 1). Muropeptides with a pentapeptide side chain are prevalent throughout the growth phase (13). However, the proportion of Gly-containing muropep­ tides doubles rapidly when cells go into the stationary growth phase (13). There is an overall decrease in monomers as the bacteria age, while the percentage of dimers, "anhydro" residues, and dipeptide monomers increases. Unique Features of H. pylori

Peptidoglycan

The structure of H. pylori PG has several unique features as shown in Table 1. The degree of crosslinking that reflects the percentage of the total number of DAP residues that are engaged in cross-linking pep­ tide bonds is defined as 0.5 X percent dimers + 0.667 X percent trimers + 0.8 X percent tetramers. In H.

strains SPl, 84-183, NCTC 11637 and

Peptide composition (% of total) Di­ peptide

- GlcNAC

1

PG fragments (% of total)

Tetrapeptide

Penta­ peptide

Mono­ mers

Dimers

31.0 36.2 26.5 31.0 77.0

58.0 53.1 51.1 47.0 14.0

73.0 64.1 64.8 68.0 58.4

27.0 35.8 35.1 32.0 39.5

E. coli

MC 4100

% Crosslinking"

Average glycan chain length

13.5 17.9 ± 0.4 17.6 ± 0.5 16.0 21.3 ± 1.0

7.0 8.5 ± 0.3 11.2 ± 0.5 7.0 34.6 ± 0.5

6

" Percent cross-linking = 0.5 X % dimers + 0 . 6 6 7 X % trimers + 0.8 X % tetramers. Since H. pylori does not possess trimers or tetramers, the degree of cross-linking in H. pylori PG is the % dimers X 0 . 5 . * Average chain length is determined from the ratio o f PG components containing 1,6-anhydromurmic acid residues to components containing reducing muramic acid residues.

CHAPTER 14

pylori murein it is 1 7 % , one of the lowest degrees of cross-linking identified to date. For comparison, the percent cross-linking of E. coli PG is 25 to 3 0 % (69, 73); Neisseria spp., 36 to 4 4 % (63); Vibrio spp., 3 0 % (46, 74); Proteus spp., 33 to 3 7 % (47); Moraxella spp., 3 7 % (54); and Pseudomonas spp., 25 to 4 5 % (21, 4 1 , 54). H. pylori PG also has an unusually high proportion of glycan chain terminating "anhydro"muropeptides, consistent with a very short glycan chain length of 9 to 11 disaccharides, one of the low­ est average lengths so far reported. In comparison, E. coli PG has an average glycan chain length of 21 disaccharide units (39). The lack of detectable trimers or tetramers in H. pylori murein indicates that crosslinking does not occur between three or more glycan chains, whereas cross-linking between three or four glycan chains is detectable in E. coli (32). No DAPDAP cross bridges were identified in H. pylori in con­ trast to E. coli. The exact function of DAP-DAP cross­ links found in E. coli is unclear, but it has been suggested that they perform unique functions since lipoprotein is found attached to muropeptide dimers with these cross-links, and not to DAP-D-Ala linked dimers (23). The absence of L - D - D A P - D A P bond sug­ gests that, unlike E. coli (28), H. pylori has no alterna­ tive mechanism to allow the formation of peptide bridges, other than the typical DAP-D-Ala cross-link­ ing. Finally, the presence of pentapeptide as the main fraction of H. pylori muropeptides indicates that it possesses little carboxypeptidase activity. The genome sequence of H. pylori supports this last conclusion (1, 70). Significance of Unique Characteristics of H. pylori Peptidoglycan In vivo H. pylori has tight spiral morphology dur­ ing log growth phase, and a coccoid form in the late

• BIOSYNTHETIC PATHWAYS

161

log phase of growth (7). In other spiral bacteria in­ cluding leptospirae, borreliae, treponemas, and Campylobacters, the coccoid forms are considered to be degenerate forms (48). However, as the mode of transmission of H. pylori remains unresolved, it has been suggested that coccoid forms are nonculturable but viable organisms that form part of a complex life cycle of the bacterium (53). Further studies are re­ quired to understand the pathological significance of low glycan chain length and the low percentage of cross-link seen in H. pylori PG. However, by compari­ son with the strong and tight murein from organisms such as Staphylococcus spp., which have nearly 9 0 % cross-linking, it is possible to hypothesize that the short glycan chain length and low peptide cross-link­ ing of H. pylori might form a PG that is weakly and loosely held together, enabling the organism to change its morphology from spiral to coccoid to spiral forms much more efficiently than other spiral organ­ isms.

BIOSYNTHETIC PATHWAY OF PEPTIDOGLYCAN

FORMATION

On the basis of protein sequence homologies, the genome of H. pylori appears to code for homologs of all the enzymes involved in the cytoplasmic synthesis of the disaccharide pentapeptide, starting with the synthesis of UDP-N-acetylmuramic acid and finishing with UDP-disaccharide pentapeptide linked to an undecaprenyl lipid carrier (Table 2 ) . The second step in building the murein sacculus is the polymerization reaction, accompanied by the insertion of the newly made material into the existing PG layer. This func­ tion is carried out by the penicillin-binding proteins ( 2 4 , 2 6 , 33). Genes coding for three penicillin-binding

Table 2. List of H. pylori 26695 putative genes that are involved in PG biosynthesis Locus

Enzyme activity*

HP0738 HP0549 HP0493 HP0743 HP1372 HP1373 HP1155 HP0740 HP1494 HP1418 HP0648

D-alanine:D-alanine ligase A (DdlA) Glutamate racemase (Glr) Phospho-N-acetylmuramyl-pentapeptide transferase (MraY) MreB MreC MreB Transferase, peptidoglycan synthesis (MurG) UDP-MurNAc-pentapeptide presynthetase (MurF) UDP-MurNAc-tripeptide synthetase (MurE) UDP-N-acetylenolpyruvoylglucosamine reductase (MurB) UDP-N-acetylglucosamine enolpyruvyl transferase (MurZ)

HP0623 HP0494

UDP-N-acetylmuramate-alanine ligase (MurC) UDP-N-acetylmuramoylalanine-D-glutamate ligase (MurD)

" Enzyme activity and putative biosynthetic function based on sequence homology.

Proposed biochemical function D-alanyl-D-alanine synthesis Conversion of L-glutamic acid to D-glutamic acid Lipid-linked MurNAc-pentapeptide synthesis Rod shape-determining protein Rod shape-determining protein Rod shape-determining protein Lipid-linked GlcNAc-MurNAc-pentapeptide synthesis UDP-MurNAc-pentapeptide synthesis UDP-MurNAc-tripeptide synthesis Catalyzes the two-step synthesis of UDP-MurNAc Involved in the formation of lipid intermediate II from lipid intermediate I and UDP-GlcNAc Catalyzes the addition of L-Ala to UDP-MurNAc UDP-MurNAc-dipeptide synthesis

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Table 3. H. pylori 26695 ORFs that show sequence motifs of PBPs" Name

ORF

Mol mass (kDa)

Proposed function

PBP1 PBP2 PBP3 PBP160 PBP211

HP0597 HP1565 HP1556 HP0160 HP0211

74.3 66.8 69.5 34.1 27.4

Transglycosylase/transpeptidase Transpeptidase Transpeptidase Not known Not known

" The classification and proposed function of those that show sequence ho­ mology to known PBPs is given. ORFs H P 0 1 6 0 and H P 0 2 1 1 do not show homology to proteins deposited in GenBank.

proteins have been identified in the genome of H. py­ lori, based on comparisons of encoded amino acid sequences (Table 3 ) . Some gram-negative organisms exhibit murein turnover. In this process substantial amounts of PG fragments are released from the murein sacculus, and some of the soluble muropeptides are reutilized for PG assembly (34, 3 5 ) . In E. coli, lytic transglycosylases and endopeptidases are responsible for releasing 1,6-anhydromuropeptides in the periplasm, and the turnover products are reutilized for PG assembly after uptake into the cytoplasm via active transport systems (35). From the analysis of the H. pylori genome, it is uncertain if murein fragments are recycled by H. py­ lori. The bacterium has orthologs of the genes sit and amiA, which encode a lytic transglycosylase and an N-acetylmuramyl-L-alanine amidase, respectively (1, 70). However, other genes coding for enzymes of the recycling pathway of murein turnover products have not been identified; for example, ampG, encoding a membrane protein required for uptake of intact muro­ peptides; ampD, coding for a cytoplasmic amidase with specificity for the 1,6-anhydromuramyl-L-Ala bond; and mpl, a specific tripeptide ligase, which forms UDP MurNAc peptides (16, 4 2 ) . Pem^illin-Binding Proteins and Other Related Proteins Penicillin-binding proteins (PBPs) are specialized acyl serine transferases involved in the assembly, maintenance, and regulation of the features of the PG structure. Most of these proteins are anchored in the bacterial inner membrane, with their active sites ac­ cessible in the periplasmic space. On the basis of size, the PBPs identified to date have been classified into the broad categories of low-molecular-weight and highmolecular-weight proteins (25). In general, high-mo­ lecular-weight PBPs have transglycosylase and/or transpeptidase activities, and low-molecular-weight PBPs have carboxypeptidase and/or endopeptidase activities (24, 2 7 , 3 3 ) ; some of the low-molecular-

weight PBPs also display transpeptidase activity (24, 25). Although PBPs fulfill different functions in PG biosynthesis, their catalytic centers have a remarkably well-conserved topology defined by three amino acid groupings, referred to as "motifs" (33). These motifs occur in the same order and with roughly the same spacing along the polypeptide chains, defining com­ mon amino acid sequence signatures, and polypeptide folding brings the three motifs close to each other (33, 67). Motif 1 is S X X K , where " S " is the essential serine residue and " X " is a variable amino acid residue; motif 2 is S/YXN/C, and defines one side of the cata­ lytic center; and motif 3 is [K,H][T,S]G, and defines the other side of the catalytic center (25, 3 3 , 6 7 ) . H. pylori Penicillin-Binding Proteins Most experimental studies to date on the PBPs of H. pylori have been conducted using labeled Blactams, which form a covalent bond with these pro­ teins. PBPs in H. pylori were first reported by Ikeda et al., who identified three proteins using [ C ] peni­ cillin G (44). More recently eight additional PBPs have been identified in H. pylori employing penicillin deriv­ atives linked to various reporter groups (14, 17, 3 8 , 52). Altogether, the combined data from the above studies indicate the presence of 11 PBPs in H. pylori ranging in molecular mass from 2 8 to 100 kDa. In the genomes of H. pylori strains 2 6 6 9 5 and J 9 9 , the genes encoding five of these PBPs have been identified and correspond to the open reading frames (ORFs) HP0597/JHP544, HP1565/JHP1473, HP1556/JHP1464, HP0160/JHP0148, and HP0211/ JHP0197 given in Table 3 ( 1 , 1 6 , 70). ORFs HP0597/ JHP544, HP1565/JHP1473, HP1556/JHP1464 rep­ resent high molecular weight proteins and show se­ quence homology to PBPs from other bacteria. They are designated PBP1, PBP2, and PBP3, respectively (Table 3 ) . ORFs HP0160/JHP0148 and HP0211/ JHP0197 represent low-molecular-weight proteins and do not show sequence homology to any known proteins ( 1 1 , 5 2 ) . The gene sequences encoding the other six putative PBPs have not been identified (14, 38). 14

Proposed Functional Roles Known monofunctional PBPs possess transpepti­ dase, carboxypeptidase, or endopeptidase activity, and multimodular PBPs have transglycosylase/transpeptidase or carboxypeptidase/endopeptidase activi­ ties (22, 2 7 , 5 6 , 67, 6 8 ) . H. pylori PBP1, PBP2, and PBP3 display sequence similarities with biosynthetic multimodular PBPs from other bacteria, suggesting that they carry out transglycosylation and/or trans-

CHAPTER 14 • BIOSYNTHETIC PATHWAYS

peptidation reactions. Hierarchical analysis of the amino acid sequences of 63 multimodular PBPs indi­ cated a close similarity between H. pylori PBP1 and £ . coli PBPlb, suggesting that the H. pylori enzyme has transglycosylase/transpeptidase activities (33). This analysis also revealed relationships between H. pylori PBP3 and E. coli PBP2, an enzyme involved in cell wall expansion and maintenance of cell shape, and H. pylori PBP3 and E. coli PBP3, which is involved in septum formation in the latter bacterium (33). Protein sequences deduced from the H. pylori ge­ nome do not show homology to enzymes with carboxypeptidase activity ( 1 , 7 0 ) . This finding is in agree­ ment with the characteristics of H. pylori murein. The very high percentage of disaccharide pentapeptide is consistent with low carboxypeptidase activity, which would remove the terminal D-Ala from the penta­ peptide chain. In the H. pylori genome there are no genes ortholog to those found in other organisms en­ coding PBP endopeptidases capable of cleaving mu­ rein crosslinks (22, 2 8 , 3 3 ) ; however, this activity is essential for the growth of H. pylori PG. This type of endopeptidase need not be a PBP; for example, in E. coli, the MepA protein cleaves PG cross-links. Analy­ ses of the H. pylori genome sequences from two differ­ ent strains reveal no proteins similar to E. coli MepA. It is possible that any of the multimodular PBPs out­ lined above could function as an endopeptidase. In addition, H. pylori shows accumulation of glucosaminyl-muramyl-dipeptide, indicating the presence of an enzyme capable of cleaving the L-D bond between DAP and D-Glu. Unique Features of the Newly Identified HP160 and HP211 Neither HP160 nor HP211 shows sequence ho­ mology to known proteins, and both of these proteins are rich in cysteine (5%) (11, 52). HP160 shows regu­ lated expression; the protein is found in both the membrane and soluble fractions and has a unique ar­ rangement of penicillin-binding motifs (52). Muta­ tional analysis of HP 160 suggests that the gene prod­ uct is essential for the growth and survival of H. pylori since it was not possible to obtain viable null mutants (50). HP211 has been shown to bind and hydrolyze penicillin derivatives, suggesting that this protein is a B-lactamase (55). Further studies are needed to under­ stand the role of these PBPs in H. pylori PG biosyn­ thesis. Amoxicillin and Aztreonam Binding Characteristics of H. pylori Penicillin-Binding Proteins Amoxicillin resistance was characterized initially by Dore et al. (17) in clinical isolates in which a PBP

163

with a molecular mass of 30 to 32 kDa could no longer be detected in amoxicillin-resistant strains (MIC, 16 to 32 |xg/ml). In these studies amoxicillin resistance was found to be unstable upon freezing and subculturing, but the resistance could be "rescued" in some strains by plating the bacteria on amoxicillin gradient plates. More recently DeLoney and Schiller reported preferential binding of amoxicillin to PBP3 (14, 15) whereas Harris et al. (38) reported preferen­ tial binding of amoxicillin to a 72-kDa PBP. DeLoney and Schiller also characterized amoxicillin resistance in H. pylori as arising from an alteration in the affinity of PBP1 for B-lactam antibiotics (15). In these studies resistance was found to be stable upon freezing and subculturing. Kusters et al. (unpublished results) also found an alteration in PBP1 from a clinical isolate with a stable amoxicillin MIC of 8 u,g/ml. Further studies will serve to establish whether amoxicillin re­ sistance in H. pylori can be transferred between strains. Aztreonam at sub-MIC induces filamentation in H. pylori (14). DeLoney and Schiller reported aztreo­ nam to bind preferentially to a PBP with a molecular mass of 63 kDa (14). In E. coli aztreonam has been shown to bind to PBP3, a transpeptidase that is spe­ cific for septum formation and cell division (45). Since genetic analysis shows 3 1 % homology between H. pylori and E. coli PBP3, it was concluded that the H. pylori enzyme may be involved in septum formation in this bacterium, in agreement with the conclusions of the hierarchical analysis of modular PBP. Genetic studies will further elucidate the role of PBP (molecu­ lar mass, 63 kDa) in H. pylori. Other Proteins with Putative Murein-Related Functions In the genome of H. pylori ORFs HP0772/ JHP0709 and HP0645/JHP0590 code for enzymes homologous to an amidase and a lytic transglycosylase, respectively (1, 7 0 ) . These are not penicillinbinding proteins but are known to perform mureinrelated functions. N-acetylmuramyl-L-alanine ami­ dases specifically cleave the amide bond between the lactyl group of muramic acid and the a-amino group of D-alanine, the first amino acid of the stem peptide (71, 72). Lytic transglycosylases catalyze the transfer of the glycosyl bond onto the hydroxyl group of the carbon 6 of the same muramic acid, thereby catalyz­ ing an intramolecular glycosyl transferase reaction and forming 1,6-anhydromuramic acid (3, 19, 4 3 ) . The functions of these non-PBPs in H. pylori have not been characterized yet. However, preliminary results from mutational analysis suggest that these gene products are essential for the growth and survival of H. pylori (49).

164

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CONCLUSIONS H. pylori colonizes a hostile gastric environment where few other organisms can survive. Its prolonged presence in this milieu is associated with gastric dis­ eases such as gastritis, ulcers, and gastric cancer (5, 2 0 , 5 8 - 6 0 ) . One of the basic features that may aid the bacterium to colonize this niche is a unique PG with a low level of cross-linking, short glycan chain length, and lack of trimers and tetramers. These fea­ tures suggest that the murein of H. pylori is loosely constructed, and thus readily susceptible to altera­ tions enabling the organism to change its morphology according to environmental needs and, in particular, to the process of colonizing new subjects. Analysis of the H. pylori genome shows the pres­ ence of genes coding for all the enzymes of the biosyn­ thetic pathway leading to the disaccharide penta­ peptide, which is the basic building block of murein. Also identified are genes encoding the putative murein synthases PBP1, PBP2, and PBP3, which exhibit strong sequence similarities to penicillin-binding pro­ teins encoding transglycosylase and transpeptidase domains from other bacteria, including E. coli, Haemophilus influenzae, and Bacillus subtilis, and which are involved in the construction of the murein sacculus, maintenance of cell shape, and cell division. There is some experimental evidence that indeed these are their functions in H. pylori. Regarding murein hydrolases required for mu­ rein turnover and recycling, as well as for cell prolifer­ ation, the data obtained from genome analysis are much more fragmentary. Only a few ortholog genes to those present in other bacteria have been found, and their functions in H. pylori remain to be charac­ terized. Several other proteins able to bind labeled B-lactams have been detected in H. pylori, but nothing is known yet about their identity and further studies are required to understand the function of these putative PBPs.

4.

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CHAPTER 14 • BIOSYNTHETIC PATHWAYS

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Helicobacter pylori: Physiology and Genetics Edited by H . L. T. Mobley, G. L. Mendz, and S. L. Hazell © 2 0 0 1 ASM Press, Washington, D.C.

Chapter 15

Evasion of the Toxic Effects of Oxygen STUART L . HAZELL, ANDREW G . HARRIS, AND MARK A . TREND

O X Y G E N AS A T O X I C SPECIES

Oxygen is an efficient terminal electron acceptor in respiratory pathways. During aerobic respiration the electron transport chain generates free radical ox­ ygen species as a result of electron leakage; this gener­ ation of toxic species is proportional to the oxygen tension (51). In addition, toxic oxygen species (TOS) may be formed exogenously, for example, by chemi­ cal processes or through radiation. TOS also result from the oxidative burst of polymorphonuclear leu­ kocytes (PMN). Infection with Helicobacter pylori in­ duces an inflammatory response (gastritis), which leads to an increase in the level of TOS in the gastric mucosa and the gastric juice (4, 2 4 - 2 6 , 59). This in­ crease in the level of toxic metabolites is probably the result of the generation of the superoxide anion ( O 2 ' ) , a reactive T O S , formed as part of the oxida­ tive burst of PMN and enzymic activities of gastric epithelial cells. There is evidence that H. pylori infec­ tion leads to increased production of 0 ' via NADPH oxidase in gastric cells, stimulated by lipo­ polysaccharide as well as xanthine oxidase, another mechanism for the generation of oxygen-derived free radicals (8, 80). In response to increased superoxide anion production in gastric tissue, changes have been detected in the level of expression of human superox­ ide dismutase (SOD) (12). Human gastric SOD exists as a cytoplasmic copper-zinc-superoxide dismutase (Cu, Zn-SOD) found in gland cells of the gastric body and antral mucosa, and as a manganese-superoxide dismutase (Mn-SOD) within mitochondria (63). An increase in the amount and activity of Mn-SOD has been observed in response to H. pylori infection and gastritis, whereas the amount and activity of the Cu, Zn-SOD remained constant or decreased slightly (39). It has been suggested that the induction of Mn-SOD is in response to increased cytokine production within the inflamed gastric mucosa (39). This situation is re­ -

-

2

versed following successful treatment of the infection (38). The data suggest that within the gastric environ­ ment H. pylori may be exposed to increased levels of TOS. In such an environment it is important for bacterial survival that the impact of such TOS be neu­ tralized. Reducing the Impact of T O S How do microorganisms manage their exposure to TOS? Several strategies may be adopted, governed in part by determinants such as whether the toxic spe­ cies are generated endogenously or exogenously. Mi­ croorganisms may neutralize T O S by mechanisms that include the enzymes SOD, catalase, peroxidases, and a variety of reductases. Also, they may modulate intracellular oxygen concentration or redox potential, thus minimizing their exposure to oxidative damage, or minimize such damage through the evolution of cellular structures resistant to oxidative damage. Fi­ nally, bacterial cells may overcome the effects of oxi­ dative damage through efficient DNA repair mecha­ nisms. There are many studies on such mechanisms in other organisms, and indeed, a great part of our understanding of these mechanisms in H. pylori is based predominantly on comparisons with the sys­ tems present in other organisms. Neutralization of T O S There are two prominent enzymes that facilitate resistance to oxidative damage in H. pylori, catalase (KatA) and SOD (43, 5 4 , 6 5 , 67, 77). In addition, there is genetic and biochemical evidence for the pres­ ence of at least two other enzyme systems involved in resistance to oxidative damage, alkylhydroperoxide reductase (Ahp) and thioredoxin-linked thiol peroxi­ dase (scavengease).

Stuart L. Hazell, Andrew G. Harris, and Mark A. Trend • School of Science, Food and Horticulture, College of Science, Technology and Environment, University of Western Sydney, Sydney, Australia.

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Catalase Catalase has been studied quite comprehensively in many eukaryotic and prokaryotic systems, begin­ ning with studies in the 1800s (53). It has been used and still is used today as a diagnostic tool in bacterial identification in medical microbiology. The major function of catalase is to protect cells from the damag­ ing effects of hydrogen peroxide ( H 0 ) , catalyzing the dismutation of H 0 into water and oxygen (equation 1). Consequently, catalase is an extremely important enzyme in an organism's response to oxida­ tive stress. 2

2

2H 0 — 2H 0 + 0 2

2

2

2

2

(1)

2

Hydrogen peroxide is generated as a by-product of aerobic respiration, which uses oxygen as a termi­ nal electron acceptor and can give rise to reactive oxy­ gen species such as 0 ' and H 0 . SOD is capable of removing the superoxide anion, but this results in the generation of more H 0 (equation 2 ) . -

2

2

2

2

2

2 0 - + 2H -> H 0 +

2

2

2

+ 0

(2)

2

Nonetheless, the amounts of hydrogen peroxide and superoxide radicals produced during aerobic res­ piration are quite small in comparison with the quan­ tity released during the respiratory or oxidative burst produced by PMN. Exposure to H 0 can be catastrophic for many organisms, yet the reactions between H 0 and or­ ganic molecules, such as proteins and DNA, remain unclear. This is largely due to the rapid formation of other reactive oxygen species (ROS), which appear to be more reactive than H 0 (33). The formation of other reactive oxygen radicals is due in part to interac­ tions between H 0 and reduced metallic ions found in all biological systems. The greatest risk that is posed to any cell, in terms of ROS, occurs when H 0 reacts with reduced iron or copper ions (83) to form hy­ droxyl radicals ( O H ) in a Fenton reaction (16). The hydroxyl radicals will react with most biological and organic molecules in oxidation reactions. Exposure to hydrogen peroxide, either through direct or indirect action, can result in DNA damage (therefore being mutagenic), lipid damage, and inhibition of the activi­ ties of enzymes and other proteins through oxidation. This is a probable explanation for the decreased growth rates observed in bacterial cell cultures in the presence of H 0 (78). "Typical" catalases characteristic of eukaryotes are homotetrameric with subunit mass between 55 and 65 kDa, and no heme prosthetic group per subunit (53), as indicated by the strong Soret band at 4 0 2 to 4 0 6 nm, with minor peaks at 5 0 0 to 5 0 5 , 535 to 540, and 6 2 0 to 635 nm (45). Typical catalases differ 2

2

2

2

2

2

2

2

2

2

2

2

from catalase-peroxidases found in a number of bac­ terial species in that they do not display peroxidase activity (32). H. pylori catalase is homotetrameric; each subunit has a mass of 58.7 kDa (as determined by the inferred amino acid sequence) and one heme prosthetic group (43, 5 4 , 65). The enzyme is a monofunctional catalase, i.e., it lacks peroxidase activity (43). The activity of the H. pylori catalase is pH inde­ pendent, with no difference between pH 5.25 and 8.95 (43). The enzyme has good thermostability, re­ taining catalytic activity after incubation at 56°C for 1 h (43). These properties are consistent with those of typical eukaryote catalases (43, 5 8 ) . Catalase is expressed in the cytoplasm and prob­ ably in the periplasm of H. pylori. There is some lim­ ited evidence supporting the presence of catalase on the cell surface, a unique occurrence in H. pylori, and possibly owing to autolytic events (43, 6 8 , 71). How­ ever, Mori et al. (57) were unable to detect catalase activity in the supernatant of culture media after 2 4 h of growth, concluding that it was unlikely that the enzyme is secreted into the surrounding environment. The sequence does not show a cleavable N-terminal signal peptide, as is the case with many other periplas­ mic proteins (41), thus the putative translocation of the enzyme to the periplasm would be Sec-indepen­ dent. H. pylori catalase is expressed during exponen­ tial growth and is not induced when the cells enter stationary phase as is the case with some bacterial catalases/catalase-peroxidases, for example, in Esche­ richia coli (43, 5 5 ) . A unique property of H. pylori catalase is an iso­ electric point (pi) in the range of 9.0 to 9.3 (43). T o date it is the only basic catalase that has been charac­ terized. Catalases produced by other organisms usu­ ally have a pi in the range of 4.5 to 5.0 (58). The basic pi of H. pylori catalase is largely due to the high lysine and arginine content in the enzyme. The release of the genome made it clear that many proteins of H. pylori have a basic pi (2, 82). The biological relevance of the pi of catalase and other proteins of H. pylori has yet to be determined. Catalase in H. pylori is distinctive because in situ there is a very rapid breakdown of hydrogen peroxide. The formation of oxygen occurs extremely fast, giving an "explosive" appearance of oxygen bubbles that is characteristic of catalase tests performed on whole H. pylori cells. The kinetic properties of the enzyme do not necessarily shed light on the activity observed when whole cells are exposed to H 0 . Helicobacter pylori catalase has a K of 43 ± 3 mM and a V x of 60 ± 3 mmol/min/mg of protein (43). The K is 3 to 10 times higher than those of other bacterial cata­ lases, suggesting that the enzyme is relatively ineffi­ cient. Although the affinity of the protein for the sub2

2

m

m a

m

CHAPTER 15

strate is not as high as those of some other catalases, it is likely that the high activity characteristic of H. pylori cells is due to the amount of enzyme present, typically accounting for > 1 % of the cells' total pro­ tein content (42, 4 3 , 5 4 ) , and/or the rapid turnover of substrate. Catalases, like other proteins, are susceptible to damage by hydrogen peroxide, but the catalase of H. pylori appears to be quite stable at very high concen­ trations of hydrogen peroxide. This property appears to be shared with only a few other catalases, for exam­ ple, of some Mycobacterium spp. (36, 4 3 , 58). It may be hypothesized that the stability of the catalase of these bacteria in the presence of high concentrations of hydrogen peroxide is an adaptation by these organ­ isms to environments comparatively rich in reactive oxygen species. The H. pylori catalase gene katA from four dif­ ferent strains of the bacterium has been sequenced (2, 54, 6 5 , 82). Not surprisingly, all sequences are almost identical with very few nucleotide variations. The in­ ferred amino acid sequence of catalase revealed the presence of an NADPH-like binding motif similar to that of bovine liver catalase and other typical cata­ lases. The residues involved in NADPH binding in bovine liver catalase are R-202, D-212, K-236 (all binding to the 0 2 ' phosphate of NADP ), and H304 (binding to the pyrophosphate group) (34). This sequence appears to be semiconserved in the H. pylori catalase; R-184, D-194, H-218 (conserved replace­ ment), and L-286 (nonconservative change). Whether this sequence allows for NADPH binding remains to be determined. However, other data suggest that H. pylori catalase may bind NADH rather than NADPH (54). The inferred amino acid sequence of the protein reveals an adenylate-binding motif ( G X G X X G ) con­ sistent with NADH binding, different from the NADPH adenylate-binding motif (GXGXXA) (72). In typical catalases the presence of NADPH is important to maintain an active enzyme. The dismutation of H 2 O 2 occurs by way of an intermediate form of catalase termed "compound I . " The formation and decomposition of this intermediate occurs too rapidly for it to be detected by spectroscopy (47). Compound I (a nominal F e state) is formed by a two-electron +

5 +

oxidation involving H 2 O 2 (equation 3 ) , which then reacts with a second molecule of H 0 , returning the enzyme to its original state ( F e ) (equation 4) (23). In the presence of excess H 0 (or with other hydrogen donors), a second intermediate, termed "compound II," is formed (equation 5). Compound II is the result of the one electron oxidation of catalase (thus forming an F e intermediate). This enzyme intermediate does not react with H 0 and thus the accumulation of compound II leads to the deactivation of catalase (11). 2

2

3+

2

2

4 +

2

2

catalase ( F e ) + H 0 — compound I ( F e ) 3+

Figure 1.

Diagrammatic representation of

katA

katA

(3)

5+

2

2

compound I ( F e ) + H 2 O 2 —»• catalase ( F e ) 5+

3+

+ H 0 2

+ I/2O2

(4)

catalase ( F e ) + X H 0 - » compound II ( F e ) 3+

4+

2

2

(X = steady flow)

(5)

Formation of compound II can be reversed or inhibited by NADPH bound to catalase ( 1 1 , 4 4 ) . Four molecules of NADPH bind to the tetrameric structure of bovine liver catalase (49). This reduced dinucleotide is not essential for the catalytic action of the en­ zyme, but it is believed that NADPH reduces com­ pound II via a one electron transfer reaction to yield NADP and the active native catalase (11). All strains sequenced have the same genes flank­ ing katA; upstream is frpB coding for an iron-binding protein, and downstream is an open reading frame (ORF) of unknown function. On the basis of sequence homology, a putative Fur-Box (ferric uptake regula­ tor) has been identified upstream of katA (54, 65). Usually, the Fur protein mediates iron repression in gram-negative bacteria, and it would appear that the expression of katA might be regulated by this putative Fur-Box (Fig. 1). Although limited studies have been performed on the regulation of katA ( 5 4 , 6 5 ) , the level of catalase activity drops when H. pylori is grown in blood-based media, as opposed to serum-based media, suggesting that iron availability may have a role in the expression of katA (43). Studies by Bereswill et al. indicate that the H. pylori Fur homolog is functional as an iron-dependent transcriptional re­ pressor (9). In Campylobacter jejuni, a bacterium of +

Fur-Box frbP

169

• EVASION OF TOXIC EFFECTS OF OXYGEN

phnA

Orf2

a n d s u r r o u n d i n g genes. A d a p t e d f r o m M a n o s et a l .

(54) w i t h

permission.

170

H A Z E L L E T AL.

the same family as H. pylori, expression of catalase (katA) is repressed by iron, and regulation of catalase appears to be mediated by both Fur and the peroxide stress regulator PerR (84, 85). These findings would support the hypothesis that the putative Fur-Box of H. pylori is functional. Catalase is not essential for growth and survival of H. pylori in vitro ( 5 4 , 6 5 , 89). Vaccine studies indi­ cate that the enzyme is a highly effective antigen, sug­ gesting that it may be essential in vivo (71). However, proof that catalase is essential in vivo remains to be established, as no catalase-negative mutants have been employed in animal model studies. SOD SOD catalyzes the dismutation of superoxide ions to hydrogen peroxide, which may be deactivated by catalase or peroxidase. The SOD of H. pylori is a typical prokaryotic iron-containing enzyme (FeSOD), consisting of two identical subunits each with an apparent molecular mass of 2 4 kDa (77). Three electromorphs or isoforms of Fe-SOD have been iden­ tified in different strains of H. pylori. These isoforms are the products of mutations leading to an altered pi (10). Unlike other bacteria that may express either an Mn-SOD or Cu, Zn-SOD, these forms of the enzyme were not detected in H. pylori by Spiegelhalder et al. (77), nor are they found in the genome (2, 82). The different types of superoxide dismutase, Cu, Zn-SOD, Fe-SOD, and Mn-SOD, appear to support various functions in resistance to oxidative stress by cells. The dimeric prokaryotic Cu, Zn-SOD, which differs from the corresponding eukaryotic SOD, is usually expressed in the periplasm of gram-negative bacteria (27, 35). The Cu, Zn-SOD of E. coli is more resistant to inactivation by H 2 O 2 than the eukaryotic enzyme and appears to be an important virulence de­ terminant conferring resistance to oxidative damage induced by the respiratory burst of phagocytic cells (6). Indeed, the Cu, Zn-SOD of Salmonella appears essential to serious systemic disease (33). In contrast, Mn-SOD is found in the cytosol and does not appear to be a critical virulence determinant. Instead, it appears to fulfill a "housekeeping" role, protecting against superoxide generated endogenously in bacteria such as Bordetella pertussis (40). Similarly, the Fe-SOD are cytosolic enzymes impor­ tant in the management of endogenously generated superoxide (37). In H. pylori the absence of a leader sequence suggests that its Fe-SOD is also cytosolic (77). Interestingly, Fe-SOD show strong structural conservation between the prokaryotic and eukaryotic enzymes, as is the case for the H. pylori enzyme (37, 77).

In members of the Enterobacteriaceae periplas­ mic Cu, Zn-SOD appears to be much more important than the cytosolic SOD as a virulence determinant (37). However, there is evidence that Fe-SOD may enhance intracellular survival of C. jejuni (67) and may also be important to the virulence of Trichomo­ nas vaginalis (87). The significance of such observa­ tions in relation to the Fe-SOD of H. pylori has yet to be ascertained. Alkylhydroperoxide reductase Alkylhydroperoxide reductase (2, 67, 82) cata­ lyzes the reduction of alkylhydroperoxide to the cor­ responding alcohol. In most bacteria alkylhydroper­ oxide reductase is a two-component system consisting of the proteins AhpF and AhpO; the latter is responsi­ ble for the peroxide reductase activity, while the acces­ sory flavoenzyme, AhpF, possesses NADH or NADPH oxidase activities. The H. pylori gene tsaA is orthologous to E. coli ahpC (69, 70). Although a homolog of ahpF has not been identified in the ge­ nome of H. pylori, there is ample experimental evi­ dence for the presence of NADH oxidase activity in the bacterium (74). Niimura et al. demonstrated that in Salmonella enterica serovar Typhimurium, in the absence of AhpF, NADH oxidase or NADH oxidaselike activities coupled to AhpC are sufficient to gener­ ate alkylhydroperoxide reductase activity (61, 62). Little is known about the alkylhydroperoxide re­ ductase of H. pylori. Yet this enzyme may be common within this family of bacteria. Baillon et al. (5) identi­ fied a homolog of ahpC in the microaerophile C. je­ juni. Like H. pylori, C. jejuni appears to lack ahpF, encoding the large accessory flavoenzyme of alkylhy­ droperoxide reductase. Importantly however, insertional mutagenesis of ahpC in C. jejuni resulted in an increased sensitivity to oxidative stresses induced by cumene hydroperoxide and atmospheric air (5). These data suggest that it is likely that alkylhydroperoxide reductase is functional in H. pylori. Tluoredoxm-linked thiol peroxidase Thiol peroxidase (scavengease) belongs to a re­ cently identified family of bacterial antioxidant en­ zymes possessing thioredoxin-linked activity (92). Di­ rect biochemical evidence for the existence of thiol peroxidase in H. pylori has been provided by an assay for antioxidant activity (88). These findings are sup­ ported by data from the genome indicating the pres­ ence of the gene HP390 (JHP991) encoding a putative thiol peroxidase (2, 82). Thiol peroxidase is usually a small protein (—20 to 30 kDa) found in both prokaryotic and eukaryotic

CHAPTER 15 • EVASION OF TOXIC EFFECTS OF OXYGEN

organisms including Haemophilus influenzae, Vibrio cholerae, E. coli, streptococci, and Entamoeba histo­ lytica (15, 2 1 , 2 2 ) . Thiol peroxidase protects from inactivation enzymes sensitive to oxidative stress such as glutamine synthetase, by removing H 0 in a metal-catalyzed oxidation system (equation 6). 2

2RSH + H 0 — RSSR + 2 H 0 2

2

2

2

(6)

The thiol specificity of the enzyme is determined by the observation that the oxidized form of thiol per­ oxidase is reactivated (converted back to its sulfhydryl form) by treatment with thiols (15, 60). This observa­ tion relates to the finding that one cysteine residue, Cys-94 in the E. coli enzyme, appears to be central to peroxidase activity (21). In E. coli oxidative stress induces higher levels of expression of the enzyme (48), which is located in the periplasm (21). It has been suggested that thiol peroxidase complements the cytosolic enzymes in protecting bacteria from oxidative damage (21). However, in the amoeba E. histolytica the enzyme is located in the cytosol, not on the surface or extracellularly (15), thus its role may include protection from both endogenously and exogenously generated reac­ tive oxygen metabolites. Management of Redox Potential The oxidation-reduction (redox) status of H. py­ lori is important, as changing the environmental oxy­ gen concentration and hence the redox status of the cell can greatly affect metabolic processes and clinical outcomes. The redox state of a cell may be defined as the sum of the oxidized and reduced molecular species present, but it is usually expressed in relation to the ratio of the oxidized and reduced thiols. Oxidation of thiols leads to an increase in the disulfide forms of both proteins and smaller compounds such as gluta­ thione (y-glutamylcysteinylglycine), the major free thiol in most cells. Reduced glutathione (GSH) plays an important role in the maintenance of the redox balance of cells, as it can scavenge free radicals and be converted to oxidized glutathione (GSSG). The cy­ cling of glutathione is critical for detoxification of free radicals in many organisms, with GSSG normally con­ verted back to GSH by the enzyme glutathione re­ ductase. However, there is little evidence that GSH is im­ portant to the maintenance of the redox balance in H. pylori. On the basis of genome analyses, the bacter­ ium does not appear to have a homolog of the gene encoding for typical glutathione reductases (2, 82). There is evidence that the major free thiol compound within H. pylori is cysteine (Jorgensen et al., unpub­ lished data). This observation is consistent with data

171

from a number of microaerobic protozoan species that lack detectable levels of glutathione and use cys­ teine as their major free thiol compound (13, 3 0 , 3 1 , 76). Cysteine appears not to be an appropriate free thiol compound for aerobic organisms, because in the presence of a metal catalyst it is oxidized much faster than glutathione. Indeed, cystine (oxidized cysteine) markedly enhances the cytotoxic response of E. coli to H 0 and may impair the cell defense machinery through thiol-disulfide exchange reactions at the cell membrane (18). This does not appear to be as critical in microaerophiles. If cysteine is the primary free thiol compound in H. pylori, cycling of oxidized cysteine, that is, the maintenance of a reduced state, may de­ pend on a thioredoxin-like reductase as has been pro­ posed for Giardia duodenalis (14). H. pylori contains two ORFs encoding putative thioredoxin reductases, designated HP0825 (JHP764) and HP1164 (JHP1091), and two ORFs encoding pu­ tative thioredoxins, designated HP0824 (JHP763) and HP1458 (JHP1351) (2, 82). Thioredoxin and thi­ oredoxin reductase form an NADPH-linked thiol-dependent redox system able to reduce proteins selec­ tively. The proteins encoded by HP0825 (JHP764) and HP0824 (JHP763) appear to be typical thiore­ doxin reductase and thioredoxin components of the thioredoxin system involved in stress response (90). The "alternative" thioredoxin reductase and thiore­ doxin encoded by HP1164 (JHP1091) and HP1458/ JHP1351, respectively, may fulfill the role of the thi­ oredoxin-like reductase of G. duodenalis necessary for the maintenance of free cysteine (14), and hence the redox state of the cell. Managing the concentration of dissolved intra­ cellular oxygen is another way to regulate the redox potential of the cell. NADH oxidases are used to regu­ late the oxygen concentration in different microaero­ bic organisms. This family of enzymes directly reduces molecular oxygen to hydrogen peroxide or water. In the genome of H. pylori no ORF homologous to typi­ cal NADH oxidases is apparent, but cytosolic NAD(P)H oxidase activities have been measured in the bacterium (75). However, it is possible that such NAD(P)H oxidase activities are the product of elec­ tron leakage from the reduced flavin cofactor of flavoprotein enzymes such as alkylhydroperoxide re­ ductase, thioredoxin reductase, glutathione re­ ductase, mercuric reductase, and dihydrolipoamide dehydrogenase (3, 17, 19, 2 0 , 52, 6 4 , 9 1 ) . In addition to the enzyme activities outlined above, the pentose phosphate pathway also plays a role in resistance to oxidative stress; among its several roles, it generates reducing power in the form of NADPH. In yeasts, mutations of enzymes of the pen­ tose phosphate pathway lead to increased sensitivity 2

2

172

HAZELL ET AL.

to oxidative stress, and the pathway is required for the maintenance of the cellular redox state (46, 7 3 ) . Indeed, in mammalian systems, glucose 6-phosphate dehydrogenase, which catalyzes the first step in the pentose phosphate pathway and which provides re­ ductive potential in the form of NADPH, has been found to be essential in protecting cells against oxida­ tive stress, yet it is not essential for pentose synthesis (66). The pentose phosphate pathway was one of the first complete biochemical pathways identified in H. pylori (56), but its role in the maintenance of the redox status has not been investigated.

GENE REGULATION AND REPAIR MECHANISMS A surprising finding in the genome of H. pylori was the absence of homologs of genes encoding the transcription regulatory sigma factors or (heat shock) and o- (stress/stationary-phase) (2, 82). Not­ withstanding the absence of genes coding for n , ho­ mologs of genes encoding GroEL, GroES, DnaK, DanJ, and GrpE were identified in the genome regu­ lated by housekeeping cr -like sigma factors (1, 2, 7, 79, 82) (reviewed further in chapter 2 9 ) . The induction of an inflammatory response by H. pylori infection leads to increased potential for oxi­ dative damage of the bacterium. While H. pylori has the enzymatic capacity to deal with such oxidative stress, no homologs of the oxidative stress regulators OxyR, SoxR, SoxS, or SOS present in other bacteria (28, 2 9 , 8 6 ) , have been found in H. pylori DNA (2, 82). Together with the absence of sigma factor cr , these data suggest that either H. pylori has adapted to an environment of constant oxidative stress or the bacterium contains novel systems of protection yet to be discovered. H. pylori appears able to perform mismatch re­ pair, as suggested by the coding capacity for methyl transferases, DNA glycosylases, and MutS and UvrD proteins, involved in error-free and error-prone re­ pairs ( 2 , 8 2 ) . The RecBCD pathway is the major path­ way for recombination in wild-type E. coli cells (50), but this system appears to be absent in H. pylori. Ho­ mologous recombination may be performed by H. py­ lori through the RecF pathway. In E. coli, this path­ way generally depends on the RecA, RecJ, RecN, RecR, RecG, and RuvABC proteins, whose genes are present in the H. pylori chromosome (reviewed fur­ ther in chapter 24); and Thompson and Blaser demon­ strated that recA H. pylori mutants were highly sensi­ tive to UV light, methyl methanesulfonate, and exposure to mutagenic antibiotics such as metronida­ zole (81). 32

s

32

70

s

C. jejuni does not encode OxyR and, as discussed above, the regulation of catalase expression in this bacterium appears to be mediated by both Fur and PerR (84, 85). It has been suggested that PerR func­ tions as a nonhomologous substitute for OxyR (84). H. pylori, like C. jejuni, does not encode OxyR, and we are left to ponder the potential for the existence of previously unidentified oxidative stress regulators encoded by its genome.

CONCLUSION H. pylori is a microaerophile that colonizes the inflamed gastric mucosa of humans. These two facts suggest the presence of a network of systems needed to manage both the oxygen to which H. pylori is ex­ posed and the oxidative stress induced by endogenous and exogenous processes. That oxygen and T O S are constant companions of H. pylori in vivo is reflected in the enzymes expressed to manage them and the regulatory and repair mechanisms developed by the bacterium to cope with this type of stress. Nonethe­ less, our understanding of how H. pylori evades and avoids toxic oxygen effects is far from complete; and despite the importance of the topic, the management of oxygen and oxidative stress in H. pylori is a rela­ tively neglected subject area. REFERENCES 1. Allan E., P. Mullany, and S. Tabaqchali. 1998. Construction and characterisation of a Helicobacter pylori clpB mutant and role of the gene in stress response. /. Bacteriol. 180:426-429. 2. Aim, R., L. Ling, D. Moir, B. King, E. Brown, P. Doig, D. Smith, B. Noonan, B. Guild, B. Dejonge, G. Carmel, P. Tum­ mino, A. Caruso, M. Uria-Nickelsen, D. Mills, C. Ives, R. Gib­ son, D. Merberg, S. Mills, Q. Jiang, D. Taylor, G. Vovis, and T. Trust. 1999. Genomic sequence comparison of two unre­ lated isolates of the human gastric pathogen Helicobacter py­ lori. Nature 397:176-180. 3. Arner, E. S. J., M. Bjornstedt, and A. Holmgren. 1995. 1chloro-2,4-dinitrobenzene is an irreversible inhibitor of human thioredoxin reductase—loss of thioredoxin disulfide reductase activity is accompanied by a large increase in NADPH oxidase activity. /. Biol. Chem. 270:3479-3482. 4. Bagchi, D., G. Bhattacharya, and S. J. Stohs. 1996. Production of reactive oxygen species by gastric cells in association with Helicobacter pylori. Free Radical Res. 24:439-450. 5. Baillon, M. L., A. H. van Vliet, J. M. Ketley, C. Constantinidou, and C. W. Penn. 1999. An iron-regulated alkyl hydroper­ oxide reductase (AhpC) confers aerotolerance and oxidative stress resistance to the microaerophilic pathogen Campylo­ bacter jejuni. J. Bacteriol. 181:4798-4804. 6. Battistoni, A., G. Donnarumma, R. Greco, P. Valenti, and G. Rotilio. 1998. Overexpression of a hydrogen peroxide-resis­ tant periplasmic Cu,Zn superoxide dismutase protects Esche­ richia coli from macrophage killing. Biochem. Biophys. Res. Commun. 243:804-807.

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26.

27.

28.

29.

30.

31.

32.

33.

34. 35.

36.

37.

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40.

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CHAPTER 15 • EVASION OF TOXIC EFFECTS OF OXYGEN

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IV. PHYSIOLOGY AND MOLECULAR BIOLOGY

Helicobacter pylori: Physiology and Genetics Edited by H. L . T. Mobley, G. L . Mendz, and S. L . Hazell © 2 0 0 1 ASM Press, Washington, D.C.

Chapter 16

Urease HARRY L. T. MOBLEY

H C0

Urease is produced by numerous taxonomically di­ verse bacterial species, including normal flora and nonpathogens. Also, urease has been demonstrated as a potent virulence factor for some species, including Proteus mirabilis (51), Staphylococcus saprophyticus (36), and Helicobacter pylori (23). Urease is central to H. pylori metabolism and virulence, is necessary for its colonization of the gastric mucosa, and is a potent immunogen that elicits a vigorous immune re­ sponse. This enzyme is used for taxonomic identifica­ tion and for diagnosis and follow-up after treatment, and is a vaccine candidate. Urease represents an inter­ esting model for metalloenzyme studies. Before the discovery of H. pylori, humans were thought to pro­ duce "gastric urease." It is now known that the source of this notable protein is this bacterium, which colo­ nizes the gastric mucosa of humans.

2

2NH

3

3

«- H

+

+ HC0 "

+ 2 H 0 «-» 2 N H 4 2

3

+

+ 20H"

Kinetic Constants and Optima The kinetic parameters of purified H. pylori ure­ ase have been calculated using rates of hydrolysis measured over a range of urea concentrations from 0 to 5 mM at 23°C; the K ranged from 0.17 to 0.48 mM ( 2 1 , 2 7 , 4 6 ) . With purified enzyme under saturat­ ing conditions, specific activities ranged from 1,100 to 1,700 u,mol of urea/min/mg of protein (21, 4 6 ) . Although H. pylori has a K that reflects a higher affinity for substrate than the ureases of other species, this appears to be appropriate to the niche of the bac­ terium. Assuming that the physiological concentra­ tions of urea to which H. pylori is exposed are the same as those found in serum (1.7 to 3.4 mM), then urease would be saturated and working at its V when the bacterium colonizes the gastric mucosa. Op­ timal enzyme activity was observed at 43°C for urease in cell lysates (69), and its activity was not inhibited by 0 . 0 2 % azide (69). m

m

m a x

ENZYMOLOGY Enzymatic Reaction Urease (urea amidohydrolase: EC 3.5.1.5) cata­ lyzes the hydrolysis of urea to yield ammonia and car­ bamate. The latter compound spontaneously decom­ poses to yield another molecule of ammonia and carbonic acid: H N-CO-NH 2

2

+ H 0

NH

2

Active Site and Catalysis The active site of the enzyme is found in the UreB subunit and comprises amino acid residues found throughout the primary structure that are brought into proximity in the tertiary structure (49). Active site residues were identified by examining the results of site-directed mutagenesis (62, 8 0 , 9 4 ) , studies of apoprotein activation (81), and structural determina­ tions by X-ray crystallography (49, 50). With a num­ bering specific for H. pylori UreB (55), residues His136, His-138, Lys-219, His-248, His-274, and Asp362 come in direct contact with the two nickel ions, urea, or a water molecule within the active site. In

3

+ H N-C(0)OH 2

H N - C ( 0 ) O H + 2 H 0 — NH 2

2

3

+ H C0 2

3

In aqueous solutions, the released carbonic acid and the two molecules of ammonia are in equilibrium with their deprotonated and protonated forms, respec­ tively. The net effect of these reactions is an increase in pH. Harry L. T. Mobley 21201.

• Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD

179

180

MOBLEY

addition, His-322 is near the active site and acts as a general base in the catalysis. The mechanism by which urea is hydrolyzed fol­ lows the scheme first described by Zerner's group for the jack bean urease (20). Urea binds in O-coordination to one nickel ion aided by His-221. As an active base, His-322 activates a water molecule bound to the other nickel ion. Attack by the metal-coordinated hydroxide on the substrate carbon atom results in a tetrahedral intermediate that bridges the two nickel sites, a proton is transferred to the intermediate with accompanying ammonia release, and water displaces the carbamate to complete the cycle. Specific peptide inhibitors of H. pylori urease ac­ tivity have been identified by screening tetradodecamer and hexamer combinatorial libraries using phage display. The 24-mer TFLPQPRCSALLRYLSEDGVIVPS and the 6-mer Y D F Y W W inhibit H. pylori urease with Kj of 4 7 and 30 uM, respec­ tively, but not the Bacillus pasteurii enzyme (44).

PROTEIN S T R U C T U R E Urease is a high molecular weight multisubunit metalloenzyme. It appears that all ureases are closely related and have similar mechanisms of catalysis. It is interesting that such a complex protein is required to carry out a simple hydrolysis.

Stds

Figure 1. Purified urease electrophoresed on an SDS-polyacrylamide gel. Protein (10 |xg) from each purification step was electro­ phoresed on a 10 to 20% polyacrylamide gradient gel and stained with Coomassie Blue (46). Lanes (from left to right): crude lysate of H. pylori, DEAE-Sepharose, phenyl-Sepharose, Mono-Q, Superose 6, high molecular weight protein standards, low molecu­ lar weight protein standards. Molecular masses of the two subunits, predicted from the nucleotide sequence, are shown in the right margin.

Purification H. pylori synthesizes an extraordinary amount of urease. The purified enzyme, however, is not signif­ icantly more active than purified ureases from other species, but it simply represents a larger proportion of total cell protein in this species. When crude lysates are electrophoresed on sodium dodecyl sulfate (SDS)polyacrylamide gels and stained with Coomassie Blue, the two urease subunits of apparent molecular sizes of 66 and 29.5 kDa appear as extremely prominent bands (46) (Fig. 1). When other urease-positive bacte­ rial species are examined in this manner, urease subunits are not produced in sufficient quantities to de­ tect such bands on the gels. The native protein has been purified from H. py­ lori by isolation from the cytosol (46) or by elution from the cell surface with low ionic strength solvents (21, 27, 86, 9 9 ) . For purifications from the cytosol, French press cell lysates are chromatographed on DEAE-Sepharose, phenyl-Sepharose, Mono-Q, and Sepharose 6 resins. With this scheme, purified urease represents 6 % of the soluble protein of crude extract and has an estimated native molecular size of 5 5 0 kDa. On the basis of subunit size, a 1:1 subunit ratio

measured by scanning densitometry of Coomassie Blue-stained SDS-polyacrylamide gels, and estimated native molecular mass, the data are consistent with a stoichiometry of (29.6-61 k D a ) for the structure of the native enzyme. The enzyme has an isoelectric point between 5.90 and 5.99 (21, 27, 9 9 ) . 6

Appearance in Electron Micrographs Purified native urease has been examined by transmission electron microscopy and appears as a round, doughnut-shaped, hexagonal particle with a darkly staining core (3, 2 2 , 99). The enzyme is 13 nm in diameter and displays threefold rotational symme­ try. Urease eluted from the bacterial cell surface copurifies with a 60-kDa chaperonin heat shock protein. This heat shock protein displays a very high native molecular weight similar to that of urease and assem­ bles into a macromolecular structure that is also simi­ lar in appearance to urease (3, 2 2 , 2 6 ) . X-Ray Diffraction Studies The crystal structure of the related urease from Klebsiella aerogenes has been solved by X-ray diffrac-

CHAPTER 16

tion (49, 5 0 ) . Owing to the high degree of homology between all bacterial ureases, it can be inferred that H. pylori urease shares a similar structure. The K. aerogenes enzyme is only about half the molecular weight of the H. pylori enzyme and contains only three copies of each subunit. Therefore, the H. pylori urease may comprise two of the quaternary structures that are resolved for the Klebsiella enzyme. However, the precise arrangement for the holoenzyme is not known. By using the known crystal structure, a con-

figuration of the H. pylori can be projected (Fig. 2 ) .

•

UREASE

181

UreA-UreB heterodimer

Nickel In common with other ureases, H. pylori con­ tains nickel ions in the active site. The nickel content of H. pylori urease has been measured with atomic absorption spectroscopy (42). All ureases, whether from plants, fungi, or bacteria, that have been ana­ lyzed also have nickel ions as a component of the ac­ tive enzyme (41). On the basis of findings of many studies, two nickel ions ( N i ) appear to be present in each active site. Because H. pylori has six copies each of the two distinct subunits (UreA and UreB), there are six active sites, and thus 12 nickel ions per fully loaded enzyme molecule. 2+

Effect of pH on Urease Exposure of whole cells of H. pylori to buffer below a pH of 4 results in loss of intracellular urease activity. In soluble enzyme preparations from lysed cells and supernatants, no urease activity is measura­ ble after incubation at a pH of < 5 for 30 min. Expo­ sure of H. pylori cells to a pH of 5 or below inhibits overall protein synthesis, including nascent urease. At a pH of 6 or 7, urease represents 1 0 % of the total cell protein (5).

GENETICS Genetic Organization The genes encoding H. pylori urease are located as a single 6.13-kb gene cluster on the chromosome of the bacterium (13, 16, 55) (Fig. 3 ) . Seven contiguous genes, all transcribed in the same direction, are neces­ sary for synthesis of an active enzyme (16, 4 5 , 4 7 ) . The genes have been designated ureABIEFGH. All of the genes except urel share homology with urease genes of other species, including Bacillus sp. TB-90 (60), K. aerogenes (56, 7 3 ) , P. mirabilis (52, 5 3 , 7 6 , 94), Ureaplasma urealyticum (9, 75), Yersinia enterocolitica (17, 92), and the jack bean (85). Figure 2. Space-filling model of the predicted urease crystal struc­ ture. The primary amino acid sequence of H. pylori urease was overlaid onto the solved crystal structure of Klebsiella aerogenes urease (49, 50). The front (A) and back (B) views of the two subunits, UreA (dark) and UreB (light) are shown. Note the two nickel atoms inserted into the enzyme active site (panel B). The holoen­ zyme is composed of six copies of the heterodimer displayed in the figure; the crystal structure of the H. pylori urease has not been determined directly (figure designed by Ron Guiles and Nereus Gunther, University of Maryland).

Structural Genes The two structural subunits are encoded by ure A and ureB, the first two genes of the gene cluster (13, 55) (Fig. 3). UreA has a predicted molecular mass of 26.5 kDa, and the predicted masses of UreB are be­ tween 60.3 and 61.0 kDa, the sequences differing for various strains. The UreA subunit of Helicobacter

182

MOBLEY

Ni S t r u c t u r a l urease subunits

2+

Accessory proteins (Ni

2 +

delivery)

Figure 3. Model for synthesis of a catalytically active urease in H. pylori. The urease gene cluster, composed of seven chromosomally encoded genes, is present as a single copy on the chromosome. The genes ureA and ureB encode the 26.5-kDa and 60.3-kDa subunits, respectively. Six copies of each subunit spontaneously self-assemble to form the catalytically inactive apoenzyme. The urease protein depicted shows three copies of each subunit and is adapted from the crystal structure of the Klebsiella aerogenes urease (49). The known molecular size of H. pylori urease (550 kDa) would require two of the depicted protein structures to be associated in some manner (46). This arrangement has not yet been solved. Accessory genes ureE, ureF, ureG, and ureH encode accessory proteins UreE, UreF, UreG, and UreH, which, by analogy to homologs of other species, serve to insert nickel ions (Ni ) into the apoenzyme in an energy-requiring reaction (71). UreE is a nickel-binding dimer. UreG carries a GTP-binding site. The gene urel is proposed to encode a urea-specific pore in the inner membrane that opens at low pH to allow passage of urea and closes at high pH to prevent access of the substrate to cytoplasmic urease (101). Two nickel ions are coordinated into the active site of each UreB subunit. Thus, each H. pylori urease contains 12 nickel ions when fully activated. Nickel ions are transported into the cell by NixA, a high-affinity membrane transport protein (70). Additional backup nickel transport proteins are also likely present. The net result of the interaction of these genes and proteins is a catalytically active urease. (Figure designed by David McGee.) 2+

species is somewhat unusual because its amino acid sequence is encoded by the single ureA gene, whereas in all other bacterial species it is always encoded by two separate genes. It could be speculated that the two smaller genes of other species may have fused to form H. pylori ureA. Expression of ureA and ureB is sufficient to produce an assembled apoenzyme (45). Under these conditions no nickel ions are inserted into the active site of the enzyme and thus no catalytic activity is present. Accessory Genes For synthesis of a catalytically active urease, the accessory genes urel, ureE, ureF, ureG, and ureH also must be expressed (16) (Fig. 3). Whereas the urel gene is unique to H. pylori, the remaining accessory genes encode proteins that share homology with gene prod­ ucts of the urease gene clusters of other bacterial spe­ cies. It is believed that these accessory proteins inter­ act with the apoenzyme and deliver nickel ions to the active site in an energy-dependent process (for a re­ view, see reference 7 1 ) . Only few genera, including gastric Helicobacter (88), have the extra urel gene in the cluster. Weeks et

al. demonstrated that Urel is an integral cytoplasmic membrane protein that may form a urea-specific pore (101) (Fig. 3). Importantly, this pore is controlled by external pH via a shift in periplasmic pH (84). There is evidence that the Urel pore opens as the medium pH drops below a pH of 6.5, allowing urea to reach cytoplasmic urease. Thus, as external pH drops, ure­ ase activity acts to neutralize the acid; as pH ap­ proaches neutrality, substrate is denied to the enzyme and the danger of excessive N H is avoided. These authors could not demonstrate Urel-mediated trans­ port in H. pylori itself. However, urea transport in Xenopus oocytes expressing urel from cRNA appears to be passive (i.e., not driven by proton motive force or ATP hydrolysis), nonsaturable, nonelectrogenic, and temperature-independent. The rate of uptake in oocytes expressing Urel was high at a pH of 5.5 and low at a pH of 7.5. However, Mendz et al. propose that Urel is not involved in urea transport in H. pylori (Mendz et al., personal communication). 3

Homology to Other Ureases On the basis of nucleotide sequence of the urease genes of H. pylori and other bacteria, it is certain that

CHAPTER 16 • UREASE

all ureases share a common ancestral gene. Despite the fact that ureases are composed of multiple copies of one (jack bean), two (all helicobacters), or three (all other bacterial species) distinct subunits, the amino acid sequences are well conserved. For exam­ ple, UreA of H. pylori shares 4 8 % and 4 2 % amino acid sequence identity with the corresponding N-terminal sequences of the jack bean urease subunit and the combined sequences of UreA and UreB of P. mirabilis, respectively (53, 5 5 , 85). The entire sequence of the structural subunits of H. pylori shares 5 8 % iden­ tity with the urease of K. aerogenes (16, 73). Residues conserved for all ureases and involved in catalysis have been discussed above. Expression of Catalytically Active Recombinant Urease A number of requirements were identified to ex­ press catalytically active H. pylori urease in E. coli at levels similar to those found in wild-type H. pylori strains (16, 4 7 ) . Expression of only UreA and UreB was sufficient to produce a normally assembled apoenzyme with no catalytic activity. If accessory genes are coexpressed, a weakly active urease could be pro­ duced, but only when Escherichia coli is cultured in minimal salts medium containing no histidine or cys­ teine (these amino acids chelate free nickel ions). By overexpressing ureA and ureB structural genes in trans to the entire gene cluster, full urease catalytic activity and protein levels could be produced in E. coli, but again only on minimal salts medium lacking histidine and cysteine. Fully active urease, comparable to the wild-type H. pylori enzyme, could be expressed in rich bacteriological medium such as Luria broth, only when NixA (high-affinity N i transporter) was coexpressed with the entire urease gene cluster and the structural genes were overexpressed (70). 2 +

Regulation of Urease Gene Expression H. pylori does not appear to regulate levels of urease expression by mechanisms such as nitrogen levels, pH, urea induction (71), and iron levels (96), which control urease expression in other species. The ure promoter contains a recognizable —10 region but not a conserved — 35 region (90). Expression appears to be regulated by selective degradation of urease-encoding mRNA. Akada and colleagues concluded that the gene cluster consists of two operons, ureAB and urelEFGH, and that primary transcripts of the latter as well as the read-through transcript, ureABIEFGH, are cleaved to produce several mRNA species (1). The urelEFGH operon may be posttranscriptionally regu­ lated by mRNA decay in response to environmental pH (1).

183

Other H. pylori genes also may modulate urease gene expression. McGee and colleagues isolated gene bank clones that either stimulated (DNA helicase) or depressed [fibA) urease expression in an E. coli back­ ground, providing evidence that flagellar biosynthesis and urease activity are linked (64). Construction of Mutants Urease mutants have been constructed by allelic exchange mutagenesis. Antibiotic resistance cassettes were inserted into cloned ureA (McGee and Mobley, unpublished results), ureB, or ureG (28), and these constructs were electroporated into wild-type H. py­ lori strains. Antibiotic-resistant H. pylori was evalu­ ated for double-crossover mutations in which the wild-type allele was exchanged for the insertionally inactivated allele. Mutants that lacked detectable ure­ ase activity were readily selected and had no apparent alteration of growth rates, demonstrating that enzyme activity was not necessary for viability in vitro. These mutants, however, are uniformly avirulent in animal models of infection when tested in pathogenesis stud­ ies; urel mutants are also avirulent (91) (see below). Urease mutants have also been isolated by random insertional mutagenesis using an integration plasmid (8).

PHYSIOLOGY Substrate Availability Urea is synthesized in the liver and is found in serum, saliva, and gastric juice at concentrations below 10 mM. It is excreted in urine at high concen­ trations ranging from 4 0 0 to 5 0 0 m M (40). A small amount of urea is supplied endogenously to H. pylori by the arginase (RocF)-mediated hydrolysis of L-arginine to L-ornithine (63). Recently, Urel has been pos­ tulated to form a urea-specific pore in the cytoplasmic membrane that opens at low pH and closes at high pH, thus regulating urea availability to cytoplasmic urease (101). Enzyme Localization and Activity of External and Internal Urease Unlike the ureases of most bacterial species, the enzyme is not strictly cytoplasmic. In aging cultures, urease can be found adherent to the cell surface or shed into the medium (82); this appears to be due to lysis of a subset of the population and readsorption of the protein onto the cell surface of viable bacteria. The importance of the external urease has been de­ bated. Two studies have shown contrasting roles of

184

MOBLEY

cytoplasmic versus surface-exposed urease. In the first study, H. pylori was cultured in a way in which the bacteria possessed either cytoplasmic and surface ure­ ase or cytoplasmic urease alone; assays were con­ ducted in the presence and absence of flurofamide, a poorly diffusible urease inhibitor (54). Bacteria hav­ ing only cytoplasmic urease were more susceptible to acid; likewise, E. coli expressing H. pylori urease cytoplasmically was also susceptible to acid. This sug­ gested that surface-exposed urease contributes to re­ sistance to transient acid exposure. In contrast, Scott et al. argued that, because external urease is inactive below a pH of 5 and internal urease has maximal activity below a pH of 5.5, internal urease is most likely responsible for acid tolerance (89). Use of Ammonia Generated by Urea Hydrolysis

pylori has developed a high-affinity system to acquire nickel ions. It appears that these ions can be trans­ ported into H. pylori by at least two mechanisms. The first is NixA, a cytoplasmic membrane-bound protein of 36,991 molecular weight, which transports nickel ions with a K of 11.3 nM (70). E. coli expressing NixA transported nickel ions with a V of 1,750 pmol/min/10 bacteria. Topology studies using 21 phoA and 21 lacZ translational fusions revealed that the protein has eight transmembrane domains and a large periplasmic loop with both the N terminus and C terminus residing in the cytosol (Fig. 4) (34). Nega­ tively charged Asp and Glu residues and His residues, which are located in the transmembrane domains, were shown to be critical for active transport of nickel ions (33). Two domains have been identified in NixA and the small family of high-affinity nickel transport proteins as essential for transport: G X H A X D A D H in helix II and G X F X G H S S W in helix III (33). Insertional inactivation of nixA in H. pylori ATCC 4 3 5 0 4 resulted in a 6 9 % decrease in the rate of nickel transport and a 4 2 % reduction in urease activity relative to the parent strain (6). These rates varied among strains but are reduced significantly in the nixA mutants. The fact that nickel transport or urease activity is not totally abolished in these mu­ tants supports the presence of a second mechanism of nickel transport that has yet to be elucidated. When a nix A mutant of H. pylori SSI and the parent strain were mixed and used orogastrically to cochallenge mice, the nixA mutant was never re­ covered by culture of gastric tissue (77). That is, the wild-type always outcompeted the nixA mutant. In­ terestingly, however, the nix A mutant was able to col­ onize the gastric mucosa of mice when inoculated alone (i.e., in the absence of the wild type), and the CFU per gram of stomach approached levels achieved in mice infected with the parental strain. These studies indicated that NixA-mediated nickel transport pro­ vides a selective advantage for H. pylori in the gastric mucosa. T

m a x

8

2

Ammonia, a preferred nitrogen source for bacte­ ria and the product of urea hydrolysis, is assimilated into protein and other nitrogenous compounds in bac­ teria by a single pathway (83). Glutamine synthetase (EC 6.3.1.2) catalyzes the reaction: N H + glutamate + ATP -»glutamine + ADP + ?, 3

Glutamine, in turn, serves as nitrogen donor for other nitrogenous compounds including alanine, glycine, serine, histidine, tryptophan, CTP, AMP, carbamoylphosphate, and glucosamine 6-phosphate. The 1,443-bp glutamine synthetase gene, gink, encodes a predicted polypeptide of 4 8 1 amino acid residues with a molecular weight of 54,317 (35). In most genera this enzyme activity is regulated posttranslationally by adenylation of the protein. The adenylation site found in most bacterial homologs has consensus sequence NLYDLP, which is replaced in H. pylori by NLFKLT (residues 4 0 5 to 410). Since the Tyr (Y) residue is the target of adenylation, and H. pylori glutamine synthetase lacks that residue in four strains examined, no adenylation occurs within this motif and the enzyme is not regulated in this manner. It was not possible to isolate glutamine synthetasedeficient mutants constructed by allelic exchange, suggesting that glutamine synthetase is essential for viability and critical for nitrogen assimilation in H. pylori (35). The enzyme appears active under all phys­ iologic conditions, consistent with the singular niche that the organism occupies. NixA and Nickel Transport All ureases contain two nickel ions in each of their active sites; H. pylori urease appears to have six active sites and, thus, 12 nickel ions. To overcome the nickel limitation that probably occurs in the host, H.

2

2

Metal-Binding Proteins That May Affect Urease Activity Nickel ions are required for catalytic activity of urease, yet a nixA mutant of H. pylori still retains some urease activity (6). It was concluded from this observation that additional proteins may play a role in nickel binding or transport. Three H. pylori pro­ teins have been identified with predicted nickel-bind­ ing or general metal-binding properties. Hpn is a small protein with a molecular size of 7.1 kDa and 60 amino acid residues (37). Nearly half (47%) of its residues are histidyls, making this protein

CHAPTER 16 • UREASE

185

NH3+

Figure 4. Topological model of NixA in the cytoplasmic membrane. The amino acid sequence of NixA is presented in singleletter code. Boxed regions indicate transmembrane domains. Filled black diamonds indicate the location of PhoA and LacZ reporter fusions by number (from amino terminus) of the last NixA amino acid residue prior to the fusion junction; enzymatic activity of reporter fusions were used to predict the topology (34). Circled residues indicate conserved motifs (among known nickel transporters) in helices II and III, plus six additional transport-critical residues. (Reprinted with permission from reference 34.)

a strong binder of nickel and zinc ions. Although it is possible to hypothesize that Hpn would serve as a sink for nickel and be used to activate urease, a mutant in which hpn was insertionally inactivated showed no reduction of urease activity following culture in vitro (37). The effects of this mutation have not been exam­ ined in vivo, and under these conditions Hpn may have an effect on urease activity. CadA, a P-type ATPase, has been identified in H. pylori (66). From the gene sequence a 686-amino acid protein is predicted that contains consensus sites for phosphorylation, ATP-binding, and, at the N termi­ nus, segments rich in histidine, methionine, gluta­ mate, and aspartate residues, which may act as a nickel-binding domain. This protein has been shown to be a heavy metal ion ( C d , Z n , C o ) exporter that uses ATP as an energy source for transport. Mu­ tation of the gene encoding the ATPase appears to 2+

2+

2+

diminish but not abolish (as originally reported) ure­ ase activity. These observations suggest that CadA contributes to high levels of urease activity by export­ ing divalent metal cations that may interfere with Ni -metalloenzyme formation (43). The HspA (heat shock protein A) of H. pylori is 118 amino acid residues long and a homolog of GroES-like heat shock proteins. It is encoded by the first gene, hspA, of a bicistronic cluster (95). Although the predicted protein is highly conserved with respect to its homologs, the last 25 residues of the C terminus include 4 cysteinyls and 6 histidyls, a potential metalbinding motif not found in any of the homologs. Mu­ tation of hspA appears to be lethal for H. pylori and thus the role of this heat shock protein in urease activ­ ity cannot be tested directly in the native organism. Expression in E. coli of hspA in trans to the H. pylori urease gene cluster results in slight elevation of urease 2+

186

MOBLEY

activity, suggesting that HspA may affect urease activ­ ity in wild-type H. pylori.

PATHOGENESIS Requirement for Colonization Although urease is not required for in vitro via­ bility of H. pylori, it is clear that the enzyme is a criti­ cal virulence determinant necessary for colonization of the gastric mucosa. A urease-negative mutant, iso­ lated after mutagenesis with nitrosoquanidine of an H. pylori strain, was used to inoculate gnotobiotic piglets. The mutant, which retained only 0 . 4 % of the urease activity of the parent strain, did not colonize any of 10 orally challenged piglets as assessed at 3 and 21 days after challenge, and no pathology was observed in these piglets (23). The parent strain, on the other hand, successfully colonized all seven piglets and caused gastritis. This work was carried further by using an isogenic pair of strains that included the parent strain and an allelic exchange mutant in which ureG had been interrupted with a kanamycin resis­ tance cassette. In these studies the piglets were treated with omeprazole, a proton pump inhibitor, which abolished acid secretion and yielded a neutral pH in their stomach. The parent strain colonized normally in numbers with a mean login CFU between 4.4 and 6.9. The urease-negative mutant was unable to colo­ nize the gastric mucosa at normal physiological pH and was recovered only in low numbers (mean login CFU, < 2 ) from omeprazole-treated, achlorhydric pig­ lets (24). These results confirmed that urease enzy­ matic activity was essential for colonization. Impor­ tantly, it also implied that the role of urease extended beyond that of just acid protection. Other studies demonstrated the inability of H. pylori urease mu­ tants to colonize the gastric mucosa of nude mice (98) and cynomolgus monkeys (97).

D E T E C T I O N OF H. PYLORI USING UREASE Urease Biopsy Test The high level of expression of urease by H. py­ lori can be employed for simple detection of the bac­ terium in gastric biopsies. Samples obtained by endos­ copy are placed in a gel containing urea and phenol red (a pH-indicating dye); if H. pylori is present, pre­ formed urease will hydrolyze the urea, raise the pH, and change the color of the phenol red from yellow to red ( 6 1 , 6 5 ) . This concept was used in the CLOtest, the first commercially available test for detecting the presence of H. pylori (61). The utility of this test

has been repeatedly validated in the literature. Re­ cently, a urease sensor has been developed for endos­ copy based on the differential output of two pH-sensitive transistors at the tip of the endoscope. It carries a urea solution that would be hydrolyzed by urease produced by colonizing bacteria (87). Urease-Positive Colonies after Culture Freshly cultured H. pylori is strongly urease posi­ tive. After 3 to 5 days of culture, pinpoint colonies can be observed and tested for urease activity. H. pylori cultured directly from endoscopic biopsy specimens on serum-based medium virtually always gives a strong urease reaction, which, together with the pres­ ence of positive oxidase and catalase reactions, is di­ agnostic for this species. Urea Breath Test Although the urease biopsy test reaction is sim­ ple, it requires obtaining biopsies by endoscopy, an invasive procedure. The urea breath test has been de­ veloped as a noninvasive procedure that serves as a sensitive and specific, although qualitative, indicator of infection. The patient is given an oral dose of la­ beled urea, either [ C]urea (39) or [ C]urea (7); if the organism is present, urea will be hydrolyzed and C 0 or C 0 will be liberated. The labeled carbon dioxide will enter the bloodstream, exchange in the lungs, and be exhaled. The exhaled C 0 is trapped and quantitated in a mass spectrometer for C 0 or a scintillation counter for C 0 . A number of mem­ bers of the normal anaerobic gut flora are urease posi­ tive and potentially could interfere with this test, but data collected thus far indicate that false-positive re­ actions are rare. 13

1 3

14

1 4

2

2

2

1 3

2

1 4

2

PCR Identification PCR amplification of H. pylori urease genes has been used in methods to establish the presence of via­ ble or nonviable H. pylori. In one method boiled supernatants from H. pylori strains are used as template for the reaction, and amplification of a 411-bp frag­ ment from ureA is used to identify specifically the bacterium ( 1 0 , 1 1 , 3 0 , 1 0 0 ) . The primers are sensitive and specific for H. pylori and do not react with other Helicobacter spp. In another method a pair of PCR primers, one of which is degenerate, is employed to amplify a 365-bp segment from an area adjacent to the 5' end of the ureA gene (102). Consistent amplifi­ cation from gastric juice was reported using this sys­ tem. PCR amplification from regions of the ureB gene

CHAPTER 16 • UREASE

has also been used to detect H. pylori in paraffinembedded biopsy samples (102).

187

UREASE IN VACCINES

tected from colonization of the gastric mucosa by H. felis. UreB, which harbors the enzyme active site, gave earlier and more complete protection from the chal­ lenge strain. In later studies, oral immunization with UreB was used therapeutically for eradication (14). In independent efforts, Ferrero et al. (29) used H. pylori and H. felis UreA and UreB, purified as maltose-bind­ ing protein translational fusions, to immunize mice with cholera toxin as the adjuvant. UreB provided more significant protection than UreA, and the H. felis-deiived subunits conferred better protection than the H. pylori urease subunits. Using the holoenzyme would have the advantage of retaining conformational epitopes, but it would be undesirable to retain ureolytic activity in preparations that would be administered by any route because of the ubiquitous presence of urea in humans. Catalyti­ cally inactive but fully assembled H. pylori urease was viewed as more desirable and was adopted as a vac­ cine candidate on the basis of observations of Hu et al. (45). They found that the H. pylori urease apoenzyme, lacking nickel ions in the active site and thus lacking enzymatic activity, could be isolated from E. coli ex­ pressing only the ureA and ureB structural subunit genes. These clones lacked the accessory genes neces­ sary for nickel ion incorporation. The apoenzyme was purified according to the precise scheme used for the first reported isolation of the native enzyme, and it eluted from the four resins used for purification (DEAE-Sepharose, Phenyl-Sepharose, MonoQ, and Superose 6) at the same fractions as those observed for the native enzyme (45). This result suggested that the apoenzyme possessed the identical charge, hydrophobicity, shape, and size of the native enzyme and thus was fully assembled lacking only the 12 nickel ions from the active site. On the basis of these data the apoenzyme was adopted as a vaccine candidate.

Because infected humans mount a significant im­ munoglobulin response to urease, it was reasoned that immunization with urease may protect against acqui­ sition of infection. A matter to be elucidated was the form of the protein that should be used in vaccination. Possible candidates included peptides derived from the predicted sequence, isolated subunits (UreA or UreB), apoenzyme, or holoenzyme; but thus far, pep­ tides have not been tested. Two groups of researchers used similar strategies, with the mouse model and an H. felis challenge as a surrogate for H. pylori. Michetti et al. found moderate but nevertheless significant pro­ tection when purified urease was used for immuniza­ tion (67). However, when the urease structural subunits UreA and UreB, purified separately as histidinetagged fusion proteins on a N i nitrilotriacetic acid column, were used for immunization, mice were pro­

Recombinant apourease encoded by clones car­ rying only ureAB has been used for immunization and also shown to provide significant protection for mice against H. felis challenge ( 5 8 , 7 4 , 79). In these studies, protection was correlated with a high secretory immu­ noglobulin A titer; in other work, antibody response was found not to be required for protection (25). The results of these investigations provided strong evi­ dence that oral immunization with urease in the pres­ ence of adjuvant is a feasible strategy for development of a vaccine for prevention of H. pylori infection in humans. Studies in monkeys with purified urease apo­ enzyme (93) or in humans using salmonella phoPI phoQ deletion mutant (38) expressing the apoenzyme demonstrated little or no protection against H. pylori infection ( 2 , 1 9 , 68). Other studies in monkeys, how­ ever, showed significant reduction in colonization (57).

TYPING SYSTEMS BASED O N UREASE GENES There is a significant variation between the DNA sequences of different H. pylori strains. The result is that for a given DNA segment there is heterogeneity between strains regarding sequences and restriction sites. This allowed the development of various typing methods based on urease genes. The first system de­ veloped was based on PCR amplification of urease genes (31, 32). PCR amplifications of the structural subunit genes ureA and ureB from clinical isolates yield 2.4-kb PCR products, which digested with Haelll produce distinct patterns on agarose gels. These patterns allow easy differentiation between strains. In another method to distinguish between strains of H. pylori the ureC gene (no longer consid­ ered a urease gene) is amplified, followed by direct DNA sequencing of the PCR product (15). Numerous base-pair changes are detected in ureC, and strains are easily differentiated. Seven subsequent reports confirmed that the urease genes ureA and ureB and the former ureC, subjected to PCR amplification and digestion with restriction endonucleases, can be used to differentiate strains on the basis of patterns on aga­ rose gels, identify the presence of multiple strains in a single biopsy specimen, check for reinfection with the same strain following eradication therapy, and identify similar strains among family members ( 4 , 1 2 , 18, 4 8 , 59, 72, 78). This general strategy has been successfully applied to other H. pylori genes.

2 +

188

MOBLEY

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CHAPTER 16 • UREASE

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CHAPTER 16 • UREASE

Nakasaki. 1994. A urease-negative mutant of Helicobacter pylori constructed by allelic exchange mutagenesis lacks the ability to colonize the nude mouse stomach. Infect. Immun. 62:3586-3589. 99. Turbett, G. R., P. B. Hoj, R. Home, and B. J. Mee. 1992. Purification and characterization of the urease enzymes of Helicobacter species from humans and animals. Infect. Immun. 60:5259-5266. 100. van Zwet, A. A., J . C. Thijs, A. M. D. Kooistra-Smid, J . Schirm, and J. A. M. Snijder. 1993. Sensitivity of culture com­

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pared with that of polymerase chain reaction for detection of Helicobacter pylori from antral biopsy specimens. /. Clin. Microbiol. 31:1918-1920. 101. Weeks, D. L., S. Eskandara, D. R. Scott, and G. Sachs. 2000. A H -gated urea channel: the link between Helicobacter py­ lori urease and gastric colonization. Science 287:482-485. 102. Westblom, T. U., S. Phadnis, P. Yang, and S. J. Czinn. 1993. Diagnosis of Helicobacter pylori infection by means of a poly­ merase chain reaction assay for gastric juice aspirates. Clin. Infect. Dis. 16:367-371. +

Helicobacter pylori: Physiology and Genetics Edited by H. L. T. Mobley, G. L. Mendz, and S. L. Hazell © 2 0 0 1 ASM Press, Washington, D.C.

Chapter 17

Ion Metabolism and Transport ARNOUD H . M . VAN VLIET, STEFAN BERESWILL, AND JOHANNES G . KUSTERS

have focused on nickel and iron cations and resistance to heavy metals and to acid. There is little information available on other cations or anions, and assignment of putative functions to different ions is only by ho­ mology to the (metallo)enzymes of other organisms. Since resistance to acid is discussed in a separate chap­ ter, H homeostasis is not treated here. H. pylori genes will be described by the HP numbering of the genome sequence of H. pylori 2 6 6 9 5 (108).

Ions play an important role in the metabolism of all organisms as reflected by the wide variety of chemical reactions in which they take part. Ions are cofactors of enzymes, catalyzing basic functions such as electron transport, redox reactions, and energy metabolism; and they also are essential for maintaining the osmotic pressure of cells. Because both ion limitation and ion overload delay growth and can cause cell death, ion homeostasis is of critical importance to all living or­ ganisms. In bacteria, this is achieved by balancing their uptake, efflux, utilization, and storage (Fig. 1). Helicobacter pylori colonizes the gastric mucosa of humans, and this niche provides a challenging mi­ lieu with continuous changes in environmental condi­ tions, including the concentration of ions. Despite a comparatively small genome of approximately 1,700 kb, H. pylori must be well adapted to this environ­ ment, as colonization is usually lifelong. The first suc­ cessful cultivation of H. pylori was reported only in 1983 (69, 117), and since then a wealth of research on this important pathogen has followed. Most of the initial investigations focused on diagnostics, treat­ ment, epidemiology, and virulence factors of H. py­ lori, and not on other fundamental issues such as physiology and metabolism. However, the publica­ tion of the complete genome sequence of two H. py­ lori strains has promoted more detailed studies of the physiology and metabolism of this interesting patho­ gen. Gastric helicobacters are unique among microor­ ganisms in their colonization of the gastric mucosa. For this reason H. pylori serves as a model organism that is well suited for the study of bacterial adaptation mechanisms and host-pathogen interactions. This review concentrates on the mechanisms used by H. pylori to maintain its ion homeostasis, empha­ sizing metal cations. T o date, most H. pylori studies

+

ION HOMEOSTASIS Ions participate in a great variety of cellular pro­ cesses and are essential for cell growth. Imbalance of the cytoplasmic concentration of ions leads to a multi­ tude of stresses, for example, those caused by changes in osmolarity, acid, metal toxicity, or oxidative dam­ age. Maintaining ion homeostasis requires both sen­ sor systems to detect the cytoplasmic ion concentra­ tion and effector systems to restore normal cell conditions, or to cope with stress caused by ion imbal­ ance. For most ions, the cell can affect homeostasis through regulation of the expression or the activity of its uptake and efflux systems. Since import of many cations appears to be relatively nonspecific, the corre­ sponding regulatory mechanisms are probably based mainly on ion-specific efflux pumps. For some ions the existence of cytoplasmic storage proteins allows for more complex homeostasis mechanisms. Ion stor­ age proteins remove excess ions from the cytoplasm and keep them in a nonreactive form, which can be accessed when the ion becomes scarce. The ion-responsive regulatory systems of bacte­ ria usually consist of a single regulatory protein that combines sensor and effector functions in one mole-

Arnoud H. M. van Vliet and Johannes G. Kusters • Department of Medical Microbiology, Faculty of Medicine, Vrije Universiteit, Amsterdam, The Netherlands. Present address: Department of Gastroenterology and Hepatology, Academic Hospital Dijkzigt, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands. Stefan Bereswill • Department of Microbiology, Institute of Medical Micro­ biology and Hygiene, University of Freiburg, Hermann-Herder-Str. 11, D-79104 Freiburg, Germany.

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( ^ TTransport rai

uxT) (uptake/efflux)

Ion Homeostasis

Figure 1. Schematic representation of the mechanisms involved in maintaining ion homeostasis.

cule. It senses the cytoplasmic ion concentration and, when activated, can induce or repress transcription of the corresponding uptake, efflux, and/or storage systems. H. pylori contains relatively few regulatory proteins (15), but these include homologs of the ironresponsive regulator Fur and the nickel-responsive regulator NikR (27, 3 0 , 113). Storage systems have been identified only for iron and possibly nickel, and they are described below. Since overload of both iron and nickel results in the generation of oxidative dam­ age, a brief description of mechanisms dealing with oxidative stress in H. pylori is included. Iron Homeostasis Regulation of iron uptake Iron availability is usually low in most environ­ ments, and thus bacteria require specific high-affinity iron acquisition systems, which are discussed in the "Ion Transport" section. Bacteria regulate their ironuptake and iron-storage systems in response to the cytoplasmic F e concentration (21). When this con­ centration becomes too high, bacteria switch off their high-affinity iron uptake systems. These iron-respon­ sive systems are mediated by the ferric uptake regula­ tor (Fur) protein, which acts mostly as an iron-acti­ vated transcriptional repressor (38). In some cases Fur can also mediate induction of transcription in re­ sponse to iron (34). Fur homologs, which are widely distributed in eubacteria, are thought to bind their operator DNA sequences only when sufficient F e is available in the cytoplasm, thus modulating the tran­ scription of the regulated genes. In the absence of FurF e complexes, the regulator stays and transcription 2 +

2 +

2 +

of Fur-regulated genes is not affected. Availability of free iron is normally restricted in the animal host, and pathogenic microorganisms often use low-iron condi­ tions as a stimulus for the concerted activation of viru­ lence factors, which can be mediated through Fur ho­ mologs (66). H. pylori contains a Fur homolog (HP1027) shown to function as a metal-responsive regulator ( 9 - 1 1 , 1 1 4 ) . The mutational inactivation of fur in H. pylori, as well as an H. pylori-adapted Fur titration assay (FURTA-Hp) (40), allowed the identification of several genes that are either directly or indirectly regu­ lated by Fur (9, 17, 4 0 , 114). In contrast with other bacteria, Fur regulates some but not all putative ironuptake systems of H. pylori. This suggests that under high iron conditions H. pylori is able to restrict but not close down completely its uptake of iron. How­ ever, when iron is scarce, H. pylori can express addi­ tional iron-acquisition systems securing sufficient iron (114). Iron storage Continuous uptake of iron creates the need for removal of iron from the cytoplasm and storage of excess iron. This protects the cell from iron toxicity and also provides for an iron deposit, which is avail­ able when iron is scarce. Bacterial iron storage pro­ teins can be divided into two classes: ferritins and bacterioferritins. Their most important structural dif­ ference is that bacterioferritins usually contain heme, whereas ferritins do not (2). Bacterioferritins are com­ posed of 2 4 subunits and are able to store approxi­ mately 4,500 iron atoms per molecule (2, 19). Re­ cently a new subgroup of bacterioferritins has been described that form an oligomer of 12, rather than 24 subunits, and only store approximately 5 0 0 iron atoms per molecule (19). H. pylori contains one ferri­ tin, the 19-kDa prokaryotic ferritin (Pfr) protein (HP0653), and one putative bacterioferritin, the HPNAP protein (HP0243). This Pfr is similar to eukaryotic H-chain ferritins and to other prokaryotic ferritins (33, 4 1 ) . It serves as an intracellular iron deposit and protects H. pylori against iron toxicity (14), and iron stored in Pfr can be released and reused to support growth under ironlimited conditions (116). The protective function of Pfr against metal overload may not be limited to iron, as shown by the increased sensitivity of a Pfr-negative mutant to manganese, copper, and cobalt (10, 14). These mutants showed a significant decrease in the iron uptake capacity (10) and increased resistance to oxidative stress (116). The H. pylori neutrophil activating Erotein (HPNAP) was originally isolated as an immunodominant

CHAPTER 17

• ION METABOLISM AND TRANSPORT

195

protein that activates neutrophilic granulocytes in vitro (39). It was subsequently shown also to mediate adhesion of H. pylori to mucin (82). The HP-NAP protein is homologous to both bacterioferritins and the DNA-binding proteins of the Dps family (109). HP-NAP binds iron, and the three-dimensional struc­ ture of HP-NAP provides strong evidence that the protein represents a member of the recently discov­ ered 12-subunit ferritin family (19). A role of HPNAP in H. pylori iron storage has been suggested, but it is yet to be demonstrated (109).

amino acid identity of 3 0 % and similarity of 5 5 % to the E. coli protein. Surprisingly, preliminary studies have shown that mutational inactivation of the H. pylori nikR gene does not affect expression of urease or the GroELS chaperone proteins, which are in­ volved in nickel metabolism of H. pylori ( 2 7 , 6 3 , 1 1 3 ) . Possible targets of H. pylori NikR are the nickel acqui­ sition system encoded by the genes nixA and abcCD (see "Nickel Transport," below). Further studies will be required to establish the precise function of NikR in H. pylori.

Regulation of iron storage

Nickel storage

Iron homeostasis requires regulation of iron stor­ age systems (Fig. 1). Expression of Pfr is induced by iron and repressed under iron-restricted conditions (14). This enables the bacteria to increase storage of iron if excess environmental iron is available and to secure availability of free cytoplasmic iron when it is scarce. Pfr expression is repressed under iron-re­ stricted conditions in wild-type strains but is constitu­ tive in the fur mutant, suggesting that Fur represses pfr transcription under iron-restricted conditions. This is unusual because in other bacteria the Fur protein is thought to be inactive in the absence of iron. How­ ever, the regulation of bacterial iron storage has not been investigated in detail, and the function of Fur still is not completely understood. H. pylori Fur also seems involved directly in downregulation of ferritin synthesis in response to other metals (9), and plays a role in nickel-responsive induction of urease expres­ sion in H. pylori (113). Although a Fur-negative mu­ tant showed no obvious growth deficiencies, it was more sensitive to increased levels of transition metals (10). This suggests that H. pylori Fur represents a more global metal-dependent regulator that orches­ trates the expression of metalloenzymes in response to the availability of different metal ions. Interest­ ingly, the H. pylori fur mutants display reduced acid resistance, underlining the central role of this regula­ tor in the control of ion homeostasis (17).

The H. pylori Hpn (HP1427) protein was ini­ tially isolated through its binding to nickel and zinc in vitro (49). Hpn is a small (7-kDa) protein that has a very high histidine and cysteine content (28 His and 4 Cys in 60 amino acids total) (49). Hpn is thought to play an important role in protection against nickel toxicity, possibly by binding excess cytoplasmic nickel. In line with a putative nickel storage or scav­ enging function, H. pylori Hpn-negative mutants are significantly more sensitive to nickel overload but not to cobalt and copper (77). These mutants were also not affected in their urease activity, indicating that Hpn is not required for nickel transport or urease apo­ protein activation (49). Although Hpn also forms complexes with zinc in vitro, resistance to zinc was not affected in the H. pylori hpn mutant (77).

Nickel Homeostasis Nickel regulation Recently, a nickel-responsive gene regulator, des­ ignated NikR, was identified in Escherichia coli (30). Similar to iron regulation by Fur, when free nickel is available NikR binds to the promoter of the E. coli nikA gene, which encodes a periplasmic nickel-bind­ ing protein and represses transcription of the nikABCDE nickel acquisition system (23, 2 4 , 30). H. pylori contains a NikR homolog (HP1338), which displays

Defense against Oxidative Stress H. pylori requires oxygen for optimal growth. However, in combination with oxygen, divalent met­ als contribute to the generation of reactive oxygen species such as superoxides and hydroxyl radicals through the Haber-Weiss and Fenton reactions (74, 110). Superoxide and hydroxyl radicals damage lip­ ids, proteins, and DNA by oxidation, and cells will attempt to remove superoxide and peroxides before they cause significant damage. In H. pylori, this is mediated by the enzymes superoxide dismutase (SodB) (HP0379), catalase (HP0875), and probably alkyl hydroperoxide reductase (HP1563) (87, 8 8 , 9 0 , 103). SodB activity seems to be essential for H. pylori, as the sodB gene could not be inactivated (12). The heme-cofactored catalase, however, is not essential in vitro, as H. pylori catalase-negative naturally occur­ ring mutants (68, 119) and mutants constructed by insertional mutagenesis (87) have been readily iso­ lated. In contrast to other bacteria, both SodB and catalase are present on the H. pylori cell surface (80) and are thought to protect H. pylori from toxic oxy­ gen metabolites produced by activated immune cells

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in the inflamed gastric mucosa. It has also been sug­ gested that secretion of SodB and catalase might also function as decoy for the evasion of the immune re­ sponse (80). A more detailed discussion of the func­ tions of these enzymes is given in chapter 15.

ION METABOLISM The adaptation of H. pylori to life in the gastric mucosa seems to have led to a more pronounced role of nickel in its metabolism, for example, in its role as an essential cofactor of urease, which mediates acid resistance of H. pylori. In contrast, relatively little is known about the role of other ions in H. pylori metab­ olism. This section gives a brief overview of H. pylori metabolic processes in which metal cations act as cofactors; this information was obtained from genome analyses and sparse experimental data. Nickel H. pylori expresses large amounts of urease, and levels can reach up to 1 0 % of total cellular protein (3). Urease contains 12 nickel atoms per molecule, and thus H. pylori has a relatively high demand for nickel. Native H. pylori urease is a multimeric protein that consists of six UreA and six UreB subunits (35). The active site of each UreB subunit contains two nickel ions (54, 78), and without these ions the urease apoenzyme is not active (57). Nickel is inserted into the urease apoenzyme by the UreE, UreF, UreG, and UreH proteins (29). Urease plays a central role in the pathogenesis of H. pylori infection (75) and catalyzes the conversion of urea into carbon dioxide and am­ monia. The latter is able to neutralize gastric acid and offer protection to H. pylori against the low pH in the stomach (64, 9 9 , 1 1 8 ) . In addition, ammonia may be used as a nitrogen source supporting growth of H. pylori (45). Another important H. pylori enzyme, likely to contain nickel, is a hydrogen uptake NiFe hydrogenase (67). Like NiFe hydrogenases from other bacteria, it is involved in electron transfer and respira­ tion and subject to anaerobic activation (67). A de­ tailed discussion of urease structure and function is given in the chapter devoted to this enzyme. Iron Iron is an essential nutrient for all living organ­ isms, with the exception of some lactobacilli. Iron is a cation that exists in the ferrous ( F e ) and ferric ( F e ) states. The redox potential of F e / F e in biomolecules spans a range from + 3 0 0 to - 5 0 0 mV, which makes iron well suited for participating in elec­ 2+

3+

2+

3+

tron transfer reactions. In addition, iron interacts chemically with oxygen, sulfur, and nitrogen ligands, allowing coordination of the iron atom in the active sites of enzymes with different redox potentials de­ pending on the protein environment surrounding the complexed iron (21, 36). Iron-containing proteins are mostly involved in basic cell metabolism. Ferroprotoporphyrin (heme) groups are essential moieties of many enzymes involved in bacterial respiration, elec­ tron transport, and peroxide reduction. Iron-sulfur proteins participate in electron transport reactions, anaerobic respiration, amino acid metabolism, and energy metabolism. Finally, iron-containing nonheme, non-iron-sulfur proteins are required for DNA synthesis, protection from superoxide, and amino acid biosynthesis (36). Potassium and Sodium Potassium is the predominant cation in the bacte­ rial cytoplasm and also is the main ion involved in the adaptation to changes in osmolarity. The first re­ sponse of bacteria when adapting to environmental high osmolarity is to increase the uptake of potassium (28). Adaptation to a low osmolarity medium leads to the efflux of potassium and other osmotic solutes through nonspecific channel proteins (28). The re­ sponse of H. pylori to either type of osmotic shock has not been extensively characterized, but there are indications that H. pylori regulates some of its heat shock chaperone proteins in response to salt and/or osmotic stress through the HspR regulatory protein (104). The function of sodium in bacterial metabolism is predominantly in symporter and antiporter systems for different nutrients. It also functions in maintaining the proton gradient necessary for the proton motive force, and thus has a role in energy production (52). Magnesium Magnesium is the most abundant divalent cation in living cells and often functions in conjunction with ATP in many enzymatic reactions. This cation is often found bound to cellular polyanions such as nucleic acids and lipids, and owing to its being mostly com­ plexed, magnesium does not contribute much to os­ motic processes (100, 102). In H. pylori the use of magnesium as cofactor has been demonstrated for its phospholipase/sphingomyelinase (N-SMase) enzyme (22). N-SMase is produced in vivo since it elicits an immune response in H. pylori-positive patients and is thought to contribute to the pathogenesis of H. pylori infection through its actions against epithelial cell membranes (22). Magnesium is a cofactor of many

CHAPTER 17

enzymes catalyzing modification, replication, and transcription of nucleic acids, and as such, the restric­ tion-modification systems of H. pylori are likely can­ didates to be magnesium-requiring enzymes (15). In other bacterial pathogens such as Salmonella enterica serovar Typhimurium and Bordetella pertussis, mag­ nesium is an important signal for the concerted expression of virulence genes (44, 9 7 ) . Indirect sup­ port for a similar function in H. pylori was derived from the effects of an aluminium-hydroxide-magne­ sium-hydroxide (co-magaldrox) combination drug that inhibits bacterial adhesion and interleukin 8 (IL8) secretion and decreases expression of HSP60 on the surface of the bacterium. Zinc Zinc metalloenzymes are for the most part in­ volved in catalytic reactions. Alcohol dehydrogenases, which use zinc as a cofactor, convert alcohol to acetaldehyde. H. pylori contains one or two zinc-alcohol dehydrogenases, which may contribute to the damage to the gastric epithelium (61, 96). The bacterium also displays zinc-dependent protease activity (120), and a membrane-bound protein (HP1069) homolog of E. coli FtsH has been identified. FtsH has a strong simi­ larity to a family of eukaryotic ATPases and has been suggested as a component of a proteolytic system in £. coli. FtsH homologs are involved in the proteolytic degradation of unstable proteins that can include both soluble regulatory proteins and membrane proteins (6, 4 8 , 7 3 , 98). In H. pylori, however, homologs of the other proteins of such a proteolytic system have not been found yet. Finally, zinc is a cofactor of the tRNA-modifying enzyme tRNA-guanine transglycosylase Tgt (HP0281), which assists in maintaining fi­ delity of translation (8, 9 3 , 9 4 ) . Trace Metals Bacteria also require trace amounts of metals such as copper, molybdenum, cobalt, and manganese, which commonly are toxic when present at high con­ centrations. The roles of most of these trace metals in H. pylori metabolism are as yet unknown. Some enzymes containing trace metals have been identified in H. pylori and are briefly reviewed here. Copper Owing to its two oxidation states, copper is well suited for participation in electron transfer reactions. It is present in the cfe-type cytochrome oxidase of H. pylori that functions as a terminal oxidase in the respi­

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ratory chain. This important enzyme also contains three iron-containing heme groups (81, 112). Molybdate This ion is taken up as molybdate (M0O4 ~ ) and converted into molybdopterin, which is the cofactor of several enzymes involved in the reduction of alter­ native electron acceptors (36, 5 0 , 92). Although the H. pylori genome does not contain orthologs of molybdoenzymes, there are at least 11 genes encoding orthologs of proteins involved in molybdopterin bio­ synthesis ( 1 , 1 0 8 ) . This suggests strongly that molyb­ denum is also essential for H. pylori, but its role in the metabolism of the bacterium requires further in­ vestigation. 2

ION T R A N S P O R T The cytoplasm of gram-negative bacteria is sur­ rounded by two membranes, an inner cytoplasmic membrane (CM) and an outer membrane (OM). Ions transported to the cytoplasm have to cross both mem­ branes. The O M contains porins with a general exclu­ sion limit of approximately 600 Da that allow diffu­ sion and transport of ions into and out of the periplasm (85). Ions that have reached the periplasm via porins cannot penetrate the CM, and specific transport systems have evolved for ion transport through the CM. Two types of ion C M transporters are commonly found in bacteria. The first class consists of multicomponent ABC-transporters, which have one or two membrane-spanning permeases, one or two ATPase proteins, and in many cases a binding protein that concentrates the substrate in the periplasm and deliv­ ers it to the permease-ATPase complex. Transport by ABC transporters uses ATP as the energy source. The second type of C M transporter is the one-component transporter, which uses either ATP or the proton mo­ tive force to supply energy for the transport. An over­ view of ion transport systems identified in H. pylori is given in Fig. 2. Nickel Transport H. pylori has a high demand for nickel as cofac­ tor for its urease enzyme and thus requires high-affin­ ity transport systems to scavenge nickel from environ­ ments usually low in nickel. The concentration of this ion in human serum is very low (2 to 11 nM), and the nickel concentration in ingested food varies signif­ icantly depending on the diet and on food sources (25, 106). Nickel might be transported by the magnesium

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© ©

(5D © ©

Figure 2 . Schematic overview of ion transport systems of H. pylori. The ion transported and the direction of transport are indicated. OM, outer membrane; CM, cytoplasmic membrane. Ion transporters are grouped depending on the direction of transport: importers at the top, and exporters at the bottom.

transporter CorA when the magnesium concentration is low, but this is thought to be of little relevance under physiological conditions (102). Specific nickel transporters have been described in other bacteria: the one-component transporters like the Ralstonia eutropha HoxN protein, and the multicomponent ABC transporters like the E. coli NikABCDE system (37). The first nickel transporter identified in H. pylori was the NixA protein (HP1077), a member of the HoxN-like one-component class of nickel transport­ ers (76). NixA is a 37-kDa protein located in the CM and contains eight transmembrane domains (42, 4 3 ) . Transport through NixA does not seem to be specific for nickel, and it has been suggested that it can trans­ port also cadmium, cobalt, and zinc (42). Nonethe­ less, the high affinity of NixA for nickel makes it very suitable to supply the high nickel demand of H. pylori (76). A nix A mutant constructed by insertional inactivation displayed significantly decreased nickel trans­ port and urease activity (4), although they were not completely abolished in this mutant, indicating that H. pylori has additional nickel transport systems. Ho­ mology searches of the H. pylori genome sequence allowed the identification of the abcC gene (HP1576) (55), which encodes a protein ortholog of the NikD ATPase of E. coli Nik, a nickel ABC transporter sys­ tem (83). The adjacent abcD gene (HP1577) encodes a protein with low homology to C M permeases. An

H. pylori abcD mutant was severely affected in its urease activity, but an abcC mutant showed the same phenotype as the nix A mutant. An abcC nix A double mutant has only residual urease activity, indicating that nickel uptake was almost completely abolished (55). The H. pylori AbcCD system lacks a homolog of the E. coli NikA periplasmic binding protein, which binds nickel ions and carries them to the C M trans­ porter. This suggests that the AbcCD system functions differently from the Nik system or that the homology between the putative H. pylori nickel-periplasmic binding protein and E. coli NikA is too low to identify an H. pylori NikA homolog. The nixA and abcCD mutational studies demon­ strate that H. pylori has two separate nickel acquisi­ tion systems, and this manifests the importance of nickel in H. pylori metabolism. A detailed discussion of NixA and nickel transport is also found in chapter 16. Iron Transport Soluble ferrous iron ions would be readily avail­ able for uptake by bacteria, but these ions are stable only under anaerobic and acidic conditions, because the presence of oxygen causes a rapid conversion to the ferric state, which is almost completely insoluble at a pH of > 7 . In tissues of human or animal hosts,

CHAPTER 17

the concentration of free iron is too low to support bacterial growth, as most iron is complexed into he­ moglobin or chelated by transferrin in serum or by lactoferrin (Lf) at mucosal surfaces. Complexed iron poses a problem for bacteria, since these complexes are too large to be transported through the porins of the O M . Thus, uptake of iron complexes requires either high-affinity transport mediated by O M recep­ tors or removal of the iron from the complex followed by transport (20, 2 1 ) . Unlike many other organisms, H. pylori does not synthesize the small iron-chelating molecules called siderophores, which make chelated or precipitated ferric iron available for acquisition by cells (36, 84). This is confirmed by analysis of the H. pylori genome sequence, which does not contain homologs of siderophore synthesis genes (15). Compared to the range of iron compounds that other bacterial pathogens like E. coli and 5. enterica serovar Typhimurium can uti­ lize, the number used by H. pylori is limited. Feeding assays indicate that H. pylori uses only very few sider­ ophores produced by other organisms (13, 32, 5 8 , 59). This limitation regarding iron acquisition may have developed owing to the absence of competition for nutrients by other microorganisms and the rela­ tively low number of iron compounds available in the human stomach. Iron sources available in the gastric mucosa are Lf-bound iron, heme compounds released from damaged tissues, and iron derived from pepsindegraded food. As the conditions in the gastric lumen and mucosa are predicted to stabilize the soluble fer­ rous iron, it is likely that, in contrast with many other bacterial pathogens, ferrous iron uptake plays an im­ portant role for H. pylori. Ferrous iron uptake Ferrous iron ions pass freely through the O M porins but require transport over the CM. In E. coli and S. enterica serovar Typhimurium, these ions are transported by the Feo system, comprising the FeoA and FeoB proteins (62, 111). The FeoB protein is lo­ cated in the C M and hydrolyzes ATP to generate en­ ergy for the transport process. The function of FeoA is not known, although in E. coli it is essential for ferrous iron transport (62). The H. pylori genome en­ codes a homolog of FeoB (HP0687), but there is no obvious FeoA homolog. FeoB is required for highaffinity ferrous iron ion uptake into H. pylori (115). Isogenic feoB mutants not expressing the protein were unable to colonize the gastric mucosa of mice, indicat­ ing the importance of FeoB-mediated iron acquisition for the bacterium (115). These results are consistent with the presence of ferrous ions in the stomach, owing to its low pH and oxygen concentration. A

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similar situation was demonstrated in E. coli, where feo mutants were unable to colonize the mouse intes­ tine (105). S. enterica serovar Typhimurium feoB mu­ tants were attenuated in an animal model when mice were infected by the intragastric route but not when infection used the intraperitoneal route (111). S. en­ terica serovar Typhimurium feoB mutants are also outcompeted by the wild-type strain in mixed intra­ peritoneal infections (111). The physiological role of the extracellular ion re­ ductase activity of H. pylori has been proposed to be the increase of the concentration of ferrous iron, by reduction of ferric iron (121). This activity appears to depend on expression of the iron-repressed riboflavin synthesis gene ribBA (HP0804), which is involved in the production of flavin-like molecules (121), since H. pylori ribBA mutants did not display reductase activity (121). Thus, it can be hypothesized that fla­ vins are cofactors of the reductase or directly mediate the reduction of iron. Interestingly, the H. pylori rib A and ribBA genes enable iron acquisition by E. coli strains deficient in siderophore-mediated iron uptake (7, 121). The iron reductase activity of H. pylori awaits further functional characterization and investi­ gation of its role in colonization in animal models. Ferric iron uptake At mucosal surfaces, most ferric iron is com­ plexed in heme compounds or chelated by Lf and can­ not cross the O M via porins owing to their size; thus, uptake of complexed ferric iron requires active trans­ port through both the O M and the CM. The transport systems involved in these processes have been investi­ gated extensively in other bacteria ( 2 0 , 2 1 ) . In general, the iron-carrier complexes are bound first to a specific O M receptor, and there the iron ions may be removed from the iron-carrier complexes. Subsequently, iron ions are transported across the O M to the periplasm, bound to a periplasmic-binding protein and trans­ ported to an ABC transporter at the cytoplasmic membrane, which translocates the ions to the cyto­ plasm. Alternatively, the complete iron-carrier com­ plex is transported through the O M , periplasm, and CM into the cytoplasm (20). It is well established that H. pylori can use Lf, ferric citrate, heme compounds such as hemoglobin, and hemin as its sole source of iron (13, 3 1 , 32, 5 8 ) . To date, the corresponding O M transport systems have not yet been fully established. Several reports describe iron-regulated outer membrane proteins (IROMPs), but none of these proteins have been identi­ fied or characterized at the molecular level. Some IROMPs are expressed in vivo, since they are recognized by antisera from H. pylori-positive human patients

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(123), and therefore might be the OM receptors for these iron compounds (31, 5 8 , 122, 123). The H. pylori genome sequences of strains 26695 and J 9 9 each contain six genes encoding putative IROMPs (1, 15, 108). Whereas in other gram-negative bacteria each iron compound usually requires a spe­ cific OM receptor, in H. pylori there seems to be re­ dundancy of IROMPs as there are three genes encod­ ing homologs of the E. coli ferric citrate receptor FecA (HP0686, HP0807, and HP1400) (20, 91) and three genes encoding homologs of the Neisseria gonorr­ hoeae low-affinity ferric enterobactin receptor FrpB (HP0876, HP0916/0915, and HP1512) (16). H. py­ lori is unable to synthesize or use the siderophore en­ terobactin as sole iron source (13, 58), suggesting that the FrpB homologs of this bacterium transport differ­ ent iron compounds. The fact that each of the H. py­ lori FrpB or FecA proteins shows stronger homology with the other members of the respective H. pylori protein family than with the corresponding E. coli or N. gonorrhoeae proteins suggests that either they have slightly different substrate specificities or that one or more gene duplication events have led to the multiple copies of the frpB and fecA genes. An H. pylori fecAl (HP0686) mutant was viable and did not show any deficiency in iron transport (115). This indicates that the FecAl protein is not essential under in vitro growth conditions and suggests that the other two FecA homologs are able to make up for the miss­ ing FecAl protein (115).

with low homology to the CeuE enterobactin trans­ port protein of Campylobacter jejuni (95). The final transport step of iron or iron com­ pounds across the CM usually is mediated by an ABC transporter. Such systems consist generally of one or two cytoplasmic membrane permeases and one or two ATP-hydrolyzing proteins (ATPases), which form the conduit in the membrane and supply the necessary energy, respectively. In contrast to the multitude of putative OM receptor genes present in the H. pylori genome, only one gene encoding an ABC transport system for iron could be identified. This system con­ sists of the permease FecD (HP0889) and the ATPase FecE (HP0888), which are homologous to compo­ nents of the E. coli ferric citrate transport system, e.g., the FecA proteins (20, 91). The H. pylori fecD and fecE genes are adjacent in the genome and are proba­ bly cotranscribed. An H. pylori fecDE mutant was not affected in its iron transport capability, manifest­ ing the redundancy of ferric iron transport systems in the bacterium (115). The fecDE genes are located downstream of the vacA gene encoding the vacuolat­ ing cytotoxin, which has been proposed to be regu­ lated by iron (107), although a link between VacA production and iron uptake has not been described yet. In conclusion, the presence of seven putative iron uptake systems indicates the importance of iron ac­ quisition for H. pylori.

The OM transport process requires energy that is supplied by the TonB/ExbB/ExbD protein complex located in the CM and the periplasm (Fig. 2 ). This complex transfers energy from the transmembrane potential to the OM receptors. This transfer requires direct contact between TonB and the OM receptor at its TonB-binding sequence (TonB-box) located in the N terminus of the protein (65, 79). Consistent with this need is the fact that all H. pylori FecA and FrpB homologs contain putative TonB-boxes. Two TonB homologs (HP1341, HP0582) and three ExbB/ExbD couples (HP1130-1129, HP1339-1340, and H P 1 4 4 5 - 1 4 4 6 ) were identified in the H. pylori ge­ nome sequence, again indicating redundancy in the iron transport systems. In agreement with this conclu­ sion is the observation that an H. pylori tonB (HP1341) mutant is viable and does not show any deficiency in iron transport (115). The three ExbB/ ExbD couples may be involved in the transport of different iron substrates, similar to the situation in Vibrio cholerae, which has two TonB-ExbB-ExbD sets that interact with different OM receptors (86). Although H. pylori cannot synthesize or use entero­ bactin, the genes HP1561 and HP1562 encode puta­ tive iron-transporting periplasmic binding proteins

Potassium Transport Potassium is the major cation in the bacterial cy­ toplasm, found at concentrations up to 0.5 M even when extracellular levels are very low (100). Potas­ sium is also the main cation involved in osmoregula­ tion and maintenance of the osmotic pressure (28). In E. coli several systems that maintain potassium home­ ostasis have been identified. These include constitu­ tive low-affinity importers and inducible high-affinity importers and exporters. The latter allow the cell to respond to changes in the extracellular potassium lev­ els (28, 100). Low-affinity, high-rate potassium importers of gram-negative bacteria such as E. coli consist of the two Trk transporters and the Kup system, whereas high-affinity low-rate potassium transport is me­ diated by the Kdp system (100). Surprisingly, none of these systems is present in H. pylori, and how H. py­ lori acquires potassium remains unknown. It is possi­ ble that potassium enters the cell through other, yet unidentified importers or through nonspecific chan­ nels. The gene HP0490 encodes a protein with low homology to bacterial orthologs of eukaryotic potas­ sium channels. Eukaryotic channels allow rapid and

CHAPTER 17

nonspecific passage of cations through the membrane in a single event (28). However, such a system can be expected to be insufficient to cater to cellular demands for potassium, and it is likely that H. pylori has other potassium uptake systems. Two highly similar potassium efflux proteins have been identified in E. coli, the KefB and KefC proteins, which mediate removal of excess potassium by acting as potassium-proton antiporters (100). H. pylori contains only a single gene (HP0471), encoding a Kef homolog, which is most closely related to KefB. Sodium Transport The intracellular sodium concentration of bacte­ ria is usually lower than that of the environment. This is mostly achieved by the export of sodium ions by sodium-proton antiporters (Fig. 2 ) . These transport­ ers assist in maintaining intracellular pH, osmolarity, and salt concentration (100). The H. pylori nhaA gene (HP1552) encodes a homolog of a sodium-proton antiporter. This gene is able to complement an E. coli mutant lacking all sodium-proton antiporter activity (60). An interesting difference between E. coli and H. pylori NhaA is their respective activities at different pH values. H. pylori NhaA shows high activity at pH values between 6 and 8.5, while E. coli NhaA is only active above pH 8. The activity of H. pylori NhaA at higher proton concentrations may reflect an adapta­ tion to the acidic conditions in the gastric mucosa. It was recently postulated that a stretch of 40 extra amino acids that is only present in H. pylori NhaA might mediate its altered pH sensitivity (60). H. pylori contains a second gene (HP1183) encoding a putative sodium-proton antiporter homolog as well as four ho­ mologs of transport systems that probably use sodium as symport or counterport ion, namely, the sodiumglutamate (HP1506), sodium-proline (HP0055), and sodium-alanine (HP0942) symporters and L-serine permease (HP0133). Magnesium Transport Bacteria require micromolar concentrations of magnesium for growth, and thus have the need for active magnesium acquisition. Magnesium uptake has been extensively studied in S. enterica serovar Typhi­ murium, which contains four magnesium transport systems, the CorA, MgtA, MgtB, and MgtE transport­ ers (102), of which only a CorA homolog (HP1344) is present in H. pylori. CorA homologs are found in almost all microbial genomes analyzed so far and are thought to be ubiquitous in the eubacteria (102). S. enterica serovar Typhimurium CorA can mediate both import and export of magnesium, cobalt, and

• ION METABOLISM AND TRANSPORT

201

nickel (102). It has also been implicated to transport ferrous iron (51). However, the affinity of S. enterica serovar Typhimurium CorA for magnesium is much higher than for cobalt and nickel (102). The role of the H. pylori CorA homolog in cation transport has not been investigated experimentally, and its cation specificity may well differ from that of its eubacterial homologs. Trace Metal Transport In most gram-negative bacteria, transmembrane ion import via transport proteins like NixA or CorA occurs with relatively little ion specificity, although with different affinities (42, 102). Some metals might enter the cell also through phosphate or other ion transporters. Bacteria require small amounts of transi­ tion metals like cobalt or copper, but when present at higher concentrations, these metals are toxic (10, 56, 101). Other transition metals like cadmium are toxic even at very low concentrations. To keep the concentration of these cations below toxic levels, bac­ teria require efflux systems next to uptake systems. This is especially important for H. pylori, as the enzy­ matic activity of its urease is very sensitive to transi­ tion metals (89). Metal resistance in bacteria is predominantly achieved by specific P-type ATPases such as CopA (101), and/or by multicomponent cation-proton anti­ porters as the R. eutropha CzcABC system (101). Analyses of the H. pylori genome sequences indicate the presence of genes encoding both types of metal resistance systems. Three H. pylori P-type ATPases have been identified so far, and two of them, CadA and CopA, have been characterized experimentally. The cadmium-zinc-cobalt efflux pump CadA The H. pylori protein CadA encoded by gene HP0791 was originally cloned by hybridization screening for the conserved phosphorylation motif of P-type ATPases (72). The protein sequence is homolo­ gous to other bacterial cadmium and copper P-type ATPases and contains eight transmembrane domains (71, 72). Mutational studies demonstrated that CadA is involved in resistance to cadmium, zinc, and cobalt, but not to copper or nickel, indicating that this ex­ porter is not specific for a single metal (56). Inactivation of cadA also reduced urease activity and nickel accumulation in some but not all the mutants (56), but the reduced urease activity was not related directly to the disruption of cadA, and the exact role of CadA in metal and urease metabolism remains to be deter­ mined (56).

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The copper efflux pump CopA The H. pylori copper resistance determinant con­ sists of two genes, cop A (HP1072) and cop? (HP1073) (5, 6, 4 6 , 4 7 , 70). The protein CopA is a P-type ATPase, exports copper ions, and is homolo­ gous to other copper-transporting enzymes. CopA is predicted to have eight transmembrane domains, sim­ ilar to CadA (72). Insertional mutagenesis of cop A made H. pylori significantly more sensitive to copper, but the resistance to cadmium, mercury, nickel, cal­ cium, and magnesium was not affected (5, 6, 4 6 , 4 7 ) . CopP is a homolog of the Enterococcus hirae CopZ protein (26). CopZ-like proteins are copper-chaperones, delivering copper to enzymes and regulators (26, 5 3 ) . As mutation of cop? did not alter copper sensitivity, the role of CopP in copper metabolism of H. pylori remains unclear. Possible functions of CopP are storage of copper (analogous to iron storage by ferritin) or chaperone activity like that of CopZ (26, 53). Other cation efflux systems In addition to cadA and cop A, H. pylori contains genes for three other putative cation efflux systems: HP1503 encoding a third P-type ATPase, designated CopA2 or Fixl, and HP0969/0970 and HP1329/ 1328, part of an incomplete czc multicomponent sys­ tem. Czc cation-efflux systems are cation-proton antiporters and usually contain a C M transporter (CzcA), a periplasm-spanning protein (CzcB), and an O M protein thought to be involved in transport across the O M (CzcC) (101). There are two separate loci encod­ ing CzcA and CzcB homologs, but a CzcC homolog has not been identified in the H. pylori genome se­ quences. Experimental evidence for any of these sys­ tems being involved in cation transport is not avail­ able yet. Mutation of czcA gene HP0969 resulted in an acid-sensitive phenotype, suggesting a role for the CzcA protein in acid tolerance (18), although the mechanism for the acid-sensitive phenotype of the czcA mutant is not known.

ModD and ModF, of unknown function (50). H. py­ lori contains genes encoding ModA (HP0473), ModB (HP0474), and ModC (HP0475) homologs, but genes ortholog to those encoding the regulator ModE as well as ModD and ModF homologs have not been found.

CONCLUSIONS During coevolution with the host, H. pylori has developed unique systems necessary for survival in the human stomach. Concerning competition for nu­ trients as well as the acidic pH, this niche differs con­ siderably from the environments most other bacteria colonize. Elements of its systems for ion metabolism and transport seem to reflect specific adaptations of H. pylori to its ecological niche, and the functions and mechanisms involved might be different from the paradigms developed for enteric bacteria like E. coli. H. pylori contains relatively low numbers of ion transport systems, except for nickel and iron. These two ions can be transported by two or more systems, indicating their importance in the physiology of H. pylori. The number of regulatory proteins involved in ion metabolism and uptake is also low, and those that have been identified seem to be involved in the regula­ tion of nickel and iron homeostasis. This is surprising, as the gastric mucosa where H. pylori resides is a chal­ lenging environment, in which conditions can vary considerably. Thus, it is possible to infer that systems contributing to ion homeostasis in H. pylori have an extra level of functionality. Further insights into the mechanisms governing ion homeostasis might allow the development of new or improved strategies to pre­ vent or cure infection of this versatile pathogen. Acknowledgments. We thank our present and past collaborators for their valuable suggestions and helpful discussions and the Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO grant 901-14-206) and the Deutsche Forschungsgemeinschaft (DFG grants Ki201/8-l, Ki201/8-2, and Ki201/9-l) for financial support.

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76. Mobley, H. L., R. M. Garner, and P. Bauerfeind. 1995. Heli­ cobacter pylori nickel-transport gene nixA: synthesis of cata­ lytically active urease in Escherichia coli independent of growth conditions. Mol. Microbiol. 16:97-109. 77. Mobley, H. L., R. M. Garner, G. R. Chippendale, J. V. Gil­ bert, A. V. Kane, and A. G. Plaut. 1999. Role of Hpn and NixA of Helicobacter pylori in susceptibility and resistance to bismuth and other metal ions. Helicobacter 4:162-169. 78. Mobley, H. L., M. D. Island, and R. P. Hausinger. 1995. Molecular biology of microbial ureases. Microbiol. Rev. 59: 451-480. 79. Moeck, G. S., and J . W. Coulton. 1998. TonB-dependent iron acquisition: mechanisms of siderophore-mediated active transport. Mol. Microbiol. 28:675-681. 80. Mori, M., H. Suzuki, M. Suzuki, A. Kai, S. Miura, and H. Ishii. 1997. Catalase and superoxide dismutase secreted from Helicobacter pylori. Helicobacter 2:100-105. 81. Nagata, K., S. Tsukita, T. Tamura, and N. Sone. 1996. A cbtype cytochrome-*; oxidase terminates the respiratory chain in Helicobacter pylori. Microbiology 142:1757-1763. 82. Namavar, F., M. Sparrius, E. C. Veerman, B. J. Appelmelk, and C. M. J. E. Vandenbroucke-Grauls. 1998. Neutrophilactivating protein mediates adhesion of Helicobacter pylori to sulfated carbohydrates on high-molecular-weight salivary mucin. Infect. Immun. 66:444-447. 83. Navarro, C , L. F.Wu, andM. A. Mandrand-Berthelot. 1993. The nik operon of Escherichia coli encodes a periplasmic binding-protein-dependent transport system for nickel. Mol. Microbiol. 9:1181-1191. 84. Neilands, J. B. 1995. Siderophores: structure and function of microbial iron transport compounds. /. Biol. Chem. 270: 26723-26726. 85. Nikaido, H. 1996. Outer membrane, p. 29-47. In F. C. Neidhardt (ed.), Escherichia coli and Salmonella: Cellular and Molecular Biology, 2nd ed. ASM Press, Washington, D.C. 86. Occhino, D. A., E. E. Wyckoff, D. P. Henderson, T. J. Wrona, and S. M. Payne. 1998. Vibrio cholerae iron transport: haem transport genes are linked to one of two sets of tonB, exbB, exbD genes. Mol. Microbiol. 29:1493-1507. 87. Odenbreit, S., B. Wieland, and R. Haas. 1996. Cloning and genetic characterization of Helicobacter pylori catalase and construction of a catalase-deficient mutant strain. /. Bacte­ riol. 178:6960-6967. 88. O'Toole, P. W., S. M. Logan, M. Kostrzynska, T. Wadstrom, and T.J. Trust. 1991. Isolation and biochemical and molecu­ lar analyses of a species-specific protein antigen from the gas­ tric pathogen Helicobacter pylori. J. Bacteriol. 173:505-513. 89. Perez-Perez, G. I., C. B. Gower, and M. J. Blaser. 1994. Ef­

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Helicobacter pylori: Physiology and Genetics Edited by H. L. T. Mobley, G. L. Mendz, and S. L . Hazell © 2 0 0 1 ASM Press, Washington, D.C.

Chapter 18

Metabolite Transport BRENDAN P . BURNS AND GEORGE L . MENDZ

transport and energy-coupling mechanism (42). Channel proteins transport via energy-independent facilitated diffusion mechanisms employing a trans­ membrane pore. Secondary carriers are the largest and most diverse category of transporters; these can be proton motive force-driven, sodium motive forcedriven, and other ion- or solute-driven exchangers. These secondary transporters are classified into three main groups: (i) uniporters catalyze the movement of a single solute, (ii) symporters couple two or more solutes in the same direction, and (iii) antiporters cou­ ple solute movement in the opposite direction (23, 44). The third type of transport system is the ATPdriven primary active transporters; they use ATP hy­ drolysis as a mode of energy coupling for the active influx and/or efflux of solutes. The last type of trans­ porters consists of translocators that phosphorylate their substrates during transport; they are part of the bacteria-specific phosphotransferase system.

In its natural habitat on the lining of the stomach gastric epithelium, Helicobacter pylori is exposed to relatively high concentrations of host breakdown products (3), and the bacterium may obtain its nu­ trients from blood supplying the epithelial cells and intercellular junctions ( 5 , 2 0 ) . Also, H. pylori appears to have both a limited and fairly basic biosynthetic capacity (21) and thus it would be expected that there are transport systems to sequester nutrients from the external environment. Information about the mecha­ nisms of specific transport mediators is important in determining the contribution of a particular metabo­ lite to overall bacterial metabolism. An understanding of the physiology and metabolism of H. pylori at the level of nutrient influx/efflux is essential to furthering our knowledge of its basic physiology, pathogenesis, and ability to survive in such a harsh environment. The acquisition of nutrients by bacteria provides the organisms with both matter and free energy, es­ sential components for maintaining and sustaining life. Many bacteria are capable of regulating the influx of nutrients and ions across the membrane by specific carrier proteins (12). Transport systems present in bacteria perform various functions in the physiology of the cells. Some are central in the maintenance of cellular homeostasis such as osmotic balance and cy­ toplasmic pH; others are involved in drug resistance, whereby an organism lowers the cytoplasmic drug concentration by direct efflux of drugs across the cy­ toplasmic membrane; but the majority of transport systems function to acquire nutrients or release meta­ bolic products (23). Through the action of specific transporters, bacterial cells can concentrate a nutrient that is in low concentration in the medium inside the cell. Each of these systems is made up of carriers or porters that facilitate substrate movement along an electrochemical gradient. Bacterial transporters have been classified in four distinct types on the basis of

This chapter describes the specific transport stud­ ies carried out in H. pylori and the information about transporters in the bacterium that has been obtained from analyses of the genome. A summary of the cur­ rent understanding of metabolite transport in H. py­ lori is presented by combining the results of physio­ logical and genomic studies. Most of the investigations of metabolite transport in H. pylori em­ ployed a centrifugation through oil method originally described by Eisenthal et al. (14). Briefly, a cell sus­ pension is added to an aqueous buffer solution con­ taining radioactively labeled substrate permeant that has been layered onto oil in a centrifugation tube. Cells and permeant are allowed to mix for a fixed amount of time, and then are centrifuged to pellet the cells through the oil and stop the transport process (53). The technique yields intervals of permeant expo­ sure as brief as 5 to 10 s (25), as separation of the cells from the permeant solution occurs immediately

Brendan P. Burns • Max von Petennkofer Institut fur Hygiene und Medizinische Mikrobiologie, Milnchen, Germany. George L. Mendz • School of Biochemistry and Molecular Genetics, The University of New South Wales, Sydney, NSW 2052, Australia.

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as they traverse the top meniscus of the oil layer. Alter­ natively, the uptake of urea was measured in one study by expression of a transport-associated gene in Xenopus oocytes (59). Genomic analyses were carried out on the fully sequenced and annotated genomes of H. pylori strains 2 6 6 9 5 (57) and J 9 9 (1). GLUCOSE T R A N S P O R T

>

Owing to the critical finding that H. pylori is capable of metabolizing glucose (30), a complete un­ derstanding of glucose metabolism in H. pylori re­ quired the investigation of the mechanisms that regu­ late the transport of this metabolite. A carriermediated entry of glucose into H. pylori cells is sug­ gested by the following properties of the transport process: (i) stereospecificity for D-glucose, demon­ strated by the exchange with external glucose, and the inhibition observed in the presence of D-glucose but not L-glucose; (ii) saturable kinetics; and (iii) tem­ perature dependence (25). Histological investigations show that the region of the gastric mucosa colonized by H. pylori is rela­ tively free of other microbial flora (48, 63); thus, in this niche the competition for nutrients by other mi­ croorganisms is limited. The lack of potential compet­ itors, the amount of glucose in its environment, and the use of other nutrients available in preference to glucose (30) may explain the relatively high K of 4.8 mM measured for the transport of 2-deoxyglucose (Fig. 1), and the concomitant low affinity of the transm

Na

Li

K

Rn

Cs

Figure 2. Effects of monovalent cations on the rates of glucose transport. Rates of 2-deoxyglucose transport into H. pylori strain NCTC 11639 cells suspended in phosphate buffers constituted with the chloride salts (150 mM) of different monovalent cations.

porters for the substrate (25). Protein carrier-me­ diated transport is generally very sensitive to changes in temperature owing to the dependence of the rates of many processes on membrane fluidity (47), and the effects of temperature on glucose transport were also investigated. The Arrhenius plots of the transport of glucose provide evidence that the process is mediated by protein transporters at low and high saccharide concentrations. The significantly different activation energies measured for 0.5 and 2 0 mM glucose, 6.8 and 51.0 k j m o l , respectively, suggest that more than one type of carrier may be involved in the trans­ port. The sigmoidal dependence of glucose transport on N a ions and the strong dependence of transport rates on this ion relative to other monovalent cations suggest that the entry of the saccharide into H. pylori cells is linked to N a (Fig. 2 ) . Inhibition of glucose transport by the sodium transport inhibitor amiloride supports this interpretation (25). Dissipation of the sodium transmembrane gradient by monensin would explain its strong inhibition of the glucose carrier. In bacteria, accumulation of solutes ordinarily is carried out by mechanisms other than by N a symport, and direct coupling of metabolite and N a translocations is infrequent (49). However, N a plays an important role in ion-coupled-solute-symport systems in organ­ isms that live in environments rich in sodium ions such as marine, halophilic, alkaliphilic, and rumen bacteria (56). Escherichia coli has carriers that can transport glutamate or proline in symport with H or N a (13, 40), but among the sugar transporters only melibiose permeases use N a as a coupling ion in this bacterium - 1

140 I

'

1

•

1

•

1

'

1

1

r

+

+

+

+

0

5

10

15

20

25

30

2-DEOXY-GLUCOSE (mM) Figure 1. Kinetics of glucose transport. Rates of transport of 2deoxyglucose into H. pylori strain NCTC 11639 cells as a function of permeant concentration. Initial rates were determined at a fixed timepoint of 20 s using the centrifugation through oil method. The values represent the average of three measurements.

+

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CHAPTER 18 • METABOLITE TRANSPORT

(22) and Salmonella enterica serovar Typhimurium (52). The sigmoidal dependence of glucose transport on sodium concentration in H. pylori suggests the possibility of a regulatory role for the cation in the uptake of glucose. Thus, the transporters of H. pylori appear to belong to a specific class of glucose carriers able to utilize the sodium ion electrochemical gradient to translocate the saccharide. The effects of pH on glucose transport were also investigated, and no significant changes in transport rates are observed between pH 6.8 and 8.2, nor for gradients established using extracellular pH between 4 and 8.5 (25). However, the inhibition of transport by carbonyl cyanide w-chlorophenyl hydrazone (25) suggests that the transmembrane H gradient may be a factor in the entry of glucose into H. pylori, since the protonophore would induce proton electrochemical equilibrium across the membrane. If H. pylori has an Na , K -ATPase that can be inhibited by ouabain, the lack of effect of this inhibitor on the transport of glu­ cose into the bacterium (25) suggests that transport of the monosaccharide does not depend on this enzyme activity, although other energy-requiring mechanisms may be operative in the translocation of glucose. Glu­ cose transport is not affected by the known inhibitors cytochalasin B, phloretin, and phloridzin; considering that these compounds are able to inhibit passive and active saccharide transport in prokaryotes and eukaryotes (2), the glucose transporters of H. pylori ap­ pear to be different from the types found in other bacteria. The presence of a highly specific glucose transport system was thus demonstrated in H. pylori. The transport of glucose is sodium-linked and unaf­ fected by inhibitors of other known bacterial glucose carriers. These features agree with its peculiar ecologi­ cal niche, and unique glucose carriers may have evolved in H. pylori. +

+

+

FUMARATE T R A N S P O R T Several amino acids including asparagine and as­ partate are deaminated at fast rates by H. pylori, and fumarate is a main product of the catabolism of these two amino acids (28). H. pylori metabolizes fumarate (27, 3 2 ) , and the presence of intermediary fermen­ tative metabolism has been established ( 8 , 2 4 , 3 1 , 3 3 ) . Conversion of pyruvate to acetate and formate pro­ vides evidence for the existence of a mixed-acid fer­ mentation pathway in H. pylori, and the accumula­ tion of lactate from pyruvate shows the presence of fermentative lactate dehydrogenase activity in the mi­ croorganism (24). Fumarate at high concentrations is an inhibitor of H. pylori fumarate reductase, a key component of fumarate respiration in the bacterium

209

(32). Owing to the importance of this metabolite in the overall metabolism of H. pylori, a detailed study characterizing fumarate transport was conducted (37). The study shows significant rates of fumarate transport at micromolar permeant concentrations. The transport has saturable kinetics, is reversible, de­ pends on temperature, and is affected by the presence of both dicarboxylic acids and inhibitors (Fig. 3) (37). Inhibition of fumarate reductase by oxantel (32) does not affect the rates of fumarate influx or efflux, indi­ cating that the transport is not coupled to the last step of anaerobic respiration and that the label is trans­ ported as fumarate. The difference between the ki­ netic parameters for the influx and efflux of fumarate suggests an asymmetry in the intake and release trans­ port systems. These results together with the data from time courses (37) provide evidence that influx and efflux of fumarate at low concentrations from H. pylori cells is a carrier-mediated secondary transport, with the driving force supplied by the chemical gra­ dient of the anion. The influx of fumarate at millimolar concentra­ tions shows nonsaturable kinetics suggesting the pres­ ence of leakage, that is, pathways other than the trans­ port system observed at micromolar permeant concentrations (51). Fumarate leakage becomes sig­ nificant at high extracellular permeant concentrations and depends on the age of the cells (37), indicating that it is associated with the general morphological changes in the H. pylori cell wall that lead to the con­ version of bacillary forms to coccoid forms (7, 5 0 ) . Thus, the data suggest that fumarate leakage may arise from a permeabilization of the bacterial mem­ brane, although it is possible also that a low-affinity, high-capacity transport system expressed late in the growth phase of the cells could cause or contribute to the leakage. The rates of transport of fumarate increase with the amount of succinate loaded into the cells, indicat­ ing that the entry of fumarate is facilitated by the ef­ flux of succinate and suggesting the presence of an antiport fumarate/succinate system. The anaerobic fu­ marate transport in E. coli is stimulated by preloading bacterial cells with dicarboxylates, and the proposed physiological role of this system is a fumarate/succi­ nate antiport under conditions of fumarate respira­ tion (15). Anaerobic fumarate respiration is present in H. pylori and its inhibition leads to cell death (27, 32). The fumarate/succinate antiport would maintain charge neutrality by exchanging dicarboxylic anions and at the same time export succinate, the principal product of fumarate respiration, thus facilitating this mode of respiration in H. pylori. This type of ex­ change process, which does not consume energy for

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Control Nigericin Monensin Valinomycin Oligonycin CCCP 2,4-DNP Furosemide Ouabain Amiloride p-CMB lodoacetamide Cyanide Azide Fluoride

0

20

40

60

80

100

120

Rates (%) Figure 3. Effects of inhibitors on fumarate transport. Rates of fumarate transport into H. pylori NCTC 11639 cells suspended in phosphate-buffered saline, pH 7, determined at a fumarate concentration of 100 u.M, 20°C, and a timepoint of 30 s.

substrate/product translocation, is important for fer­ mentative bacteria, which generate limited amounts of metabolic energy from their catabolism (44). This exchange mechanism would be very appropriate for H. pylori, in which several fermentative pathways have been identified (27, 3 1 , 33). The low-affinity fu­ marate transport system of H. pylori may reflect the ability of the bacterium to obtain fumarate readily from amino acids (27, 32) and thus would not have an essential requirement for uptake of this anion. At the same time the dicarboxylate transporter could be employed for the efflux of succinate produced by an­ aerobic respiration in exchange for other extracellular substrates. The dependence of fumarate influx rates on al­ kali metal ions (37) suggests that the transport of the anion into H. pylori cells is linked to monovalent cat­ ions. The inhibition of fumarate influx by dissipation of the sodium transmembrane gradient with monen­ sin supports this interpretation. Because fumarate entry is not inhibited by the sodium transport inhibi­ tor amiloride (37), the results suggest that the system transports fumarate-alkali cation complexes. In this case, to maintain an electroneutral exchange process in the fumarate/succinate antiport, the efflux of succi­ nate would have to take place with succinate-cation complexes. The absence of inhibitory effects by furo­ semide (37) indicates that the fumarate carrier or car­ riers are different from the N a , K , 2 C 1 carrier. The +

+

-

incorporation of radioactive carbon atoms from fu­ marate into the biomass of the bacterium shows that besides the utilization of this metabolite in fumarate respiration for bioenergetic purposes, the carbon skel­ eton of the dicarboxylate is employed also in biosyn­ thesis. Most of the incorporated label goes into lipid (53%) and protein synthesis ( 2 9 % ) , suggesting a dis­ tribution of the fumarate carbon skeleton through in­ termediates of the Krebs cycle (43).

ARGININE T R A N S P O R T L-arginine and six other amino acids are an essen­ tial requirement for growth of H. pylori (41, 45), and several amino acids including L-arginine, L-asparagine, L-aspartate, and L-serine are utilized at fast rates in liquid cultures (29). Catabolism of L-arginine to l ornithine is observed in suspensions of metabolically competent cells or lysates, and the utilization of Larginine takes place via the arginase pathway (24). It has been established also that the bacterium has a complete urea cycle (29). The transport of arginine into the cells was investigated with the aim of obtain­ ing a better understanding of the nitrogen metabolism of H. pylori (34). L-arginine influx into H. pylori occurs at a fast rate and is saturable, and the rate of transport is tem­ perature dependent. The substrate specificity of the

CHAPTER 18 • METABOLITE TRANSPORT

&

40

211

undertaken indepth studies of urea transport. A study with wild-type strains, wreJ-negative mutants, and wreJ-negative mutants with a second mutation in one of the structural genes of urease were analyzed for transport of radioactive urea (26). The transport of this metabolite has saturable kinetics, with an appar­ ent K for influx and efflux of 2 0 0 and 195 uM, respectively, indicating that the transport is reversible (26). Experiments of urea transport at different tem­ peratures showed that the process is temperature de­ pendent. This urea transport system is able to recog­ nize ureido compounds with different substituents, and compounds with alkyl groups compete for trans­ port more strongly than those with sulfur substituents (26). The data suggest that urea influx and efflux at low concentrations from H. pylori cells are carriermediated secondary transport, with specificity for ureido groups and with the driving force supplied by the chemical gradient of urea. Rates of urea transport were also determined at pH values between 3.3 and 7.3, and no significant changes in transport rates were observed for mutants with the ureA and ureB, the urel, or the ureA, ureB, and urel genes knocked out (26). The results with mutants with the urel gene knocked out suggested urea transport is independent of Urel, and the rates measured at different pH in cells with or without Urel supported this conclusion. In a series of different stud­ ies (46, 59), it was strongly postulated that Urel was involved in urea transport. These workers found that Urel expression in Xenopus oocytes resulted in a pHdependent urea uptake. The uptake was specific for urea, nonsaturable, and temperature independent. It is therefore possible that two urea uptake systems op­ erate in H. pylori, a pH-dependent and a pH-independent one, although the results with bacteria are at variance with the urea transport measured in Xeno­ pus oocytes discussed in chapter 2 5 . m

3.2

3.3

3.4

3.5

3.6

3.7

1/T (1000/K) Figure 4. Effects of temperature on arginine transport. Arrhenius plots of arginine transport into H. pylori strain NCTC 11639 cells. Initial rates were determined at a permeant concentration of 0.5 mM, 20°C, and a fixed timepoint of 30 s using the centrifugation through oil method. The values represent the average of three mea­ surements.

transport was studied in competition experiments by measuring the effects of analogs on L-arginine trans­ port rates, since a metabolite transported via the same system would affect the rate of transport of L-arginine. No significant effects are measured for L-arginine ana­ logs and other amino acids, indicating that the trans­ port system is highly specific. In particular, the lack of effects observed with D-arginine shows that the transport system is stereospecific for the L-isomer (34). The saturable nature of L-arginine transport and its dependence on temperature (Fig. 4) suggest the presence of one or more carrier proteins, and the com­ petition experiments with L-arginine analogs indicate that these transporters are highly specific (34). The lack of inhibition observed with L-lysine and L-ornithine indicates that the H. pylori carriers are different from those of the LAO (lysine-arginine-ornithine) transport system commonly found in other prokaryotes and in eukaryotes.

NUCLEOTIDE T R A N S P O R T UREA T R A N S P O R T It has been proposed that urea is central in main­ taining intracellular nitrogen balance and protecting H. pylori from the harsh conditions of the stomach, and the organism has been shown to metabolize urea at very fast rates (29). The urease gene cluster encodes seven different genes, of which urel encodes a mem­ brane protein with homology to putative amide trans­ porters (11). The transport of urea across the cyto­ plasmic membrane would be intrinsically linked to its metabolism, and consequently several groups have

The acquisition of nucleotides is a vital cellular process, since they have central roles as building blocks of DNA and RNA, structural components of major coenzymes, and storage of chemical energy. Several studies have been undertaken to characterize the transport of these compounds by H. pylori. A study on the incorporation of uracil and uridine and UPRTase activity indicates that the bacterium is capa­ ble of obtaining pyrimidine nucleotides by salvage of precursors (35), although compared to other gramnegative bacteria, H. pylori shows a very limited abil­ ity to incorporate preformed pyrimidine bases or nu-

212

BURNS AND MENDZ

cleosides, suggesting a pyrimidine metabolism that may be similar to that of Neisseria spp. The salvage route for pyrimidines in H. pylori was investigated by measuring the incorporation of orotate, uracil, uridine, cytosine, deoxycytidine, thy­ mine, and thymidine and the activities of several ki­ nases (35). It is of interest to note that although the bacterium did not appear to take up either deoxynucleoside in any significant amounts, thymidine ki­ nase activity could be detected. The role of this activ­ ity in H. pylori is unclear, since in other organisms it is common that exogenous thymidine is incorporated into DNA much more efficiently than would be ex­ pected from the activities measured for the H. pylori enzymes. Under the experimental conditions em­ ployed, much larger rates of utilization were mea­ sured for orotate than for uracil, cytosine, thymine, or any of the pyrimidine nucleosides tested (35). In contrast, the pyrimidine precursors aspartate and bi­ carbonate are taken up readily by H. pylori cells (35). The high percentage of carbon atoms from these pre­ cursors incorporated by the cells that end up forming part of genomic DNA suggests that the de novo path­ way is the ordinary route for pyrimidine nucleotide biosynthesis in H. pylori. This conclusion was further supported by a recent study showing that the first committed step of de novo pyrimidine biosynthesis was essential for the survival of the bacterium (6) (see chapter 13). The uptake of purine nucleotides and precursors by H. pylori was also investigated, and it was found that labeled precursors of purine salvage pathways such as adenine, guanine, and hypoxanthine are in­ corporated at significant rates (36). The nucleosides adenosine, guanosine, and deoxyadenosine are incor­ porated at much smaller rates; and any incorporation of deoxyguanosine was below the detection levels of the radioactive tracer experimental techniques em­ ployed (36). Salvage routes for purine nucleotide syn­ thesis in H. pylori were investigated by measuring the incorporation of adenine and guanine and the activi­ ties of the corresponding phosphoribosyltransferases. Adenine and guanine were incorporated at rates of 64.50 and 27.50 pmole h ( 1 0 cells) . The bacter­ ium was found also to salvage adenosine, guanosine, and deoxyadenosine, although the incorporation of these nucleosides takes place at a much lower rate than for the bases (36). The nucleosidase activities measured decreased in this order: adenosine, guano­ sine, deoxyadenosine, deoxyguanosine. The high activities of adenosine nucleosidase and adenine phosphoribosyltransferases, the lower activity of adenosine phosphotransferase, and the lack of adeno­ sine kinase activity led these authors to suggest that the principal route for adenosine utilization was via _ 1

6

-1

the salvage of the purine ring after hydrolysis of aden­ osine to adenine and phosphorylation by adenine phosphoribosyltransferases. The significant amounts of nucleobases incorporated by H. pylori and the rela­ tively high activities measured for the transferases (36) indicated that the bacterium can salvage purines efficiently through this pathway.

NICKEL TRANSPORT Divalent nickel ions are a necessary cofactor for the active site of urease and for expression of the cata­ lytically active form of the enzyme (19). H. pylori lives in a niche in which free divalent cations such as N i are likely to be bound by cellular components such as nucleic acids or histidine and cysteine-rich proteins (39), and human serum contains average concentra­ tions of less than 10 nM N i (54). Therefore, H. py­ lori must have in place systems to acquire N i at low free concentrations and bound on macromolecules. A number of detailed studies have shown that N i is transported into H. pylori by a high-affinity transport system (16, 39). The gene nixA was cloned into E. coli, and cells containing the clone transported N i at a rate of 1,250 pmol m i n 1 0 cells, signifi­ cantly higher than that of negative controls. The transport system in H. pylori has a very high affinity for N i (K = 11.3 nM), suggesting that it possesses a mechanism to scavenge the ion even at very low concentrations, such as those most likely present in the gastric mucosa. NixA was also shown to function in complex media in which N i is normally chelated (39). Given the importance of urease in H. pylori physiology, the dependence of its function on the ac­ quisition of the metal cofactor further underlies the importance of transport systems in the overall H. py­ lori physiology. Transport of N i ions has been dis­ cussed in some detail in chapters 16 and 17. 2+

2 +

2 +

2+

2+

- 1

- 8

2+

t

2+

2 +

IRON TRANSPORT Iron is an essential nutrient for bacteria, required for such metabolic roles as the biosynthesis of cyto­ chromes and some of the components of the electron transport chains. However, the human mucosa is a relatively iron-deficient niche, brought about by the iron withholding defense system against infection (60). For this reason, many bacteria have developed mechanisms of acquiring iron from heme, the largest source of iron in humans (61). It has been suggested that a heme uptake system has significant advantages for H. pylori (62), particularly since the scavenging system of host mucosal cells is not very efficient, and

CHAPTER 18 • METABOLITE TRANSPORT

heme compounds would be readily accessible to H. pylori after erythrocyte lysis. Worst et al. (62) identified a functional TomB protein as essential for the transport of iron and/or heme across the membrane. It is an active transport process and not just heme leakage into the cells. The bacterium appears to have one constitutively ex­ pressed uptake system and others that are regulated and can be utilized in iron-restricted conditions (62). The actual mechanism of heme transport is not com­ pletely understood, but it was suggested that rather than transport the intact molecule, heme may be de­ graded by a heme-oxygenase system during its trans­ port into the cytoplasm (62). Analysis of the H. pylori genome suggested that it possesses other iron-acquisition systems, including both F e and F e citrate transporters (1, 5 7 ) . The role of these transporters was investigated, and iron transport in a feoB mutant is more than 10-fold lower than that in the wild type (58). Complementation of the feoB mutation fully restored both F e and F e transport and biphasic kinetics of F e transport in the wild type, which suggests the presence of highand low-affinity uptake systems (58). It was con­ cluded that the FeoB-mediated F e uptake is a major pathway for H. pylori iron acquisition. Interestingly, the system in H. pylori lacks homologs of the two regulatory proteins, FecR and Feci, and it is not or­ ganized in a single operon (1, 5 7 ) . Mutants in feoB showed that this gene is necessary for mouse coloniza­ tion (58), indicating that FeoB is essential for in vivo survival. A discussion of these transporters, including genomic analyses, can be found in chapter 17. 2 +

3 +

2 +

3 +

2 +

2 +

213

tein CopA, involved in copper export, contains six cysteine residues (17). It was hypothesized that these additional residues may provide extra channels for ion transport that would allow H. pylori to react to changes in C u in the stomach. The data also indicate that the genes for C u import and export are located within different operons of H. pylori. It was con­ cluded that separate genetic control over import and export would be advantageous to H. pylori (17), since the bacterium is likely to require a more efficient sys­ tem for import than for export. The characteristics of CopA are reviewed in chapter 17. 2+

2+

NOVEL DRUG T R A N S P O R T E R SYSTEMS Owing to the importance of H. pylori as a human pathogen, and the continuing problems with the effi­ cacy of current treatment regimens, there is a need to investigate alternative mechanisms of drug treatment. A study targeted aspartate carbamoyltransferase (AC­ Tase) (5), the enzyme that catalyzes the first commit­ ted step in the de novo formation of pyrimidine nu­ cleotides and is a key regulatory enzyme in bacteria. The drug employed was N-phosphonoacetyl-L-aspartate (PALA), a synthetic, transition-state bisubstrate analog of the intermediate of the ACTase-catalyzed reaction (10). H. pylori ACTase activity was com­ pletely inhibited in situ at nanomolar concentrations of PALA and thus represented a potential target (4). However, it was found that the compound did not have an effect on cell growth and viability, initially suggesting that PALA may not be able to enter H. pylori cells. Transport experiments with [ H]PALA indicate that it is taken up by the bacterium via a saturable system (5), and thus was available in vivo to act on the ACTase. PALA transport is saturable with a K of 14.8 mM and a V 19.1 nmol m i n (u.1 of cell w a t e r ) , suggesting that the uptake of this compound is con­ trolled by a specific carrier or carriers. It is highly unlikely that this is a specific uptake system for PALA, but rather a broad phosphonate-substrate uptake sys­ tem, and this conclusion is supported by the finding that phosphonoacetate inhibits PALA transport in H. pylori (5). Interestingly, PALA is degraded by the bac­ terium, and it is possible that the products of this me­ tabolism, particularly succinate and fumarate, are uti­ lized in further cell catabolism (5). Thus, such a system of drug detoxification appears quite novel, not only breaking down the potentially toxic drug, but also in providing a source of carbon and/or phospho­ rous for the bacterium. It could be hypothesized then that this transport system also is indirectly involved in nutrient transport and uptake. 3

COPPER T R A N S P O R T The levels of intracellular copper must be tightly regulated by H. pylori, since this ion is at the same time essential for cell viability at trace levels and highly toxic at higher amounts. It has been suggested that copper may play a role in pathogenesis (55), as a deficiency of these ions in local gastric epithelial cells accompanies H. py/on'-associated gastritis. Thus, H. pylori appears to possess a system to acquire trace levels that seem to be necessary for cellular function. Ge and Taylor (18) identified a cop operon in­ volved in copper export. Although C u transport was not measured, the intracellular levels of C u were determined and compared between mutants of the cop operon. There are significantly higher levels of C u co/7-negative mutant cells (530 ng per mg of dry cell weight) compared to the wild type (320 ng per mg of dry cell weight), implicating the cop operon in C u export (18). Another study found that the pro­ 2+

2+

2+

2+

m

- 1

m a x

-1

214

BURNS AND MENDZ

M E T A B O L I T E T R A N S P O R T AND T O T A L G E N O M E ANALYSIS

An interesting feature is that H. pylori lacks the phosphoenolpyruvyte:sugar phosphotransferase sys­ tem (PTS). The characterization of a glucose transport system has already been described, and a glucose/galactose transporter system is evident in the genomes ( 1 , 5 7 ) . Nonetheless, it is likely that carbohydrate me­ tabolism is not as significant in the bacterium. The transporters of H. pylori with a substrate specificity for carbohydrates are only 1.9% (42), and therefore the bacterium depends on other sources such as amino acids for carbon. This view is supported by the fact that 1 8 % of its transporters are capable of translocat­ ing amino acids. H. pylori also displays a relatively high number of energy-generating systems (42), and this may be due in part to the presence of an incom­ plete TCA cycle (43) and the absence of a complete Embden-Meyerhof-Parnas pathway (see chapters 10 and 12). Analyses of the complete H. pylori genomes re­ vealed the presence of four open reading frames cod­ ing for proteins with high sequence similarities to carboxylic acid transporters of other microorganisms (1, 57). The sequence HP0143 codes for a protein with 5 7 . 7 % similarity to the a-ketoglutarate/malate translocator (SODiTl) of Haemophilus influenzae (57).

The identification and characterization of the transport capabilities of a bacterium employing ge­ nome analyses should indicate significant features of its physiology, including its preferred niche and sources of nutrition and its most common metabolic pathways. The number of transport systems in H. py­ lori is approximately proportional to genome size, with 3.2 transport systems per 100 kb of genomic DNA. H. pylori has 5 . 4 % of total encoded genes cor­ responding to transport systems, which is signifi­ cantly lower than that of most eubacterial genomes thus far sequenced, which have approximately 1 0 % of their genomes corresponding to transport systems (42). This suggests that although the physiology of H. pylori is overall not considerably complex, it has the capacity to perform many of its functions de novo. The predicted types and frequencies of specific protein families identified by analysis of the two published H. pylori genomes (1, 57) are shown in Table 1. This information reveals that the bacterium displays a lim­ ited repertoire of transporter protein families, with a total of 53 families.

Table 1. Type of transport protein families in H. pylori as identified by genome analysis" Name of family Channel proteins Chloride channel Secondary active transporters Major facilitator superfamily Amino acid/polyamine/choline Resistance/cell division Gj-dicarboxylate uptake Lactate permease Inorganic phosphate transporter Sodium:solute symporter Neurotransmitter:Na symporter Glutamate :Na symporter Monovalent cation:proton antiporter Nucleoside uptake permease Serine/threonine permease Divalent anion:Na symporter Primary active transporters ATP-binding cassette (ABC) +

+

+

F-ATPase P-ATPase Unclassified Nicotinamide mononucleotide uptake permease L-lysine exporter Ferrous iron uptake " Reprinted from reference 4 2 with permission.

Typical substrate(s)

No. of transport families

Chloride

1

Sugars, drugs, metabolites, organic cations, nucleosides, carboxylates, ions Amino acids, choline, polyamines Drugs, metal ions, lipooligosaccharides Dicarboxylates Lactate Phosphate Amino acids, sugars, vitamins, nucleosides Neurotransmitters Glutamate K , Na / H Nucleosides Amino acids Sulfate/phosphate/carboxylates

7

+

+

+

Sugars, amino acids, drugs, metal ions, metabolites, vitamins, proteins, carbohydrates H /Na M /M +

+

+

2+

Nicotinamide mononucleotide Lysine Ferrous uptake

2 3 1 2 1 1 2 1 2 1 1 2 15 1 3 1 1 1

CHAPTER 18 • METABOLITE TRANSPORT

The sequences HP1091, HP0724, and HP0140 code for proteins with 6 9 . 7 % similarity to the a-ketoglutarate permease (kgtP), 7 5 . 3 % similarity to the anaero­ bic C4-dicarboxylate transport protein (dcuA), and 7 8 . 2 % similarity to the L-lactate permease (IctP) of E. coli, respectively (57). The significant similarity be­ tween the H. pylori and the anaerobic E. coli C -dicarboxylate transporters is consistent with the low affin­ ity of both systems (15). A comparison of several putative phosphonate uptake genes of the phn operon in E. coli (9), with the complete genome sequence of H. pylori (1, 5 7 ) , revealed an open reading frame in H. pylori with 72.2% identity to the phn A gene of E. coli. However, recent results suggest phnA does not have a role for phosphonate metabolism in E. coli (38), and therefore the exact type of carrier involved in the transport of phosphonates and the drug PALA described above for H. pylori remains to be established. Not surprisingly for a human pathogen, analysis of the H. pylori ge­ nomes reveals that 1 5 . 1 % of transporters probably catalyze drug efflux, which are potential mechanisms for antibiotic resistance. 4

CONCLUSIONS The number of detailed studies on transport in H. pylori is limited, but it is evident that the bacterium possesses very tightly regulated and specific transport systems. The activity of its transporters represents one of the obvious links between H. pylori and its host, and the types of carriers found reflect both the biosyn­ thetic capacity of the microorganism and the possible coevolution with its host. In addition, it should be noted that owing to the lack of homologs with charac­ terized functions, over 4 0 % of H. pylori genes have no known function, so it is possible that the bacterium may have novel transport systems. It will be interest­ ing to ascertain whether new transport families emerge that diverge extensively with respect to sub­ strate specificity or the number of molecular species they transport, which should further our understand­ ing of the evolutionary processes that allow some fam­ ilies but not others to diversify in function. Also evident from the wealth of information gained from physiological transport studies is the need to complement in silico analyses of total ge­ nomes with detailed specific transport studies. Ge­ nome analyses can supply information on the basis of sequence and/or structure similarity with known sequences, but provide little indication of the specific­ ity or unique characteristics of a particular transport system. A gene encoding a putative transporter that appears to be missing in the genome may actually be

215

present but may have low sequence similarity to en­ zymes of similar functions, may be part of a multi­ functional protein, or simply may not yet have been correlated with a gene sequence. Conversely, the pres­ ence of a gene does not necessarily mean that the cor­ responding protein is functional, as evolutionary pres­ sure may have led to loss of function or disruptive mutations with a subsequent lack of transcription and/or translation. The combination of specific trans­ port studies and total genome analysis has provided us with information not only enhancing our under­ standing of H. pylori physiology, but also serving to identify novel membrane targets for therapy. REFERENCES 1. Aim, R. A., L. S. Ling, D. T. Moir, B. L. King, E. D. Brown, P. C. Doig, D. R. Smith, B. Noonan, B. C. Guild, B. L. dejonge, G. Carmel, P. J. Tummino, A. Caruso, M. Uria-Nickelsen, D. M. Mills, C. Ives, R. Gibson, D. Merberg, S. D. Mills, Q.Jiang, D. E. Taylor, G. F. Vovis, and T. J. Trust. 1999. Genomicsequence comparison of two unrelated isolates of the human gastric pathogen Helicobacter pylori. Nature 397:176-180. 2. Baldwin, S. A. 1992. Mechanisms of active and passive trans­ port in a family of homologous sugar transporters found in both prokaryotes and eukaryotes, p. 169-217. In J. J. H. H. M. de Pont (ed.), Molecular Aspects of Transport Proteins. Elsevier, Amsterdam, The Netherlands. 3. Blaser, M. J. 1997. The versatility of Helicobacter pylori in the adaptation to the human stomach. /. Physiol. Pharmacol. 48:307-314. 4. Burns, B. P., S. L. Hazell, and G. L. Mendz. 1997. In situ properties of aspartate carbamoyltransferase activity in Heli­ cobacter pylori. Arch. Biochem. Biophys. 347:119-125. 5. Burns, B. P., S. L. Hazell, and G. L. Mendz. 1998. A novel mechanism for resistance to the antimetabolite N-phoshonoacetyl-L-aspartate by Helicobacter pylori. J. Bacteriol. 180: 5574-5579. 6. Burns, B. P., S. L. Hazell, G. L. Mendz, T. Kolesnikow, D. Tillett, and B. A. Neilan. 2000. The Helicobacter pylori pyrB gene encoding aspartate carbamoyltransferase is essential for survival. Arch. Biochem. Biophys. 380:78-84. 7. Catrenich, C. E., and K. M. Makin. 1991. Characterization of the morphologic conversion of Helicobacter pylori from bacillary to coccoid forms. Scand. J. Gastroenterol. 26:58-64. 8. Chalk, P. A., A. D. Roberts, and W. M. Blows. 1994. Metabo­ lism of pyruvate and glucose by intact cells of Helicobacter pylori studied by C NMR spectroscopy. Microbiology 140: 2085-2092. 9. Chen, C , Q. Ye, Z. Zhu, B. L. Wanner, and C. T. Walsh. 1990. Molecular biology of carbon-phosphorus cleavage. /. Biol. Chem. 265:4461-4471. 10. Collins, K. D., and G. R. Stark. 1971. Aspartate transcarbamy­ lase. Interaction with the steady state analogue N-(phosphonacetylH-aspartate. /. Biol. Chem. 246:6599-6605. 11. Cussac, V., R. L. Ferrero, and A. Labigne. 1992. Nucleotide expression of Helicobacter pylori urease genes in Escherichia coli grown under nitrogen-limiting conditions. /. Bacteriol. 174:2466-2473. 12. Dawes, I. W., and I. W. Sutherland. 1992. Microbial Physiol­ ogy. Blackwell Scientific, Oxford, United Kingdom. 13. Deguchi Y., I. Yamato, and Y. Anraku. 1990. Nucleotide se13

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quence of gltS, the Na /glutamate symport carrier gene of Escherichia coli B. /. Biol. Chem. 265:21704-21708. Eisenthal, R., S. Game, and G. D. Holman. 1989. Specificity and kinetics of hexose transport in Trypanosoma brucei. Biochim. Biophys. Acta 985:81-89. Engel, P., R. Kramer, and G. Unden. 1992. Anaerobic fumar­ ate transport in Escherichia coli by an /ttr-dependent dicarbox­ ylate uptake system which is different from the aerobic dicar­ boxylate uptake system. /. Bacteriol. 174:5533-5539. Fulkerson, J. F., Jr., and H. L. T. Mobley. 2000. Membrane topology of the NixA nickel transporter of Helicobacter pylori: two nickel transport-specific motifs within transmembrane helices II and III. /. Bacteriol. 182:1722-1730. Ge, Z., K. Hiratsuka, and D. E. Taylor. 1995. Nucleotide se­ quence and mutational analysis indicate that two Helicobacter pylori genes encode a P-type ATPase and a cation-binding pro­ tein associated with copper transport. Mol. Microbiol. 15: 97-106. Ge, Z., and D. E. Taylor. 1996. Helicobacter pylori genes hpcopA and hpcopP constitute a cop operon involved in cop­ per export. FEMS Microbiol. Lett. 145:181-188. Hawtin, P. R., H. T. Delves, and D. G. Newell. 1991. The demonstration of nickel in the urease of Helicobacter pylori by atomic absorption spectroscopy. FEMS Microbiol. Lett. 77: 51-54. Hazell, S. L., A. Lee, L. Brady, and W. Hennessy. 1986. Campylobacter pyloridis and gastritis: association with inter­ cellular spaces and adaptation to an environment of mucus as important factors in colonization of the gastric epithelium. /. Infect. Dis. 153:658-663. Hazell, S. L., and G. L. Mendz. 1997. How Helicobacter pylori works: an overview of the metabolism of Helicobacter pylori. Helicobacter 2:1-12. Lopilato, J., T. Tsuchiya, and T. H. Wilson. 1978. Role of Na and Li in thiomethylgalactoside transport by the melibiose transport system of Escherichia coli. ] . Bacteriol. 134: 147-156. Maloney, P. C. 1994. Bacterial transporters. Curr. Opin. Cell Biol. 6:571-582. Mendz, G. L. 1996. Elucidation of metabolic pathways em­ ploying one- and two-dimensional NMR spectroscopy. Bull. Magn. Reson. 17:138. Mendz, G. L., B. P. Burns, and S. L. Hazell. 1995. Characteri­ sation of glucose transport in Helicobacter pylori. Biochim. Biophys. Acta 1244:269-276. Mendz, G. L., B. P. Burns, E. M. Holmes, S. L. Hazell, S. Skouloubris, and H. de Reuse. 2000. Characterization of urea transport in Helicobacter pylori. In Abstr. 100th Gen. Meet. Am. Soc. Microbiol. American Society for Microbiology, Washington, D.C. Mendz, G. L., and S. L. Hazell. 1993. Fumarate catabolism by Helicobacter pylori. Biochem. Mol. Biol. Int. 31:325-332. Mendz, G. L., and S. L. Hazell. 1995. Amino acid utilization by Helicobacter pylori. Int. J. Biochem. CellBiol. 27:1085-1093. Mendz, G. L., and S. L. Hazell. 1996. The urea cycle of Helico­ bacter pylori. Microbiology 142:2959. Mendz, G. L., S. L. Hazell, and B. P. Burns. 1992. Glucose utilisation and lactate production by Helicobacter pylori. ] . Gen. Microbiol. 139:3023-3028. Mendz, G. L., S. L. Hazell, and B. P. Burns. 1994. The EntnerDoudoroff pathway in Helicobacter pylori. Arch. Biochem. Biophys. 312:349-356. Mendz, G. L., S. L. Hazel, and S. Srinivasan. 1995. Fumarate reductase: a target for therapeutic intervention against Helico­ bacter pylori. Arch. Biochem. Biophys. 321:153-159. Mendz, G. L., S. L. Hazell, and L. van Gorkom. 1994. Pyruvate +

14.

15.

16.

17.

18.

19.

20.

21.

22.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

+

+

23. 24.

25.

26.

27. 28. 29. 30. 31.

32.

33.

44. 45.

46.

47.

48.

49. 50.

51.

52.

metabolism in Helicobacter pylori. Arch. Microbiol. 162: 187-192. Mendz, G. L., E. M. Holmes, and R. L. Ferrero. 1998. In situ characterization of Helicobacter pylori arginase. Biochim. Biophys. Acta 13882:465-477. Mendz, G. L., B. M. Jimenez, S. L. Hazell, A. M. Gero, and W. J. O'Sullivan. 1994. De novo synthesis of pyrimidine nu­ cleotides by Helicobacter pylori. ] . Appl. Bacteriol. 77:1-8. Mendz, G. L., B. M. Jimenez, S. L. Hazell, A. M. Gero, and W.J. O'Sullivan. 1994. Salvage synthesis of purine nucleotides by Helicobacter pylori. J. Appl. Bacteriol. 77:674-681. Mendz,G. L.,D.J. Meek, andS. L.Hazell. 1998. Characteriza­ tion of fumarate transport in Helicobacter pylori. J. Membr. Biol. 165:65-76. Metcalf, W. W., and B. L. Wanner. 1993. Evidence for a fourteen-gene, phnC to phnP locus for phosphonate metabolism in Escherichia coli. Gene 129:27-32. Mobley, H. L., R. M. Garner, and P. Bauerfeind. 1995. Helico­ bacter pylori nickel-transport gene nixA: synthesis of catalyti­ cally active urease in Escherichia coli independent of growth conditions. Mol. Microbiol. 16:97-109. Nakao, T., I. Yamato, and Y. Anraku. 1987. Nucleotide se­ quence of putP, the proline carrier gene of Escherichia coli K12. Mol. Gen. Genet. 208:70-75. Nedenskov, P. 1994. Nutritional requirements for growth of Helicobacter pylori. Appl. Environ. Microbiol. 64: 3450-3453. Paulsen, I. T., M. K. Sliwinski, and M. H. Saier, Jr. 1998. Microbial genome analyses: global comparisons of transport capabilities based on phytogenies, bioenergetics and substrate specificities. /. Mol. Biol. 277:573-592. Pitson, S. M., G. L. Mendz, S. Srinivasan, and S. L. Hazell. 1999. The tricarboxylic acid cycle of Helicobacter pylori. Eur. J. Biochem. 260:258-267. Poolman, B., and W. N. Konings. 1993. Secondary solute transport in bacteria. Biochim. Biophys. Acta 1183:5-39. Reynolds, D. J., and C. W. Penn. 1994. Characteristics of Heli­ cobacter pylori growth in a defined medium and determination of its amino acid requirements. Microbiology 140:26492656. Scott, D. R., E. A. Marcus, D. L. Weeks, A. Lee, K. Melchers, and G. Sachs. 2000. Expression of the Helicobacter pylori urel gene is required for acidic pH activation of cytoplasmic urease. Infect. Immun. 68:470-477. Shinitzky, M. 1984. Membrane fluidity and cellular functions, p. 1-51. In M. Shinitzky (ed.), Physiology of Membrane Fluid­ ity, vol. I. CRC Press, Boca Raton, Fla. Sipponen, P., M. Siurala, and C. S. Goodwin. 1993. Histology and ultrastructure of Helicobacter pylori infections: gastritis, duodenitis and peptic ulceration, and their relevance as precan­ cerous conditions, p. 37-62. In C. S. Goodwin and B. J. Worsley (ed.), Helicobacter pylori Biology and Clinical Practice. CRC Press, Boca Raton, Fla. Skulachev, V. P. 1988. The sodium world, p. 293-326. In Membrane Bioenergetics. Springer-Verlag, Berlin, Germany. Sorberg, M., M. Nilsson, H. Hanberger, and L. E. Nilsson. 1996. Morphologic conversion of Helicobacter pylori from bacillary to coccoid form. Eur. J. Clin. Microbiol. Infect. Dis. 15:216-219. Stein, W. D. 1990. Coupling of flows of substrates: antiporters and symporters, p. 173-219. In Channels, Carriers and Pumps. Academic Press, San Diego, Calif. Stock, J. W., and S. A. Roseman. 1971. Sodium-dependent sugar co-transport system in bacteria. Biochem. Biophys. Res. Commun. 44:132-138.

CHAPTER 18 • METABOLITE TRANSPORT

53. Strauss, P. R., J. M. Sheehan, and E. R. Kashket. 1976. Mem­ brane transport by murine lympocytes. /. Exp. Med. 144: 1009-1018. 54. Sundermann, F. W. 1993. Biological monitoring of nickel in humans. Scand. J. Work Environ. Health. 19(Suppl. l):34-38. 55. Taha, A. S., I. M. Huxman, R. H. Park, and A. D. Beattie. 1995. Assessment of gastric mucosal and mucus layer elemen­ tal trace metals in H. pylori gastritis using the novel techniques of electron energy loss spectroscopy (EELS). Gut 36:7521. 56. Tolner, B., M. E. van der Rest, G. Speelmans, and W. N. Konings. 1992. Sodium coupled transport in bacteria, p. 43-50. In E. Quagliariello and F. Palmieri (ed.), Molecular Mecha­ nisms of Transport. Elsevier, Amsterdam, The Netherlands. 57. Tomb, J.-F., O. White, A. R. Kerlavage, R. A. Clayton, G. G. Sutton, R. D. Fleischmann, K. A. Ketchum, H. P. Klenk, S. Gill, B. A. Dougherty, K. Nelson, J. Quackenbush, L. Zhou, E. F. Kirkness, S. Peterson, B. Loftus, D. Richardson, R. Dodson, H. G. Khalak, A. Glodek, K. McKenney, L. M. Fitzegerald, N. Lee, M. D. Adams, E. K. Hickey, D. E. Berg, J. D. Gocayne, T. R. Utterback, J. D. Peterson, J. M. Kelley, M. D. Cotton, J. M. Weidman, C. Fujii, C. Bowman, L. Watthey, E. Wallin, W. S. Hayes, M. Borodovsky, P. D. Karp, H. O. Smith, C. M. Fraser, and J. C. Venter. 1997. The complete genome

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sequence of the gastric pathogen Helicobacter pylori. Nature 388:539-547. Velayudhan, J., N. J. Hughes, A. A. McColm, J. Bagshaw, C. L. Clayton, S. C. Andrews, and D. J. Kelly. 2000. Iron acquisi­ tion and virulence in Helicobacter pylori: a major role for FeoB, a high-affinity ferrous iron transporter. Mol. Microbiol. 372:274-286. Weeks, D. L., S. Eskandari, D. R. Scott, and G. Sachs. 2000. A H -gated urea channel: the link between Helicobacter pylori urease and gastric colonization. Science 287:482-485. Weinberg, E. D. 1984. Iron withholding: a defense against infection and neoplasia. Physiol. Rev. 64:65-102. Welch, S. 1992. Iron metabolism in man, p. 25-40. In S. Welch (ed.), Transferrin: The Iron Carrier. CRC Press, Boca Raton, Fla. Worst, D. J., J. Maaskant, C. M. Vandenbroucke-Grauls, and J. G. Kusters. 1999. Multiple heme-utilization loci in Helico­ bacter pylori. Microbiology 145:681-688. Wyatt, S. T., and S. F. Gray. 1989. Detection of Campylo­ bacter pylori by histology, p. 63-68. In B. J. Rathbone and R. V. Heatley (ed.), Campylobacter pylori and Gastro­ duodenal Disease. Blackwell Scientific, Oxford, United Kingdom. +

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Chapter 19

Protein Export DAG ILVER, RINO RAPPUOLI, AND JOHN L . TELFORD

Export of proteins is necessary for a variety of essen­ tial bacterial functions including expression of viru­ lence factors on the cell surface, release of effector proteins to the extracellular milieu, nutrient acquisi­ tion, and organelle biogenesis. To export a protein, Helicobacter pylori as a gram-negative bacterium faces the triple barrier of transporting the polypeptide first across the inner membrane (IM), then through the periplasmic space, and finally across the outer membrane (OM). The task of passing through the per­ iplasm is not trivial since proteins may fold and form disulfide bonds in this space before reaching and crossing the O M . A number of different pathways have been char­ acterized for protein export in gram-negative bacteria. The general secretory pathway (GSP) exports proteins carrying an amino-terminal signal sequence in a step­ wise manner, across the I M first, and then across the OM. Proteins secreted via the different terminal branches of the GSP require the Sec system to cross the IM but use different approaches to get through the O M . The Sec-independent pathways are able to transfer proteins directly from the cytoplasm to the outside of the bacteria. In addition, Sec-independent pathways that use other strategies to cross both mem­ branes are known (Table 1). This chapter describes what is known about protein export in H. pylori from the perspective of the general mechanisms for protein export in gram-negative bacteria.

translocated via the Sec system carry an amino-termi­ nal signal sequence that is cleaved by a signal pepti­ dase when the protein is released into the periplasmic space. The genome of H. pylori strain 26695 has 1,590 open reading frames (62), of which 517 have a putative signal sequence indicating that these proteins could be secreted via the GSP (2). This figure may be an overestimate since it is based on sequence similari­ ties to a peptide motif derived from the characteristics of known signal peptides. Nevertheless, it indicates that a substantial proportion of proteins synthesized by H. pylori are destined to cross at least the IM. In comparison, Haemophilus influenzae has a similar number of putative genes (1,680), but only has 330 predicted signal peptides (8). Homologs of essential Sec-related genes besides secA and a set of signal peptidases are found in H. pylori. At the same time, some factors important for the sec system in Escherichia coli are not found in H. pylori. One of these is the SecB chaperone. The presence of a signal sequence on a protein slows down the folding of the whole protein, allowing chaperones like SecB to bind and prevent premature folding that would interfere with export. SecB binds to a group of proteins targeted for secretion and also guides them to exit sites in the membrane. A factor reported to direct the interaction of SecB with this set of proteins is the trigger factor, and a homolog of this factor is found in the H. pylori genome. Apparent orthologs to secB are also missing in some of the other bacterial genomes recently sequenced, despite the presence of other components of the Sec system (61). Taken to­ gether, this makes it likely that a protein with a "SecBlike" function is present in H. pylori even though no open reading frame has been annotated as secB.

T H E GENERAL S E C R E T O R Y PATHWAY Sec-Dependent Secretion across the Inner Membrane The GSP of gram-negative bacteria is a group of secretory pathways unified by the common require­ ment of the Sec system for translocating proteins across the IM (for a review, see reference 18). Proteins Dag liver, Rino Rappuoli, and John L. Telford

An alternative route for targeting to the mem­ brane is via the signal recognition particle (SRP). It recognizes the signal sequence and, together with cell division FtsY protein, targets mainly hydrophobic

• IRIS, Chiron S.p.A., Via Florentina 1, 53100 Siena, Italy.

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Table 1. Summary of protein export in H. pylori Transport and system Transport across inner membrane Translocation system Sec system TAT system Transport across outer membrane Secretion system Auto transporter Chaperone/usher Type I Type II Type III TypelV Flagellar export machinery Membrane vesicles Altruistic autolysis

Comments

Presence

Yes Yes

Homologs to all components except SecB and SecE H. pylori tatA can complement E. coli tatA

Yes

VacA exported, possibly also VacA paralogs Not reported Not reported Not reported Not reported CagA exported Atypical scattered chromosomal organization MVs containing VacA are taken up by host cells Autolysis reported for surface location of urease and Hsp but questioned by other researchers

>

? ? ?

Yes Yes Yes Yes?

proteins with multiple membrane-spanning regions to the cell membrane for export. The SRP is composed of the Ffh protein and a 4.5S-RNA species. Both ffh and ftsYhomologs are present in H. pylori, suggesting the presence of this pathway. A second "missing" gene in H. pylori is secE. E. coli SecE, SecY, and SecG make up the membrane-located protein translocase unit together with the auxiliary SecD and SecF. In E. coli only SecE and SecY are essential translocase components; thus, it is surprising to find homologs in H. pylori to all E. coli translocase components except SecE. Secretion across the Outer Membrane Autotransporters Autotransporters constitute one terminal branch of the GSP, frequently employed to export virulence factors. After Sec-dependent transfer of the protein to the periplasm, the proteins pass the OM without assistance of other factors. Autotransporters have been identified in a number of bacteria. The most well-known example is the IgA protease of Neisseria gonorrhoeae (44). The typical autotransporter is or­ ganized in three functional domains: (i) an aminoterminal signal sequence for Sec-dependent secretion over the IM, (ii) a passenger domain constituting the functional domain to be secreted, and (iii) a carboxyterminal transporter domain (B-domain), which in­ serts in the OM and makes it possible for the passen­ ger domain to cross the OM and reach the bacterial surface. The B-domain is thought to form a B-barrel pore structure, analogous to bacterial porins, through which the passenger domain crosses the OM. A linker region connects the passenger domain to the B-do­

main, possibly with a role to guide the passenger through the channel (36). The passenger domain is either retained on the bacterial surface or proteolytically released from the OM-associated transporter domain. The concept of functionally separate passen­ ger and transport domains is strengthened by experi­ ments showing that the passenger domain can suc­ cessfully be substituted with foreign proteins that become secreted (32, 5 8 ) . Few cysteine residues are found in the passenger domain, but often two cysteinyls are located close to each other in the C-terminal region of the polypeptide. The general interpretation for this lack of cysteine res­ idues has been that formation of disulfide bonds by the passenger domain would interfere with export through the B-barrel to the bacterial surface. This opinion has support from work on secretion of for­ eign proteins (32, 5 8 ) . However, there seem to be ex­ ceptions to this rule, and recent research showed that export of a functional disulfide-bonded antibody fragment fused to the IgA protease B-domain is indeed possible (64). VacA of H. pylori The vacuolating cytotoxin, a major virulence fac­ tor of H. pylori (12, 4 2 , 4 9 , 5 9 ) , belongs to the auto­ transporter group of secreted proteins. It has a typical autotransporter organization with an N-terminal sig­ nal sequence and a passenger domain connected to the C-terminal transport domain via a linker region. The mature VacA toxin ( ~ 9 0 kDa) is released from the bacteria, but the C-terminal transport do­ main is retained in association with the OM (59). The B-domain of VacA has amino acid sequence homol-

CHAPTER 19 • PROTEIN EXPORT

ogy with B-domains of other autotransporters and an insertional knockout of the B-domain of VacA inhib­ its secretion of the passenger domain (49). In addition, the B-domain of VacA has been identified as a func­ tional transport domain by its ability to translocate an alternative passenger protein to the surface of H. pylori (21). The VacA toxin is further discussed in this chap­ ter 9 and also in chapter 9. Besides vac A, only the genes HP0289, HP0610, and HP0922 in the genome of H. pylori strain 26695 show homology to auto­ transporters (62). The three genes also have weak ho­ mology with the passenger domain of vacA. This raises the question whether these proteins are also vir­ ulence factors and what function they might have in pathogenesis. Missing Pathways The second terminal branch of the GSP is the chaperone/usher pathway, responsible for export and assembly of (adhesive) virulence-associated structures such as P and type 1 pili of uropathogenic E. coli (60). For export through this pathway only two accessory proteins are needed for transfer across the OM: a chaperone guiding the protein through the periplas­ mic space, assisting in folding and preventing the pro­ tein from premature interactions, and an usher pro­ tein for translocation through the O M . No component of this pathway has been found in H. py­ lori and, in addition, no surface organelles besides the flagella have been described. The third terminal branch of the GSP is type II secretion, also called the main terminal branch of the GSP. The type II secretion pathway is closely related to the biogenesis of type IV pili and is considerably more complex than the autotransporter pathway, re­ quiring 12 to 16 accessory proteins (for a review, see reference 46). No example of this export mechanism has been described for H. pylori, nor have homolo­ gous genes to those encoding type II proteins been identified in the annotated genomes of H. pylori strains 26695 or J 9 9 . It is not very surprising not find type II secretion candidate genes absent from the H. pylori genome, since, in fact, only a few of the bacte­ rial genomes sequenced have components for type II secretion in their annotated sequences.

SEC-INDEPENDENT E X P O R T The T A T Secretion System A new Sec-independent system for translocation of proteins across the IM has recently been identified,

221

and orthologs of the essential genes of this system have been found in the H. pylori genome (48). Like the Sec system, it requires an amino-terminal signal peptide but with specific different features. Most no­ ticeable is the requirement of two consecutive arginine residues preceding the hydrophobic core region of the signal peptide. This feature served to give the new system the name "twin arginine translocation," or Tat-system. The system was first found in chloroplasts of maize where it constitutes a ApH-driven transport system for protein transport across the thylakoid membrane. The H f c l 0 6 protein is necessary for this pathway in maize, and homologs to hfcl06 are found in a wide variety of bacterial genomes (for a review, see reference 13). In E. coli a set of genes homologous to hfcl 06 has been found; two of them, tatA and tatB, are organized as part of an operon, and a third named tatE is in an unlinked position. The gene tatB is essential for the pathway to function, because a knockout of this gene completely blocks Tat-dependent secretion. Neither tatA nor tatE is es­ sential, although efficiency is reduced in knockouts of either gene. In a double tatAltatE knockout the pathway is inactive. Interestingly, a tatA knockout in E. coli can be functionally complemented by the H. pylori tatA homolog (48). However, E. coli tatB can­ not be complemented by H. pylori tatB, which is in agreement with the fact that the tatB genes of the two bacteria are much more divergent than tatA. Never­ theless, it is unusual that the most essential gene of the pathway is the least conserved between the two bacteria. Sec-dependent translocation across the IM re­ quires the substrate protein to be unfolded. On the other hand, it has been reported that proteins trans­ ported by the Tat pathway in most cases bind redox cofactors, which seem to fold and even oligomerize before crossing the membrane ( 4 7 , 5 4 ) . A motif search in the H. pylori genome reveals a number of genes encoding proteins with the twin arginine motif typical of the Tat system in their signal sequences, and as expected, among them are genes coding for proteins using redox cofactors. Type I secretion and ATP-binding cassette protein exporters The Sec-independent type 1 pathway transports proteins directly from the cytoplasm to the outside of bacteria without an intermediary periplasmic state. Proteins exported by this pathway display a carboxyterminal signal sequence that is not cleaved during export. Structurally, the system consists of three pro­ teins: (i) an IM-located ATP-binding cassette (ABC) exporter containing an ATP-binding cassette; (ii) a

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membrane fusion protein that connects the ABC ex­ porter to the third component; and (iii) an O M pro­ tein. In this system the interactions of the three pro­ teins create a channel through the periplasm. The prototypical type I secreted protein is the a-hemolysin of E. coli (for a review, see reference 6). The H. pylori genome contains genes encoding potential ABC ex­ porters, or more correctly, members of the ABC superfamily with the ATP-binding cassette (41, 62). Most ABC transporters are, however, not involved in protein export, but in the efflux and influx of a diverse group of substrates. To date, none of the potential ABC transporters in H. pylori has been proposed to be involved in protein export. Type HI secretion The Sec-independent type III secretion system translocates virulence factors directly into the cytosol of eukaryotic target cells. The secretion machinery is a large structure composed of approximately 20 com­ ponents, reaching over the bacterial inner and outer membranes and possibly also the eukaryotic host cell membrane. Components of the type III secretion ma­ chinery implicated in transfer over the IM show ho­ mology to components of the flagella export appara­ tus (37), while pore-forming type III components involved in secretion through the O M are homolo­ gous to O M components of the type II secretion path­ ways. The prototypical type III export pathway is the system for secretion and translocation of Yersinia outer proteins ("Yops") into the target cell cytosol. The genes encoding the machinery for type III secre­ tion are clustered, and in most cases, have a different G + C content relative to that of the surrounding ge­ nome, indicating that the region has been acquired as a pathogenicity island (PAI) by horizontal gene trans­ fer (for a review, see reference 25). No type III secre­ tion system has been identified in the annotated ge­ nomes of H. pylori 26695 or J 9 9 (2, 62). Type IV secretion A type IV secretion system, the cag pathogenicity island, plays a major role in H. pylori pathogenesis. Type IV secretion systems, like type III systems, are capable of direct secretion of complex proteins and nucleoproteins across both membranes into the extra­ cellular milieu or directly into the eukaryotic host cell cytosol. Whereas type III systems are related to the flagellar export system, type IV systems contain core components of bacterial conjugation machines that have been adapted to export complex structures. The prototypical type IV secretion system of Agrobacterium tumefaciens transfers an oncogenic nucleoprotein

into plant cells. Other pathogens like Bordetella per­ tussis use type IV secretion systems to export protein effector molecules (for a review, see reference 10). The cag PAI is named after the cytotoxin-associated gene cagA, which was the first of its genes identi­ fied. The CagA protein is a strongly immunogenic an­ tigen but is not expressed by all H. pylori strains. Early work revealed a close relationship between serum an­ tibody response to CagA and the more severe forms of H. py/on-related disease (11). In the ensuing years, the association of anti-CagA seropositivity with pep­ tic ulcer, gastric cancer, and atrophic gastritis has been well established (7). It was subsequently discov­ ered that strains that do not express CagA lack the cagA gene (65) and a flanking region of the genome of about 40 kb containing approximately 4 0 genes (1, 9). On the basis of differences in G + C content, it was concluded that this pathogenicity island had been acquired as foreign DNA by a subset of H. pylori strains. Strains that lack the cag region are defective in several components of pathogenicity, including the capacity to induce interleukin (IL)-8 expression in epi­ thelial cells and host cell tyrosine phosphorylation. Individual mutations in several genes within the cag PAI resulted in defects in these functions ( 9 , 5 2 ) . Based on these results and sequence similarities of some of the cag genes with genes identified in other pathoge­ nicity islands, it was concluded that the cag PAI codes for a type IV secretion system. This system is now known to be responsible for the direct transfer of CagA from the cytoplasm of the bacterium to the cytosol of the host target cell (3, 39, 5 1 , 5 7 ) . Once transferred, the protein becomes phosphorylated on tyrosine residues, and it is believed that this process triggers actin reorganization and pedestal formation. Since CagA is not necessary for the changes in gene expression resulting in production of IL-8, it can be concluded that other effector mole­ cules present in the cag system are either exported to the host cell or presented on the bacterial surface. It is not known at this time if any other factors beside CagA are exported to the host cell via the type IV secretion system. It is clear, however, that secretion through the cag type IV system is important for H. pylori colonization and virulence. Flagellar Export Motility is mediated by the sheathed flagella in H. pylori (22) and is necessary for colonization in animal models (17). In the context of this chapter the flagellar export apparatus potentially is interesting owing to its similarity to type III export systems. The prototype flagellum is anchored to the bac-

CHAPTER 19 • PROTEIN EXPORT

terium by a flagellar basal body that spans both inner and outer membranes and is structurally similar to the type III secretion apparatus (34). Flagellar subunits are secreted through a channel within the basal body and polymerize at the distal end of the growing flagellum. Recently, in a different system, it has been demonstrated that the flagellar transport apparatus of Yersinia enterocolitica also functions as a protein export system for a pathogenesis-related phospholi­ pase, placing the flagellar export/assembly system under a new light (66). T o date, a similar phenomenon has not been reported for H. pylori. Based on homolo­ gies to better defined systems, the genome of H. pylori 26695 contains more than 4 0 genes suggested to en­ code either structural components of flagella or pro­ teins involved in its biogenesis and regulation (62). Two structural H. pylori genes have been demon­ strated to encode flagellin FlaA and FlaB (35), and a third structural gene encodes FlgE, the hook protein joining the flagella filament to the basal body (38). Most of what is known about the flagellar export ap­ paratus (the flagellar basal body) of H. pylori is based on the presence of genes homologous to others encod­ ing parts of the flagellar export machinery in other bacteria. Knocking out flil and fliQ, which are pro­ posed to encode components of the transport appara­ tus, rendered H. pylori aflagellated and nonmotile ( 2 6 , 4 5 ) . The organization of genes involved in flagel­ lar synthesis in H. pylori is remarkedly different from that of other bacterial genomes. In H. pylori flagellar genes are scattered over the genome, contrary to their clustered presence in other bacterial genomes.

LESS CONVENTIONAL MECHANISMS F O R PROTEIN E X P O R T Altruistic Autolysis The proteins discussed in this section, urease, heat shock proteins, and superoxide dismutase, are found only in the cytoplasm of most bacterial species. In H. pylori these proteins are found also on the bacte­ rial surface and in the growth media (19, 2 3 , 56). Since in other species they are not exported proteins and also lack signal sequences for export by the GSP, the mechanisms through which these proteins are ex­ creted are intriguing, especially in the case of urease. This enzyme hydrolyzes urea to ammonia and carbon dioxide. The ammonia formed can neutralize hydro­ chloric acid of the gastric juice, creating a more suita­ ble microenvironment around the bacteria. It can be used also for metabolic purposes by the bacterium and has toxic effects on epithelial cells. Urease activity is necessary for pathogenesis in animal models (15,

223

16) and is the basis for the highly reliable urea-breath test for detecting H. pylori infection. Urease and the heat shock protein HspB are lo­ cated in the cytoplasm as well as on the bacterial sur­ face both in vitro and in vivo (14). The urease located on the bacterial surface is reported to be catalytically active since inactivation of extracellular urease with a membrane-impermeable urease inhibitor makes H. pylori susceptible to acid (33). The enzyme is a 5 5 0 kDa multimer consisting of six ureA and six ureB subunits. For activity it needs nickel ions and a complex of accessory proteins to import the ions, to incorpo­ rate them into the apoenzyme, and to produce the active holoenzyme. An obvious obstacle for export of an active urease is its large size, unless it can be ex­ ported as subunits. Altruistic autolysis, the "unselfish" autolysis of some bacteria for the benefit of the whole population, has been reported for some pathogenic bacteria. Au­ tolysis of Streptococcus pneumoniae appears to en­ hance virulence (4) and is genetically regulated (40). Autolysis of Neisseria gonorrhoeae is suggested to be a way for the bacteria to release DNA that can be taken up by other nonlysed bacteria, i.e., transforma­ tion, leading to increased antigenic variation of pili subunits (27, 5 3 ) . Autolysis has been reported also for H. pylori. It has been suggested that enzymatically active urease and HspB are exported in this way from lysed bacteria and adsorbed to the surface of other living bacteria (43). Kinetic studies of protein release from H. pylori demonstrate that VacA can be detected at early timepoints as a major Helicobacter protein in the culture media, while most H. pylori proteins appear in the media at later times in combination with a lowering of turbidity of the broth media, suggesting cell lysis (50). However, autolysis as a major mechanism for export of urease and HspB has been questioned. In­ stead, export by specific pathways has been suggested for UreA, UreB, HspA, and HspB on the basis of re­ ported quantitative differences in the relative amount of these proteins found in the growth media compared with their relative abundance in whole H. pylori bac­ teria (63). The reasoning behind this proposal is that if the proteins were released by lysis, their relative abundance would be the same in intact bacteria as in culture supernatant. Since this does not correspond to the observations, the data support specific export. However, to make this hypothesis more consistent, the relative stability of these proteins must be deter­ mined to rule out protein degradation as a contribut­ ing factor to the results. Other investigators have sug­ gested that superoxide dismutase is also selectively released from the bacteria (50). In conclusion, there

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ELVER ET AL.

is no clear consensus on how these factors are ex­ ported, and the question remains on the mechanisms for export if not by autolysis. Budding Outer Membrane Vesicles—a Tool for Protein Export The cell wall of gram-negative bacteria has a fea­ ture not seen among gram-positive organisms, namely, the constant shedding during growth of mem­ brane vesicles from the bacterial outer membrane. Outer membrane vesicles (MV) are formed by bud­ ding from the O M , thus entrapping periplasmic com­ ponents in the process. From this perspective, M V can be regarded as small (50- to 250-nm) units of gramnegative cell wall, complete with O M and periplasmic contents (for a review, see reference 5). Even though M V were observed and reported 30 years ago, their existence and importance have become better recog­ nized during the last years. M V of H. pylori bind to target cells followed by uptake and localization to membranous structures. The integrity of M V located intracellularly is maintained for an extended period (20, 5 5 ) . For a protein to be exported via an M V it must first be located to the periplasm or O M . This would most likely, but not necessarily, involve a Secdependent transfer across the IM. The H. pylori VacA toxin is a Sec-dependent autotransporter protein. It is secreted as a soluble oligomer into the growth media but is also found associated with the O M and M V when cultured in vitro and in vivo (20, 31). Purified M V from H. pylori induce vacuole formation in cul­ tured epithelial cells (20, 31). The purified toxin has to be activated by acid to be able to induce vacuoles in eukaryotic cells. This is not the case for the toxin on the bacterial surface or the toxin associated with M V that do not need to be activated by acid treat­ ment. The finding of VacA toxin associated with M V suggests an alternative way for export/delivery of the toxin. Surprisingly, a number of proteins found on the surface H. pylori cells are not associated with its MV. Urease B and HspB could not be detected by Western blot analyses of MV, nor could urease activ­ ity be detected in the M V (31). It is unclear whether the proteins are selectively excluded from the M V or simply less strongly associated. The most intensively studied M V are those of Pseudomonas aeruginosa. They were shown to con­ tain protease, phospholipase C, alkaline phosphatase, and autolysin, all of which are examples of periplas­ mic proteins ( 2 8 - 3 0 ) . The membrane composition of M V is similar to the bacteria from which they derive. Some surface features of the M V must, however, be different from those of the bacterial cell wall from which they originate. The curvature of M V is very

different from that of the bacterial cell wall, thus mak­ ing it likely that physical properties and composition of the vesicle membrane and the bacterial O M some­ how differ to accommodate the necessary higher cur­ vature. An important difference observed in P. aerugi­ nosa M V is that instead of containing both A- and Bband lipopolysaccharide (LPS) as the bacterial OM, the vesicle membrane only contains B-band LPS. Clus­ tering of B-band LPS into the small domains of vesi­ cles that are being formed has been suggested to allow for the higher curvature of the M V , and also as a driving force in formation of vesicles (30). Recent work on M V shed from enterotoxigenic E. coli (24) demonstrated that active heat-labile enterotoxin can be shed from bacteria in M V , and that vesicles are enriched in toxin compared to the per­ iplasm of the bacteria from which they derived. Heatlabile toxin was found both inside and on the surface of the vesicles. Vesicule formation in enterotoxigenic E. coli has been proposed to occur at specific sites in the O M with specific protein composition, since the relative abundance of membrane proteins differs be­ tween bacteria and M V . Work with M V produced by Shigella flexneri demonstrated the potential of M V for delivery of effector molecules into the host cell cytoplasm. S. flexneri M V containing the membraneimpermeable antibiotic gentamicin were taken up by mammalian cells, and the antibiotic effect was de­ tected in the host cell cytoplasm (29). There are potential advantages of M V as vehicles for delivery of effector proteins. M V can carry a selec­ tion of factors from the bacterial O M . They may pro­ tect proteins from degradation and scavenging func­ tion by the host to which soluble secreted proteins are subjected. In addition, if the M V display adhesive factors recognizing structures on a target cell, they can be regarded as targeted vehicles for local delivery of high concentrations of virulence factors, thus avoiding the problems of dilution related to secreted proteins. H. pylori appears to make ample use of these strategies.

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CHAPTER 19 • PROTEIN EXPORT

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Helicobacter pylori: Physiology and Genetics Edited by H. L. T. Mobley, G. L. Mendz, and S. L. Hazel] © 2 0 0 1 ASM Press, Washington, D.C.

Chapter 20

Alternative Mechanisms of Protein Release STEVEN R . BLANKE AND DAN Y E

are released from H. pylori via the formation of outer membrane vesicles, which appear to have the ability to be taken up into mammalian cells.

Helicobacter pylori must be able to modulate proper­ ties of its immediate environment to colonize effec­ tively and to persist within the human stomach (12, 2 1 , 34, 86). An important aspect of environmental remodeling is the bacterium's ability to control and facilitate movement of both small and large molecules from the intracellular compartment to the extracellu­ lar environment. Proteins destined for the extracellu­ lar milieu must be translocated through a differen­ tiated cell envelope composed of inner and outer membranes that sandwich one or more peptidoglycan layer(s) and the periplasmic space ( 1 0 , 1 3 , 1 8 , 2 6 , 39, 40, 4 5 - 4 7 , 6 3 , 7 5 , 80, 88, 9 0 , 96). In gram-negative bacteria, most proteins are secreted by either type I or type II mechanisms (reviewed in another chapter of this volume). However, recent data suggest that in addition to these important and well-studied secretion pathways, some H. pylori strains use alternative strategies, including type IV secretion (16, 20), bacte­ rial autolysis (60), and the generation of outer mem­ brane vesicles (8, 62, 99), to release proteins from the bacterial cell. Each of these alternative mechanisms is fundamentally different from the others in terms of the cellular machinery involved, overall mechanisms, and fate of the transported proteins. At the same time, all three mechanisms represent potentially important approaches for facilitating adaptation of H. pylori to the unique environment of the human gastric mucosa.

TYPE IV SECRETION: ADAPTATION O F CONJUGATTVE CELLULAR MACHINERY A number of pathogenic bacteria have adapted their conjugation machinery for protein secretion (16, 20). Bacterial conjugation machinery not only facili­ tates the transport of proteins to the extracellular ma­ trix; in the case of H. pylori, effector molecules are injected directly into recipient eukaryotic cells (16, 20). This mechanism of bacterial conjugation machin­ ery-mediated secretion is now widely known as type IV secretion. Most of our current knowledge of the conjugation apparatus is derived from the Agrobacterium tumefaciens T-DNA transfer machine, which delivers oncogenic nucleoprotein particles into plant cells (19, 4 3 , 57, 5 8 , 6 1 , 7 8 , 1 0 1 , 102). Bacterial pathogens with known or putative type IV systems include Bordetella pertussis ( 2 0 , 9 8 ) , Legionella pneu­ mophila (83, 85), Rickettsia prowazekii (4), and Brucella spp. (71). The proteins comprising the type IV secretion systems are encoded by genes generally associated with pathogenicity islands, suggesting horizontal transfer of these conjugative systems (20). As dis­ cussed elsewhere in this volume, the genes associated with the H. pylori type IV apparatus are encoded within the cag (cytotoxin-associated gene) pathoge­ nicity island (1, 17). The 145-kDa effector protein CagA is directly secreted from bacterial cells into epi­ thelial cells via the cag-encoded type IV translocation apparatus. The strong correlation of the presence of the cag pathogenicity island with H. pylori strains as­ sociated with gastric disease, as well as the potential role of cag in bacterial pathogenesis, has been dis-

The purpose of this chapter is to review recent results concerning alternative strategies that H. pylori strains employ to introduce proteins into the environ­ ment. The chapter is divided into three sections. The first section describes the cellular machinery compris­ ing the type IV secretion system of H. pylori. The second section discusses evidence that H. pylori undergoes autolysis as a mechanism for releasing cy­ toplasmic proteins directly into the extracellular envi­ ronment. Finally, the third section reviews recent data supporting the theory that cellular envelope proteins

Steven R. Blanke and Dan Ye • Department of Biology and Biochemistry, University of Houston, TX 77204.

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BLANKE AND YE

cussed in detail elsewhere in this volume. This chapter focuses on the cag genes linked to the H. pylori type IV secretion apparatus. Components of the Type IV Secretion Apparatus The conjugation machines of gram-negative bac­ teria are composed of two surface structures, the mat­ ing channel through which the cargo is transferred and the conjugative pilus for contacting recipient cells, along with membrane-associated ATPases (37, 98). High-resolution structural data are not available for any mating channel, although various conjugative pili have been visualized by lower resolution tech­ niques such as electron microscopy (48). Most of the current information contributing to the model of bac­ terial conjugation machines derives from studies fo­ cused on interactions between specific protein subunits of the A. tumefaciens T-pilus transporter, as well as localization of individual transporter components in the assembled secretion machinery. The vir genes that encode the type IV secretion apparatus in A. tu­ mefaciens can be divided into three functional groups that encode the following: (i) proteins comprising the mating channel, (ii) cytoplasmic membrane ATPases, and (iii) proteins that localize exocellularly to form the T-pilus or other adhesive structures (20, 58). Al­ though these components are hypothesized to form a large supramolecular complex, there is not yet direct evidence for a physical association between the mat­ ing channel and the conjugative pilus. Unraveling the structure of the H. pylori secre­ tion apparatus represents a rich area for future re­ search. Information about the functions and proper­ ties of specific Cag proteins have been derived primarily from sequence homologies with the Vir pro­ teins of the A. tumefaciens conjugation machine (1, 17). It is striking that of the 31 genes identified within the H. pylori pathogenicity island, only six cag gene products demonstrate substantial amino acid similari­ ties to the vir gene products (Fig. 1A). On the basis of biochemical and genetic studies of the A. tumefaciens secretion apparatus (58), the locations of the six H. pylori Cag proteins are predicted via analogy to their Vir homologs (Fig. IB) to be either part of the mating channel, or, alternatively, one of the putative ATPases (Table 1). There are no Cag homologs to Vir proteins, which are involved exocellularly in contact or adhe­ sion to host cells. The lack of additional homology between Vir and Cag proteins suggests that the H. pylori cag secretion apparatus may be substantially different from the type IV apparatus of A. tumefa­ ciens. Alternatively, it cannot be ruled out that other Cag proteins may be able to carry out similar or iden­

tical functions to the Vir proteins in the A. ciens secretion apparatus. Homologs of A. tumefaciens components

tumefa­

mating channel

The VirB6-VirB10 gene products from A. tumef­ aciens, as well as one or more of the three ATPases, are believed to be channel subunits (19). VirB7, an outer membrane lipoprotein (35), is homologous to H. pylori CagT; and VirB9 is a homolog to H. pylori Cag-528 (1, 17). VirB7 forms homodimers with neighboring VirB7 and interacts with VirB9 via reac­ tive cysteines present in each protein that are oxidized to form disulfide linkages (3, 8 7 ) , The VirB7-VirB9 heterodimer has been localized to the outer membrane and appears to be essential for stabilizing other VirB proteins during assembly of the A. tumefaciens trans­ fer apparatus (35). By analogy to VirB7 and VirB9, CagT and Cag-528 may also be localized to the H. pylori outer membrane, and serve a role for assembly of the type IV secretion machinery in H. pylori. H. pylori Cag-527 is homologous to A. tumefa­ ciens VirBlO (Fig. 1A) (1, 17). VirBlO is believed to interact directly with VirB9 based on yeast two-hy­ brid analysis (24). In support of this hypothesis, VirB9 is essential for chemically cross-linking VirBlO into higher ordered multimers predicted to be homotrimers (6). It is thus reasonable to hypothesize that Cag527 and Cag-528 (the VirB9 homolog) may interact within the H. pylori "mating channel" and carry out the same or similar functions during H. pylori type IV secretion as their homologs do in A. tumefaciens (Fig. IB). Although VirBlO is proposed to bridge the cytoplasmic and outer membrane VirB subcomplexes (6), no evidence yet exists to support such a role for Cag-527. Interestingly, there is no clearly identified H. py­ lori homolog to VirB6, which is a highly hydrophobic protein thought to span the cytoplasmic membrane several times (23). VirB6 is potentially the best candi­ date for a channel-forming protein in A tumefaciens. Presumably, if the H. pylori type IV transfer appara­ tus is similar to that of the conjugation machine of A. tumefaciens, such a putative channel protein must also exist in H. pylori but may be more specifically tailored to the translocation requirements of this gas­ tric bacterium. Homologs of A. tumefaciens

membrane ATPases

Type IV secretion requires functional cyto­ plasmic membrane ATPases. H. pylori CagE and Cag525 are required for type IV translocation of effector molecules into mammalian cells and have considera-

CHAPTER 20 • ALTERNATIVE MECHANISMS OF PROTEIN RELEASE

229

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A. tumefaciens

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H. pylori

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525

527 528

Figure 1. H. pylori homologs of the A. tumefaciens conjugation machine. (A) Genetic organization of the A. tumefaciens conjugation machine. H. pylori type IV transporter components with homology to the Vir proteins are aligned underneath their homologs. (B) A model of the A. tumefaciens conjugation machine with the location of the H. pylori homologs in (A) indicated by arrows with dashed lines. This model is compiled from biochemical data described in the text predicting both the localization and interactions of the individual Vir proteins.

Table 1. H. pylori type IV system Apparent mol wt (kDa)

Localization

CagE

101

Inner membrane?

CagT

34

Outer membrane?

HP528

63

HP527

207

Inner and outer membrane? Inner membrane

HP525

38

Inner membrane

HP524

90

Inner membrane?

Protein

Biochemical interaction and/or function

Vir homolog

References

Required for N F - K B activation and IL-8 induction; nucleoside triphosphate hydrolases (putative) Lipoprotein (putative); chaperone (putative); required for the stability of other components (putative) Interacts with other components (putative)

VirB4

67, 93

VirB7

20, 58

VirB9

20, 58

Interacts with other components (putative); thermostability (putative) Nucleoside triphosphate hydrolases; form hexameric assemblies Nucleoside triphosphate hydrolases (putative); required for CagA transportation

VirB 10

20, 58

VirBll

53, 54

VirD4

20, 58

230

BLANKE AND YE

ble homology to the putative ATPase proteins of A. tumefaciens VirB4 and VirBl 1 ( 1 , 1 7 , 5 4 ) . These VirB proteins contain conserved Walker A nucleotidebinding motifs required for function ( 1 9 , 9 1 ) . Purified forms of VirB4 and VirBl 1 possess weak in vitro ATPase activities (19). Both proteins appear to assem­ ble minimally in vivo as homodimers. Dimerization of VirB4 is mediated by a domain in the amino-terminal third of the protein. In contrast, dimerization of VirBl 1 is mediated by domains located in each half of the protein (22, 7 7 , 1 0 0 ) . VirBl 1 and the H. pylori homolog Cag-525 have been shown to form higher ordered homohexameric rings in solution, further supporting the view that they may share similar or identical properties and functions (53, 77). Another putative A. tumefaciens ATPase, VirD4, is believed to be important for DNA processing and transfer reactions by discriminating for transferred DNA (42). The H. pylori homolog of VirD4 is HP524, which is required for transport of the H. pylori protein effector molecule CagA into mammalian cells (89), suggesting that this family of putative ATPases may not be specific for the type of molecule transported by the type IV secretion machinery. Homologs of A. tumefaciens components

exocellular T-pilus

The third component of the A. tumefaciens secre­ tion apparatus is composed of the exocellular VirB proteins localized to form the adhesive and contact structure for direct interactions with host cells (20, 58). There are no cag gene products that share signifi­ cant sequence homology to A. tumefaciens proteins comprising the scaffold of the T pilus (1, 17). The lack of clearly identifiable homologs may represent differences in adaptive requirements for H. pylori and A. tumefaciens, which interact with gastric epithelial cells and plant cells, respectively. The proteins that comprise the exocellular structure required for con­ tact and adhesion of the H. pylori type IV machine remain undefined, and their identification and charac­ terization represent important areas for future investi­ gations.

BACTERIAL AUTOLYSIS: AN "ALTRUISTIC" MECHANISM F O R H. PYLORI ADAPTATION WITHIN A HOSTILE ENVIRONMENT? Nascent proteins targeted for secretion by type I or type II mechanisms are transported rapidly subse­ quent to their expression in the cytoplasm (70, 74). Consequently, proteins destined for secretion by these mechanisms are not generally localized simultane­

ously within the cytoplasm and extracellular milieu. In contrast, typical cytoplasmic H. pylori proteins, such as urease and catalase, have been found in the cytoplasm, localized to the outer membrane, and/or in the extracellular medium (14, 3 3 , 73). This phe­ nomenon has been observed in vitro, but it is thought that extracellular transport of H. pylori cytoplasmic proteins also occurs in vivo. Supporting this hypothe­ sis are the observations that administration of urease, catalase, and other proteins confers protection against H. pylori challenge in animal models (11, 2 8 , 30, 4 1 , 59, 64, 6 8 , 76). Recent studies have suggested that specific H. py­ lori cytoplasmic proteins are localized to the outer membrane by a mechanism of bacterial autolysis (33, 73). According to this model (Fig. 2), individual bacte­ ria are lysed by a highly regulated and controlled pro­ cess that results in the release of cytoplasmic proteins from the bacterial cytosol. Notably, a number of other pathogenic bacteria, including Streptococcus pneu­ moniae and Neisseria gonorrhoeae, convincingly have been demonstrated to undergo a regulated program of autolysis (60, 69, 7 9 , 84, 85). In H. pylori, some of the released cytoplasmic proteins are then adsorbed to the surface of the outer membrane of neighboring bacteria. Such a model has been called "altruistic au­ tolysis" since the lysis of a fraction of the bacterial population presumably benefits the remaining viable bacteria (5, 32, 3 3 , 7 3 ) . This type of mechanism has been suggested by a number of investigators to ex­ plain how vaccination with a cytoplasmic protein such as urease can confer protection against chal­ lenges with Helicobacter spp. in animal models, as described (11, 30, 4 1 , 5 9 , 68). Because H. pylori is noninvasive, autolysis might also facilitate presenta­ tion of virulence factors and immunogens to the host (5, 32, 3 3 , 7 3 ) . Experimental Evidence for Autolysis Evidence supporting a model of altruistic autoly­ sis is based primarily on recent H. pylori urease locali­ zation studies (5, 3 2 , 3 3 , 7 3 ) . All H. pylori isolates generate large quantities of a potent urease, which catalyzes the hydrolysis of urea in the stomach to pro­ duce ammonia and bicarbonate ions, as detailed else­ where in this volume. H. pylori urease has been cate­ gorized as a virulence factor because the enzyme is required for bacterial colonization of the gastric mu­ cosa (65). In addition, urease functions in the utiliza­ tion of exogenous urea as a nitrogen source for amino acid synthesis (97). Although urease is localized in the cytoplasm of H. pylori and other bacterial species, cryoimmunoelectron microscopy showed the enzyme to be found both in vitro and in vivo on the cell surface

CHAPTER 20 • ALTERNATIVE MECHANISMS OF PROTEIN RELEASE

©

231

El

Gastric mucosa colonization

Figure 2. H. pylori altruistic autolysis. This model predicts that H. pylori responds to specific environmental signals to activate genetically programmed bacterial autolysis. Specific protein autolysins degrade the H. pylori peptidoglycan layer to lyse the cell envelope and release cytoplasmic proteins such as urease and catalase. Released urease is subsequently adsorbed to the surface of the neighboring bacteria and is important for H. pylori survival in the acid environment and colonization of the gastric mucosa.

under specific conditions (73). The release of urease and other cytoplasmic proteins from H. pylori has been demonstrated now in vitro by three independent groups (73, 8 1 , 82), and an autolytic mechanism has been proposed to explain their location (73, 81). How does cytoplasmic urease localize to the H. pylori outer membrane surface? Urease does not pos­ sess an amino-terminal signal peptide for targeting to the type II (Sec-dependent) secretion pathway. Re­ combinant H. pylori urease expressed in E. coli shows cytoplasmic activity, demonstrating the efficacy of ex­ pressing a functional protein in a heterologous system (55). However, no urease was detectable on the outer surface of E. coli, suggesting that the mechanism em­ ployed by H. pylori for surface localization of the en­ zyme is not ubiquitous to all gram-negative bacteria. In H. pylori, autolysis is sensitive to the phase of bacterial growth. Urease, catalase, and the heat shock protein HspB are localized only in the cytoplasmic compartment during exponential growth phase (ca. 24 h) in H. pylori in vitro cultures (73). However, bacteria cultivated for 72 h demonstrated significant amounts of surface-bound and extracellular urease, suggesting that urease is localized on the outer surface only in older bacteria. Catalase and HspB are also

localized outside the cytoplasm in older or subcultured preparations, suggesting that the extracellular release of cytoplasmic proteins with age is not a pro­ cess that is exclusive to urease. The presence of an autolytic mechanism for re­ lease of some H. pylori proteins remains controversial because of observations not properly explained by this mechanism. H. pylori yielded differential release patterns of proteins from the cytoplasm to the extra­ cellular supernatant, and kinetic studies indicated that proteins were not released at the same rate (94). Col­ lectively, these data were interpreted to support a role for specific secretion mechanism(s) rather than autol­ ysis for the release of cytoplasmic proteins (94). None­ theless, autolysis of some gram-negative bacteria by a regulated and controlled mechanism is gaining ac­ ceptance (60, 6 9 , 7 9 , 84, 8 5 ) , and the current data supporting H. pylori autolysis warrants additional in­ vestigation of this problem. Autolysis is a strategy for survival in a hostile environment The presence of cytoplasmic proteins on the sur­ face of the H. pylori outer membrane both in vitro

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and in vivo is consistent with a mechanism of bacterial autolysis. In addition, the dependence of cytoplasmic protein release on the phase of bacterial growth in vitro suggests that the process is regulated. H. pylori autolysis has been termed "altruistic" because surface-bound urease is important for bacte­ rial survival in acid. Using membrane-impermeable (flurofamide) and -permeable (acetohydroxamic acid) inhibitors, it was demonstrated that decreasing only surface-bound urease activity had almost as great an effect on H. pylori acid resistance as decreasing both surface and cytoplasmic urease activity (55). More­ over, H. pylori lacking surface-associated urease was unable to withstand exposure to acid (55). Specifi­ cally, H. pylori cultures grown for 2 4 h did not survive the acid environment in the presence of urea, whereas cultures, which had been grown and passaged every 72 h, demonstrated a marked ability to survive acid treatment (pH 2 in the presence of 5 mM urea). In support of the importance of surface-bound urease to bacterial acid resistance is the observation that E. coli strains expressing only cytoplasmic urease activity were not resistant to acid (55), although alternative explanations for sensitivity of these bacteria to acidic conditions are possible. Collectively, these data sug­ gest that surface-bound urease performs an essential function in H. pylori acid resistance, leading to the conclusion that the release of this protein by some bacteria has beneficial effects on the overall popula­ tion. Machinery for autolysis An autolytic mechanism influenced by environ­ mental factors and regulated by genetic factors re­ quires cellular components that contribute directly to autolysis. In other bacterial species, autolysis requires disruption of the cell wall peptidoglycan layers by spe­ cific proteins called autolysins. These proteins can be penicillin-binding proteins or non-penicillin-binding proteins. They have been identified for S. pneumon­ iae, which undergoes genetically regulated autolysis both in vivo and in vitro (72). S. pneumoniae autolysis occurs under conditions of nutrient starvation or anti­ biotic treatment. Direct genetic evidence for the role of autolysins comes from the inability of autolysindeficient mutants to undergo autolysis during the sta­ tionary phase of growth (72) and from restoration of the autolytic phenotype of S. pneumoniae mutants by genetic complementation with a gene encoding the autolysin (7, 79). The importance of autolysis in bac­ terial pathogenesis is suggested from results showing that autolysin-negative S. pneumoniae strains are less virulent in animal models than wild-type strains (7, 38, 79). N. gonorrhoeae apparently undergoes auto­

lysis as a mechanism of releasing DNA for uptake by other bacteria (27). The mechanism of N. gonor­ rhoeae autolysis also appears to be regulated. Signifi­ cantly, autolysin-deficient mutants are not lysed under conditions that generally promote autolysis in this bacterium (27). An autolytic mechanism in H. pylori would re­ quire the bacterium to produce factors that function as autolysins. Genome analyses of H. pylori predict open reading frames encoding a putative lytic trans­ glycosylase and an amidase (2, 92). In addition, the genes HP597, HP1556, and HP1565 encode proteins with sequence similarities to penicillin-binding pro­ teins from Escherichia coli, Haemophilus influenzae, and Bacillus subtilis (92), but in the H. pylori genome no sequence similarities are detected to lower molecu­ lar weight penicillin-binding proteins with carboxy­ peptidase or endopeptidase activities that may be in­ volved in peptidoglycan degradation. Recent work with biotin- and digoxigenin-labeled antibiotics served to identify numerous putative penicillin-bind­ ing proteins with an assortment of molecular weights ( 2 5 , 2 9 , 4 4 ) . A novel penicillin-binding protein (PBP4) has been discovered in both the soluble and mem­ brane fractions of H. pylori preparations (56), whose expression increases significantly during mid- to lateexponential phase of H. pylori. The sequence of PBP4 has highly conserved penicillin-binding motifs ar­ ranged in a novel fashion, and it is hypothesized that PBP4 has endopeptidase activity to degrade peptidog­ lycan (56). These features of PBP4 suggest a possible link to autolysis. Although evidence is accumulating that H. pylori may release cytoplasmic proteins into the extracellu­ lar milieu in a regulated fashion and that the bacter­ ium may possess putative autolysins, many aspects of the altruistic autolysis model remain untested. In particular, it will be important to establish the signifi­ cance of an autolytic mechanism to colonization and survival of the bacterium in its unique environment. Notwithstanding that H. pylori apparently releases cytoplasmic proteins in vitro as a function of the growth phase, the signals that may trigger autolysis in vivo remain unidentified. Nonetheless, altruistic autolysis remains an intriguing hypothesis to explain bacterial adaptation to hostile conditions.

OUTER MEMBRANE VESICLES: BUNDLING CELL ENVELOPE PROTEINS FOR DELIVERY? Recently, it has been shown that H. pylori re­ leases small vesicles from its outer membrane by a process that bears striking similarity to the release of membrane vesicles by a number of other bacterial

CHAPTER 20 • ALTERNATIVE MECHANISMS OF PROTEIN RELEASE

OM

233

PS IM C

Figure 3. H. pylori outer membrane vesicles budding. The release of outer membrane vesicles by H. pylori is an alternative mechanism for the delivery of bacterial toxins and antigens to the gastric mucosa. These small vesicles are 50 to 300 nm in diameter. They contain VacA and other proteins such as porins within the trilayered membrane (OM, outer membrane; PS, periplasm; IM, inner membrane; C, cytoplasm).

pathogens (36), namely, Neisseria meningitidis, H. in­ fluenzae, Borrelia burgdorferi, Pseudomonas aerugi­ nosa, and Campylobacter jejuni (9, 15, 4 9 - 5 2 , 62, 66, 99). H. pylori strains examined from gastric biop­ sies of infected patients generate small trilayered membranous vesicles, and it is hypothesized that they derive from the bacterial outer membrane (Fig. 3). Significantly, these outer membrane vesicles fre­ quently contain numerous antigens and virulence fac­ tors. The production of these vesicles potentially rep­ resents an important mechanism for bacterial pathogens to modulate their environment within the host. Production of Outer Membrane Vesicles To investigate whether outer membrane vesicles are produced by H. pylori cultured in vitro as well as in vivo, bacteria were examined by ultrastructural and immunochemical methods (36). These studies pro­ vided insights on the composition and production of H. pylori vesicles. Outer membrane vesicle formation appears to be a multistep process (Fig. 3). Vesicles originate as "blebs" that protrude from the body of the cell as outward expansions of the periplasmic space surrounded by the outer membrane. The ob­ served protruding blebs are likely to represent the

early stages of vesicle formation. The blebs continue to bud, with concomitant focal expansion of the per­ iplasmic space, and finally, vesicles are released from the cell. The vesicles are much smaller than the bacte­ ria from which they originated, but still of significant size, ranging from 50 to 300 nm in diameter. H. py­ lori-derived outer membrane vesicles accumulate in the culture supernatants over time. The vesicles contain outer membrane proteins such as porins (36). Interestingly, outer membrane vesicles did not contain detectable urease activity or cross-reacting material, suggesting that their forma­ tion is a fundamentally different process than the pro­ posed autolytic mechanism described above. More­ over, vesicle formation occurs during H. pylori exponential growth, while the release of cytoplasmic proteins by the putative autolytic mechanism de­ scribed above occurs during the stationary phase. Outer membrane vesicles and environmental adaptation Outer membrane blebs have been found in every examined biopsy of H. pylori-infected patients, whether the outer membranes of bacteria were adja­ cent to the gastric epithelium or not (36). Besides being distributed around bacteria, the vesicles adhere

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to the luminal surface of gastric superficial-foveolar epithelium. The outer membrane blebs and vesicles contain vacuolating toxin (VacA), which is an impor­ tant virulence factor in H. py/on'-mediated disease pathogenesis; and vesicles containing VacA were de­ tected inside cytoplasmic tubulovesicles or vacuoles of the epithelium. These vesicles were found also within dilated endosomes and the vacuoles formed by pro­ gressive dilation and fusion of these structures (31). These observations suggest an in vivo role for outer membrane vesicles (36). The role of outer membrane-derived vesicles in H. pylori growth and survival is poorly understood. In common with other gram-negative bacteria, vesicle formation in H. pylori may represent an important mechanism for the delivery of various virulence fac­ tors and antigens to host tissues that otherwise would be insoluble and/or susceptible to degradation in the extracellular matrix. Antigens derived from outer membrane vesicles might traverse the gastric epithelial monolayer and ultimately reach the lamina propria, thus providing a mechanism for antigen presentation resulting in local and systemic immune responses (31). Many interesting problems concerning H. pylori outer membrane vesicles remain unsolved, for exam­ ple, ascertaining the way in which the release of outer membrane vesicles is regulated, determining the gene products that specifically facilitate membrane blebbing, and establishing the fate of the H. pylori cell from which the membrane vesicle is derived.

CONCLUSIONS Bacterial secretion remains a fertile area of re­ search. H. pylori has classical type I and type II gramnegative bacterial secretion pathways, but it is becom­ ing apparent that this bacterium uses alternative mechanisms for exporting proteins to the outside en­ vironment. The ability of a noninvasive bacterium such as H. pylori to modulate its environment is an important strategy for colonization and survival within the human stomach. Each of the mechanisms described above has the potential of allowing H. py­ lori to carry out important functions within the host. The type IV secretion system facilitates direct trans­ port of protein effectors from the bacterium into the host cell. This adaptation of the conjugal machinery of many gram-negative bacteria bypasses the need for active uptake by the host cell. Bacterial autolysis prob­ ably involves mechanisms of peptidoglycan degrada­ tion to release cytoplasmic contents into the extracel­ lular medium, which can then be strategically relocalized onto the outer membrane of other bacte­ ria. In the case of H. pylori, localization of urease to

the outer membrane appears to assist its survival in the acidic environment of the gastric mucosa. Finally, the release of outer membrane vesicles may allow H. pylori to bundle up cellular envelope proteins and de­ liver them to gastric epithelial cells and potentially to other locations distal to the site of colonization. Clearly, these three mechanisms represent remarkable adaptation strategies of H. pylori. Acknowledgments. Research funding from the National Institutes of Health (ROI AI45928), the American Heart Association (98B 6472), and the Robert A. Welch Foundation (E-1311) is gratefully acknowledged.

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lytic effect of membrane vesicles from Pseudomonas aerugi­ nosa on other bacteria including pathogens: conceptually new antibiotics. /. Bacteriol. 178:2767-2774. Kadurugamuwa, J. L., and T. J. Beveridge. 1997. Natural release of virulence factors in membrane vesicles by Pseudom­ onas aeruginosa and the effect of aminoglycoside antibiotics on their release. /. Antimicrob. Chemother. 40:615-621. Kadurugamuwa, J. L., and T. J. Beveridge. 1999. Membrane vesicles derived from Pseudomonas aeruginosa and Shigella flexneri can be integrated into the surfaces of other gramnegative bacteria. Microbiology 145:2051-2060. Rolling, G. L., and K. R. Matthews. 1999. Export of virulence genes and Shiga toxin by membrane vesicles of Escherichia coli 0157:H7. Appl. Environ. Microbiol. 65:1843-1848. Krause, S., M. Barcena, W. Pansegrau, R. Lurz, J. M. Carazo, and E. Lanka. 2000. Sequence-related protein export NTPases encoded by the conjugative transfer region of RP4 and by the cag pathogenicity island of Helicobacter pylori share similar hexameric ring structures. Proc. Natl. Acad. Sci. USA 97:3067-3072. Krause, S., W. Pansegrau, R. Lurz, F. de la Cruz, and E. Lanka. 2000. Enzymology of type IV macromolecule secre­ tion systems: the conjugative transfer regions of plasmids RP4 and R388 and the cag pathogenicity island of Helicobacter pylori encode structurally and functionally related nucleoside triphosphate hydrolases. /. Bacteriol. 182:2761-2770. Krishnamurthy, P., M. Parlow, J. B. Zitzer, N. B. Vakil, H. L. Mobley, M. Levy, S. H. Phadnis, and B. E. Dunn. 1998. Helicobacter pylori containing only cytoplasmic urease is sus­ ceptible to acid. Infect. Immun. 66:5060-5066. Krishnamurthy, P., M. H. Parlow, J. Schneider, S. Burroughs, C. Wickland, N. B. Vakil, B. E. Dunn, and S. H. Phadnis. 1999. Identification of a novel penicillin-binding protein from Helicobacter pylori. J. Bacteriol. 181:5107-5110. Lai, E. M., O. Chesnokova, L. M. Banta, and C. I. Kado. 2000. Genetic and environmental factors affecting T-pilin ex­ port and T-pilus biogenesis in relation to flagellation of Agrobacterium tumefaciens. J. Bacteriol. 182:3705-3716. Lai, E. M., and C. I. Kado. 2000. The T-pilus of Agrobacterium tumefaciens. Trends Microbiol. 8:361-369. Lee, C. K., K. Soike, J. Hill, K. Georgakopoulos, T. Tibbitts, J. Ingrassia, H. Gray, J. Boden, H. Kleanthous, P. Giannasca, T. Ermak, R. Weltzin, J. Blanchard, and T. P. Monath. 1999. Immunization with recombinant Helicobacter pylori urease decreases colonization levels following experimental infec­ tion of rhesus monkeys. Vaccine 17:1493-1505. Lewis, K. 2000. Programmed death in bacteria. Microbiol. Mol. Biol. Rev. 64:503-514. Li, P. L., I. Hwang, H. Miyagi, H. True, and S. K. Farrand. 1999. Essential components of the Ti plasmid trb system, a type IV macromolecular transporter. /. Bacteriol. 181: 5033-5041. Li, Z., A.J. Clarke, and T.J. Beveridge. 1998. Gram-negative bacteria produce membrane vesicles which are capable of kill­ ing other bacteria. /. Bacteriol. 180:5478-5483. Lory, S. 1992. Determinants of extracellular protein secretion in gram-negative bacteria. /. Bacteriol. 174:3423-3428. Marchetti, M., B. Arico, D. Burroni, N. Figura, R. Rappuoli, and P. Ghiara. 1995. Development of a mouse model of Heli­ cobacter pylori infection that mimics human disease. Science 267:1655-1658. Marshall, B. J., L. J. Barrett, C. Prakash, R. W. McCallum, and R. L. Guerrant. 1990. Urea protects Helicobacter (Campylobacter) pylori from the bactericidal effect of acid. Gastroenterology 99:697-702.

66. Mayrand, D., and D. Grenier. 1989. Biological activities of outer membrane vesicles. Can. J. Microbiol. 35:607-613. 67. Munzenmaier, A., C. Lange, E. Glocker, A. Covacci, A. Moran, S. Bereswill, P. A. Baeuerle, M. Kist, and H. L. Pahl. 1997. A secreted/shed product of Helicobacter pylori acti­ vates transcription factor nuclear factor-kappa B. /. Immu­ nol. 159:6140-6147. 68. Myers, G. A., T. H. Ermak, K. Georgakopoulos, T. Tibbits, J. Ingrassia, H. Gray, H. Kleanthous, C. K. Lee, and T. P. Monath. 1999. Oral immunization with recombinant Helico­ bacter pylori urease confers long-lasting immunity against Helicobacter felis infection. Vaccine 17:1394-1403. 69. Novak, R., and E. Tuomanen. 1999. Pathogenesis of pneu­ mococcal pneumonia. Semin. Respir. Infect. 14:209-217. 70. Nunn, D. 1999. Bacterial type II protein export and pilus biogenesis: more than just homologies? Trends Cell Biol. 9: 402-408. 71. O'Callaghan, D., C. Cazevieille, A. Allardet-Servent, M. L. Boschiroli, G. Bourg, V. Foulongne, P. Frutos, Y. Kulakov, and M. Ramuz. 1999. A homologue of the Agrobacterium tumefaciens VirB and Bordetella pertussis Ptl type IV secre­ tion systems is essential for intracellular survival of Brucella suis. Mol. Microbiol. 33:1210-1220. 72. Paton,J.C,P.W.Andrew,G.J.Boulnois,andT.J.Mitchell. 1993. Molecular analysis of the pathogenicity of Streptococ­ cus pneumoniae: the role of pneumococcal proteins. Annu. Rev. Microbiol. 47:89-115. 73. Phadnis, S. H., M. H. Parlow, M. Levy, D. liver, C. M. Caulkins, J. B. Connors, and B. E. Dunn. 1996. Surface locali­ zation of Helicobacter pylori urease and a heat shock protein homolog requires bacterial autolysis. Infect. Immun. 64: 905-912. 74. Pugsley, A. P. 1993. The complete general secretory pathway in gram-negative bacteria. Microbiol. Rev. 57:50-108. 75. Pugsley, A. P., C. d'Enfert, I. Reyss, and M. G. Kornacker. 1990. Genetics of extracellular protein secretion by gramnegative bacteria. Annu. Rev. Genet. 24:67-90. 76. Radcliff, F. J., S. L. Hazell, T. Kolesnikow, C. Doidge, and A. Lee. 1997. Catalase, a novel antigen for Helicobacter pylori vaccination. Infect. Immun. 65:4668-4674. 77. Rashkova, S., X. R. Zhou, J. Chen, and P. J. Christie. 2000. Self-assembly of the Agrobacterium tumefaciens VirBl 1 traffic ATPase. /. Bacteriol. 182:4137-4145. 78. Ream, W. 1998. Import of Agrobacterium tumefaciens viru­ lence proteins and transferred DNA into plant cell nuclei. Subcell. Biochem. 29:365-384. 79. Ronda, C , J. L. Garcia, E. Garcia, J. M. Sanchez-Puelles, and R. Lopez. 1987. Biological role of the pneumococcal amidase. Cloning of the lytA gene in Streptococcus pneumoniae. Eur. ] . Biochem. 164:621-624. 80. Sandkvist, M., and M. Bagdasarian. 1996. Secretion of re­ combinant proteins by gram-negative bacteria. C«rr. Opin. Biotechnol. 7:505-511. 81. Schraw, W., M. S. McClain, and T. L. Cover. 1999. Kinetics and mechanisms of extracellular protein release by Helico­ bacter pylori. Infect. Immun. 67:5247-5252. 82. Scott, D. R., D. Weeks, C. Hong, S. Postius, K. Melchers, and G. Sachs. 1998. The role of internal urease in acid resis­ tance of Helicobacter pylori. Gastroenterology 114:58-70. 83. Segal, G., J. J. Russo, and H. A. Shuman. 1999. Relationships between a new type IV secretion system and the icm/dot viru­ lence system of Legionella pneumophila. Mol. Microbiol. 34: 799-809. 84. Shockman, G. D. 1992. The autolytic ('suicidase') system of Enterococcus hirae: from lysine depletion autolysis to bio-

CHAPTER 20 • ALTERNATIVE MECHANISMS OF PROTEIN RELEASE

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90. 91.

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93.

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Helicobacter pylori: Physiology and Genetics Edited by H. L. T. Mobley, G. L. Mendz, and S. L. Hazell © 2 0 0 1 ASM Press, Washington, D.C.

Chapter 21

Motility, Chemotaxis, and Flagella GUNTHER

S P O H N AND V l N C E N Z O

For many pathogenic bacteria, flagellum-dependent motility and chemotaxis are crucial factors in the pro­ cess of colonization of the host organism and estab­ lishment of a successful infection (reviewed in refer­ ences 36 and 39). The flagella of Helicobacter pylori have been extensively studied, and convincing evi­ dence is available demonstrating the key role of these organelles in the colonization of the human gastric mucosa by this fastidious pathogen. Experiments with gnotobiotic piglets have established a correlation be­ tween the motility state of some H. pylori isolates and their ability to colonize the gastric epithelium ( 1 1 , 1 2 ) . The characterization of isogenic mutants deficient in specific flagellar proteins has confirmed the impor­ tance of an intact flagellar apparatus and the resulting motility for H. pylori pathogenicity (8, 13, 17, 2 1 , 28, 2 9 ) . Structural components of the flagellum as well as secretory and regulatory proteins involved in the synthesis of the flagellar apparatus and the control of chemotaxis have been analyzed in recent years, and our knowledge regarding the mechanisms of H. pylori motility is rapidly increasing. This chapter summa­ rizes the available experimental and genomic data on flagellar function and provides an overview of our current knowledge of this field. The emphasis will be on the regulation of flagellar gene transcription and chemotaxis, while a more detailed description of the morphology of the H. pylori flagellum will be given in chapter 7.

T H E FLAGELLA H. pylori cells normally possess a unipolar bun­ dle of two to six sheathed flagella that enable the bac­ teria to move in their ecological niche represented by the mucous layer of the gastric epithelium (45). Each flagellum is about 3 u.m long and exhibits a typical

SCARLATO

bulb-like structure at its distal end that represents a dilation of the flagellar sheath (20). The sheath itself is an extension of the outer membrane and is thought to protect the acid-labile flagellar structure from the attack of the stomach acid (20). The H. pylori flagella are composed of three structural elements like those of enteric bacteria: a basal body, which is embedded in the cell wall and contains the proteins required for rotation and che­ motaxis; an external helically shaped filament that works as a propeller when rotated at its base; and a hook that serves as a joint between the basal body and the flagellar filament (Fig. 1). Table 1 shows that in H. pylori more than 5 0 putative proteins are pre­ dicted to be involved in expression, secretion, and as­ sembly of this complex flagellar apparatus (3, 4 7 ) . At least 2 0 of these proteins constitute the structural components of the flagellar basal body, hook, and filament (Table 1). T o date, only components of the filament and the hook have been characterized in some detail. The filament is a copolymer of the flagel­ lin subunits FlaA (21, 32) and FlaB (46). The FlaA is the predominant subtype and FlaB the minor subtype, localized close to the basis of the flagellum (30). Both flagellins have similar molecular mass (53 kDa) and share considerable amino acid homology ( 5 8 % iden­ tity), but the respective genes are unlinked in the chro­ mosome (46). Studies with isogenic mutants of either flaA or flaB revealed that both genes are necessary for full motility (28) and for establishment of a persistent infection in the gnotobiotic piglet model (13). The flgE gene encodes the structural protein of the hook, and the fliD gene encodes a hook-associated protein (HAP2), which is localized at the tip of the flagellar filament and which promotes the incorporation of the flagellin monomers into the growing flagellar filament (Fig. 1). These genes have been characterized; mutants in flgE are nonmotile and aflagellate (38), and mu-

GuntherSpohn • Department of Molecular Biology, IRIS, Chiron S.p.A., Siena, Italy. Vincenzo Scarlato • Department of Molecu­ lar Biology, IRIS, Chiron S.p.A., Siena, Italy, and Department of Biology, University of Bologna, Bologna, Italy.

239

240

SPOHN AND SCARLATO

Outer membrane Peptidoglycan layer Cytoplasmic membrane

Figure 1. Flagellar and chemotaxis proteins and their putative locations and interactions. The protein components of the major elements of theflagellum,the external filament, the hook, the basal body, and the motor-switch complex are shown. The externalflagellarproteins that form the rod, hook, and filament are secreted by a specific export apparatus forming at the cytoplasmic side of the MS ring. The proton gradient at the cytoplasmic membrane drives a proton flow that energizes flagellar motor rotation. The direction of rotation is determined by the interaction of FliM in the motor-switch complex with the chemotaxis regulator CheY, whose activity is regulated by the other components of the chemotaxis system. Symbols: H , protons; P, phosphate; C H 3 , methyl group. +

tants in fliD produce truncated flagella and are se­ verely impaired in motility and their ability to colo­ nize the gastric mucosae of mice (29). A protein of the flagellar sheath was also characterized and shown to be identical to HpaA, an N-acetylneuraminyllactose binding hemagglutinin (15, 2 7 ) . Another structural gene identified on the basis of sequence homologies with characterized genes is fliF encoding the subunits of the MS ring (Fig. 1), the first complex to be assembled in the course of flagellar morphogenesis (26). This complex is formed on the cytoplasmic side of the inner membrane, where it serves as a construction base for the flagellar rod made of the FlgB, FlgC, and FlgG proteins (24) and as an anchor for the motor switch proteins FliM, FliN, and FliG (18, 19) and the motor rotation proteins Mot A and MotB (10, 44) (Fig. 1). The genes encoding all of these proteins have been found in the H. pylori ge­ nome as well as the flgl and flgH genes encoding the subunits of the P and L rings, (26), which form in the periplasmic space and the outer membrane, respec­

tively (Fig. 1). In addition to FliD, the hook-associated proteins FlgK and FlgL (22), the FlaG polar flagellins, and additional paralogs of the sheath protein HpaA are also encoded by the genome. All these proteins are likely to become incorporated into the H. pylori flagella. Biosynthesis and Assembly Most of the flagellar apparatus is localized be­ yond the cytoplasmic membrane and therefore many of the numerous flagellar proteins have to cross the membrane to reach their final destination. The pro­ teins constituting the P and L rings, which anchor the flagellum in the periplasm and the outer membrane, respectively, are secreted via the conventional signalpeptide-dependent Sec pathway, whereas the axial components of the flagellar apparatus, including the structural proteins of the filament, the hook, and the rod, which connects the hook to the basal body, are believed to be secreted by a specialized flagellum-spe-

CHAPTER 21 • MOTILITY, CHEMOTAXIS, AND FLAGELLA

Table 1. Flagellar genes identified in the two published genome sequences" Function

Gene/protein Structural Basal body HP1031/JH393 HP584/JH531 HP352/JH326 HP815/JH751 HP816/JH752 HP351/JH325 HP325/JH308 HP246/JH231 HP1558/JH1466 HP1559/JH1467 HP1585/JH1492 HP1557/JH1465 HP1092/JH333 HP1030/JH394 Hook HP753/JH690 HP870/JH804 HP908/JH844 HP907/JH843 HP1119/JH1047 HP752/JH689 HP295/JH280 Filament HP115/JH107 HP601/JH548 HP797/JH733 HP492/JH444 HP410/JH971 HP751/JH688 HP327/JH310 Biosynthetic HP1575/JH1483 HP1419/JH1314 HP809/JH745 HP770/JH707 HP685/JH625 HP173/JH159 HP1041/JH383 HP684/JH625 HP353/JH327 HP1035/JH389 HP1420/JH131S Regulatory Chemotaxis HP19/JH17 HP99/JH91 HP103/JH95 HP82/JH75 HP599/JH546 HP1067/JH358 HP392/JH989 HP393/JH988 HP616/JH559 HP391/JH990 HP1274/JH1195 Transcription HP244/JH229 HP703/JH643 HP714/JH652 HP1032/JH392

Flagellar Flagellar Flagellar Flagellar Flagellar Flagellar Flagellar Flagellar Flagellar

motor switch protein (fliM) switch protein (fliN) motor switch protein (fliG) motor rotation protein (motA) motor rotation protein (motB) basal-body M-ring protein (flip) basal-body L-ring protein (flgH) basal-body P-ring protein (flgl) basal-body rod protein (proximal rod

protein) (flgC) Flagellar basal-body Flagellar basal-body Flagellar basal-body Flagellar basal-body FliY protein (fliY)

rod protein (flgB) rod protein (flgG) protein (fliE) rod protein (flgG)

Flagellar protein (fliS) Flagellar hook protein (flgE) (38) Flagellar hook protein homolog (flgE') Hook assembly protein (flgD) Flagellar hook-associated protein 1 (HAPl//ZgK) Flagellar hook-associated protein 2 (HAP2//KD) (29)

241

cific pathway. This pathway is constituted of a num­ ber of flagellar biosynthetic proteins that assemble into a structure at the MS ring of the flagellum that binds the flagellar proteins and transfers them actively into the growing flagellum (Fig. 1). The members of this system share homology to components of the widely distributed contact-dependent type III secre­ tion systems, which in other organisms have been found to be involved also in the secretion of virulence factors (5, 9). In H. pylori the export apparatus is assembled from the proteins encoded by the fliH, flil, fliQ, fliL, fliP, fliR, fib A (fib A), and flhB genes (Table 1). To date, only the flil, fliQ, flhA, and flhB genes have been analyzed to some extent. Both flil and flhA (fibA) code for members of the LcrD/FlbF family of motility and virulence-associated proteins, and knockout mutants in both of these genes were shown to be nonmotile and aflagellate ( 2 5 , 4 0 , 4 1 ) . Similarly, the FliQ and FlhB genes were shown to be essential for assembly of the flagellum and for motility of the bacteria (17).

Flagellar hook-associated protein 3 (HAP3//ZgL) Flagellin B (flaB) ( 4 6 ) Flagellin A (flaA) ( 2 1 , 3 2 ) Flagellar sheath adhesin bpaA ( 1 5 , 2 7 ) Paralog of bpaA Paralog of bpaA Polar flagellin (flaG) Polar flagellin (flgaG) Flagellar biosynthetic protein (flhB) Flagellar biosynthetic protein (fliO) (17, 4 0 ) Flagellar biosynthetic protein (fliL) Flagellar biosynthetic protein (flhB) (17) Flagellar biosynthetic protein (fliP) Flagellar biosynthetic protein (fliR) Flagellar biosynthetic protein (flhA/flbA) (41) Flagellar biosynthetic protein (flip) Flagellar export protein (fliH) Flagellar biosynthetic protein (flhF) Flagellar export protein A T P synthase (flil) (25, 40)

Chemotaxis protein (cheV) Methyl-accepting chemotaxis protein (tipA) Methyl-accepting chemotaxis protein (tlpB) Methyl-accepting chemotaxis transducer (tlpC) Methyl-accepting chemotaxis protein Chemotaxis protein (cheV) (7, 16) Histidine kinase/chemotaxis protein (cheAY) (17) Chemotaxis protein (cheV) Chemotaxis protein (cheV) Purine-binding chemotaxis protein (cheW) Paralyzed flagella protein (pflA)

Regulation of Flagellar Gene Transcription In Salmonella and Caulobacter crescentus the transcriptional regulation of flagellar gene expression has been extensively studied (reviewed in references 34 and 50). In these organisms, expression of the genes required for flagellar biosynthesis is tightly reg­ ulated by alternative sigma factors and specific tran­ scriptional activators. In H. pylori only a few flagellar genes have been analyzed regarding their transcrip­ tional regulation. Table 2 shows those H. pylori genes and putative operons for which transcriptional start points have been mapped experimentally and for which a specific promoter sequence could be assigned; the table also shows those genes and operons for which a putative promoter has been deduced on the basis of homology with the consensus recognition se­ quence of one of the three sigma factors encoded by the H. pylori genome. The analysis shows that the three sigma factors cr , cr , and o , deduced from the genome sequence analysis, participate in the tran­ scription of flagellar genes, indicating that transcrip­ tion of H. pylori flagellar genes is highly regulated like in other organisms studied (Table 2 ) . 80

54

2 8

Rod and hook functions: a -dependent genes 54

Histidine kinase (6) Response regulator (flgR) (42) Alternative sigma factor sigma 5 4 Alternative sigma factor sigma 2 8 (fliA)

A closer inspection of the genes regulated by RNA polymerase containing the alternative sigma fac­ tor cr (Table 2) reveals that the proteins encoded by these genes are likely to be localized in the same region of the flagellum. The flgB, flgC, and flgG genes are 54

° Characterized genes and proteins are in bold letters. Numbers labeled "HP" refer to the sequence published by Tomb et al. ( 4 7 ) , those labeled " J H " to the one published by Aim et al. (3).

242

SPOHN AND SCARLATO

Table 2. Putativeflagellargenes and operons and associated promoter sequences Gene/operon

ORFs

cheV-A tlpC tlpA-O tlpB-O flaB" O-fliR-A flgl -0-hp244 flgL flgH-A-flaG O-fliF-fliG-fliH-A-A-O A-O-A-O-O-O-cbeVcheAY-cheW O-A-hpaA' hpaA' fliN bpS99-A-A flaA cbeV-A-A-A-A fliP-fliP' O-O-A-O-flgR A-O-A-rpoN flaG-fliD-fliS A-flhB bpaA A-A-A-fliL O-A-motA-motB-O c

19-20 82 99-100 103-102 115 174-172 246-244 295 325-327 350-356 399-391

d

oa cr , tr o^

0.4 H

0.2 H

0.0 0.0

0.2

0.4

0.6

0.8

1.0

I

I

1.2

1.4

1.6

H. pylori J99 start position (Mb) Figure 2. Genomic location of the conserved orthologous genes between H. pylori J99 and 26695. The nucleotide position of the initiation codon of each orthologous gene-pair ( • ) in J99 and 26695 was plotted. The disruption of the conserved gene order of the orthologs is easily detected. The genes affected by the artificial breaks introduced to align the orthologous genes from the two genomes are encircled and numbered according to the inversion and/or translocation number given in Fig. 1. Due to the resolution, the smallest inversion (number 5) cannot be detected, but its location is indicated with an arrow. The locations of the plasticity zones (PZ) are indicated. Due to their large size, the location of the coincident plasticity zone is represented by a gap in the diagonal "orthologous gene line. " The other plasticity zone (split by rearrangement 3) in 26695 is located at approximately 0.45 Mb on the 26695 chromosome.

Nucleotide comparisons and allelic diversity Sequencing studies of several genes {cagA, vacA, flaA, flaB, cysS, ureC/glmM, and tnpAlB genes from IS605) have demonstrated that the nucleotide diver­ sity of orthologous genes between independent H. py­ lori isolates is relatively high and that it was extremely rare that orthologous genes from two strains of H. pylori would contain the same sequence (25, 3 3 , 4 2 , 50, 94). Indeed, it has been shown (1, 5 3 , 94) that recombination between gene alleles within the H. py­ lori population is extremely common, and any geo­ graphically based clonal groupings are very weak. Methods that detect nucleotide diversity, other than direct sequencing, have also been used to measure the genetic diversity of H. pylori isolates, including RAPD-PCR and PCR-based RFLP (4, 5, 14, 39). These data, many of which sample the whole genome,

also demonstrate that it is rare for two H. pylori iso­ lates to possess the same pattern. For this reason, al­ though the gene order was surprisingly highly con­ served between the genomes of H. pylori J 9 9 and 26695, it was not unexpected to observe significant nucleotide diversity between the orthologous genes. However, due to the redundancy in the genetic code, there was a higher level of divergence in the genes than in the respective encoded proteins. Consistent with literature studies, no two orthologous genes be­ tween J 9 9 and 2 6 6 9 5 shared 1 0 0 % identity whereas 41 proteins (2.9%) were 1 0 0 % identical. Indeed, only eight orthologous genes (0.6%) had an identity level greater than 9 8 % , whereas 3 1 0 proteins (22.2%) shared this level of identity (21). The average nucleo­ tide identity for all orthologous genes was 9 2 . 6 % , although the average nucleotide identity for the or­ thologs with a predicted function is higher, at 9 4 . 0 % .

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The expected increase in the predicted protein simi­ larity due to silent nucleotide changes is evident in both classes, being 93.4 and 9 5 . 4 % for all the or­ thologous proteins and those with a predicted func­ tion, respectively (9) The H. py/on'-specific genes have the lowest level of identity at 88.4% (9). The lengths of all the orthologous proteins were also compared as a further measure of divergence. Of the 4 2 9 proteins classified in metabolic functional groups (amino acid, cofactor, nucleoside and fatty acid biosynthesis, DNA replication, energy metabolism, transcription, and translation), only 4 9 (11.4%) were different in size, and then by an average of 3.6 amino acid residues. In contrast, the length divergence of the orthologous proteins with no predicted function was an average of 2 9 . 4 residues and occurred in 2 8 % of the proteins (157 out of 5 5 5 ) , with the H. py/on'-specific genes contributing the majority of the diversity (9). It is pos­ sible that the nucleotide differences are a result of DNA mismatch during replication as both J 9 9 and 2 6 6 9 5 lack an identifiable mutH or mutL ortholog, the mutator genes that, in combination with MutS, are responsible for efficient correction of purine/pyrimidine mismatches during replication (9). This lack of a complete MutHLS system has been suggested to be reason for the high frequency of transition muta­ tions in orthologous H. pylori genes (102). Codon usage was analyzed to determine whether the drift in the amino acid coding triplets endowed J 9 9 and 2 6 6 9 5 with a different bias toward particular amino acids. While it was evident that H. pylori does possess an overall bias toward certain amino acids, this bias did not differ significantly between the two strains at a genomic level although it was evident in individual orthologous genes (10). The bias toward (G + C) nucleotides in the third "wobble" position of the coding triplets was higher than the entire genome at 42.7 and 4 2 . 0 % for J 9 9 and 2 6 6 9 5 , respectively. This was unexpected as the bias in this position is usually more extreme to compensate for the fixed po­ sitions in the coding triplet. For example, in low (G + C ) % organisms like Borrelia burgdorferi (29%) or Campylobacter jejuni (31%), the (G + C) bias in the wobble position is considerably lower at around 20%. The rate of nucleotide sequence variation ob­ served between J 9 9 and 2 6 6 9 5 would provide the in­ dividuality that has been detected with different fin­ gerprinting methods such as RAPD-PCR, repetitive sequence element (REP)-PCR, and oligofingerprinting (4, 5, 14, 32, 3 9 , 4 5 , 5 3 , 6 3 , 72, 87, 97, 101). Simi­ larly, this variation would affect physical PFGE maps of the genomes due to alterations in the restriction endonuclease recognition sites. For example, a single silent nucleotide change is responsible for six of the

seven additional Notl restriction sites found in the J99 genome compared to 2 6 6 9 5 (8). These changes coupled with the possibility of large chromosomal rearrangements may provide a distorted impression of the macrodiversity level of H. pylori genomes. However, all of the techniques that sample the nucleo­ tide variation evident between H. pylori isolates will remain invaluable as a rapid and precise method for the epidemiological identification and fingerprinting of H. pylori strains. Comparative functional analyses Central intermediary and general metabolism. Detailed analyses of the predicted metabolic function of H. pylori annotated from the genomic sequences have recently been reviewed (21, 6 0 ) . Analyses of the genomes of J99 and 2 6 6 9 5 show that H. pylori does not appear capable of using complex carbohydrates as energy sources. Glucose appears to be the only car­ bohydrate utilized by H. pylori and is metabolized via the Entner-Douderoff pathway. The glycolytic/gluco­ neogenic pathway is likely to be used for anabolic biosynthesis rather than catabolic energy production. These predictions from genomic analysis are sup­ ported by experimental evidence from several labora­ tories (21). Both literature reports and genomic analysis sug­ gest that the primary sources of pyruvate in H. pylori are lactate, L-alanine, L-serine, and D-amino acids rather than glucose or malate. Pyruvate can be con­ verted to acetyl coenzyme A by a pyruvate oxidore­ ductase. Acetate formation by fermentation of pyru­ vate has been reported for H. pylori, and the genome of J99 contains homologs to the pta (phosphate acetyltransferase) and ackA (acetate kinase) genes. Inter­ estingly, in strain 2 6 6 9 5 pta has a frameshift mutation that would inactivate the gene product. The reverse reaction that would convert acetate to acetyl-coA can be carried out in 2 6 6 9 5 by an acetyl-coA synthetase (HP1045) (Table 2 ) , a gene that is not found in H. pylori J99. H. pylori has the ability to ferment pyru­ vate to ethanol (83, 84) via an alcohol dehydrogenase although J 9 9 contains a second unique paralogous enzyme (JHP1429) (Table 2 ) , suggesting a redun­ dancy in this pathway. Genomic analysis identifies homologs to enzymes of the tricarboxylic acid cycle in H. pylori, which suggests that it possesses a branched noncyclic pathway. However, literature reports of ex­ perimentally derived enzymatic activities do not cor­ relate well with the genome annotations (21). Further efforts are needed before the metabolic features of H. pylori are completely understood. All the enzymes required for the biosynthesis of biotin, folate, heme, molybdopterin, pantothenate, pyridoxyl phospate, riboflavin, and thioredoxin are

CHAPTER 27 • THE GENOME

301

Table 2. Genes unique to either H. pylori J99 or 26695 No. of genes in H. pylori strain: Type of gene J99 H. py/orf-specific with no known function Conserved hypothetical with no known function Genes with predicted function DNA restriction/modification DNA recombination/repair DNA replication Cell envelope Cellular processes Energy metabolism Phospholipid metabolism Total unique genes

55 9 25" 14 1 2 4 2 2 0 89

26695 70 21 26" 14 2 2 2 4 1 1 117

" The total genes with predicted function are shown in their predicted functional groups

present in both H. pylori genomes. De novo pyrimi­ dine biosynthesis has been experimentally shown in H. pylori, and all the genes encoding the necessary enzymes have been identified in the genome. In the case of de novo purine biosynthesis from formate, gly­ cine, or serine, most of the necessary enzymes do not appear to be present, but homologs encoding putative salvage and interconversion pathways have been iden­ tified. H. pylori contains orthologs of all the enzymes required for the initiation and elongation of fatty acid biosynthesis. However, in addition to the acyl-carrier protein contained in both genomes, strain 26695 con­ tains a unique second paralog (HP0962) (Table 2) that possesses an extended N-terminal domain. Geno­ mic analyses predict that H. pylori ]99 and 26695 can synthesize eight amino acids, while the remaining amino acids are likely to be transported into the bac­ terium using the large number of transporters en­ coded within the genome (21). Transcription and translation. The transcrip­ tion and translation processes in H. pylori appear to bear a lot of similarities to those of other gram-nega­ tive bacteria. One significant difference is the fusion of the rpoB and rpoC genes encoding the B and B' subunits of RNA polymerase that is evident in both H. pylori genomes (21). The same is true in Wolinella, but not in Campylobacter and other related genera, where the coding regions overlap (106). The fusion of the two subunits in H. pylori and Wolinella sp. may have been the result of a frameshift mutation, and recently the separation of rpoB and rpoC by insertion mutagenesis to yield two polypeptides results in viable H. pylori that retains the ability to colonize and prolif­ erate in C57BL/6 mice (81). Analyses of the genomes of both J 9 9 and 2 6 6 9 5 indicate that there are only three sigma factors present in H. pylori, rpoD, rpoN,

and fliA. No homologs to the stationary-phase sigma factor, RpoS, or the heat shock-specific RpoH are evi­ dent. Two-component systems are a highly conserved mechanism of controlling bacterial gene expression, often in response to external stimuli. Both J 9 9 and 26695 genomes have four orthologs of histidine ki­ nase sensor proteins and seven distinct orthologs of DNA-binding response regulators. Both genomes contain homologs to the termination factors NusA, NusB, and Rho, and the paucity of transcriptional termination stem-loop structures is evidence that ter­ mination in H. pylori is likely to be Rho-dependent (21). Both H. pylori strains have 36 tRNA genes in the same relative physical location and all the expected tRNA synthetases except for glutaminyl- and aspara­ ginyl-tRNA synthetases (8). Two copies of the gene encoding glutamyl-tRNA synthetase, gltX, are pres­ ent in J 9 9 and 2 6 6 9 5 , one of which may function as a glutaminyl-tRNA synthetase. Alternatively, H. pylori may perform an in situ transamidation of gluta­ mate to glutamine (and possibly also aspartate to as­ paragine) via the gatABC gene products, which in Bacillus subtilis encode a glutamyl-tRNAGln amidotransferase, demonstrated to functionally replace glu­ taminyl-tRNA synthetase activity (19). DNA replication, restriction, and modifica­ tion. H. pylori contains only five identifiable genes (dnaE, dnaN, dnaQ, dnaX, and holB) that encode core subunits of DNA polymerase III, which although representing a less complex holoenzyme than E. coli, is consistent with the complexity observed in many of the sequenced organisms to date. Both H. pylori J 9 9 and 2 6 6 9 5 contain multiple homologs to DNA topoisomerase I (top A). The orthologous top A gene lies adjacent to the flagellin B subunit (92) whereas

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the two J99-unique and the single 26695-unique top A genes are all located within the plasticity zones of the respective genomes (Table 2 ) . The plasticity zone in both strains also contains an orthologous gene that resembles the xerCD family of integrases/recombinases, and J 9 9 contains an additional unique xerCD homolog. H. pylori possesses an unusually large number of genes with homology to restriction and modification enzymes. Interestingly, many of these genes appear to be strain-specific. This is consistent with subtractive hybridization studies between H. pylori J I 66 and 2 6 6 9 5 that demonstrated that many of the unique genes that were found in strain J I 66 encoded putative DNA restriction/modification enzymes (2). This re­ sult, taken together with the analysis of the presence or absence of the J 9 9 or 2 6 6 9 5 unique genes in addi­ tional strains, suggests that every H. pylori isolate may contain its own specific complement of these genes (8). Genomic analysis allows these gene prod­ ucts to be classified as type I, II, or III restriction or modification enzymes, and recent studies have begun to look at the nucleotide sequence specificity of these enzymes as well as at their expression levels. Recently, efforts have begun to clone and express the DNA re­ striction and modification genes from H. pylori 26695 and J 9 9 and test for activity. Many of the re­ striction genes do not appear to be active, although the cognate methylases were. H. pylori 26695 has four active restriction genes with specificities repre­ senting isoschizomers of Ahdl, Mbol, Hinil, and Mboll. H. pylori J 9 9 has four active restriction genes with specificities representing isoschizomers of Tsp4Sl, Bsajl, Hhal, and a novel specificity (GCWGC) called Hpy991 (57). Interestingly, all the active genes identified were strain-specific. While both H. pylori J 9 9 or 2 6 6 9 5 contained many unique DNA restriction-modification genes, which comprised ap­ proximately 6 0 % of the functionally assigned strainspecific genes, there were 2 5 genes that did appear to be orthologous (21). Among these were 10 type II methyltransferases, all of which lacked an immedi­ ately recognizable, tandemly oriented cognate restric­ tion enzyme partner. However, all were flanked on at least one side by a gene of unknown function, often that was specific to H. pylori. If these methyltransfer­ ases are found to be conserved between other H. py­ lori strains, it may support the hypothesis that one method of gene regulation in H. pylori is by methyla­ tion (8). Outer membrane protein genes and lipopolysac­ charide biosynthesis. The outer membrane protein profile of H. pylori strains differs significantly from other gram-negative species as no major outer mem­

brane proteins (OMPs) predominate, but rather mul­ tiple lower-abundance OMPs are observed (7). Analy­ sis of the H. pylori J 9 9 and 2 6 6 9 5 genomes have identified five paralogous gene families ranging in size from 3 to 33 members, and two of these families con­ tained members that were specific for either H. pylori J 9 9 or 26695 (7). These gene families comprise ap­ proximately 5 . 5 % of the coding capacity of each strain. Some of the proteins in the large family (Hop/ Hor proteins) (7) have been shown to be porins (22, 26) and/or adhesins for gastric epithelial cells ( 4 4 , 7 1 ) , and this unusual set of OMPs may be a reflection of the adaptation of H. pylori to the unique gastric environment where it is found. Intriguingly, both H. pylori J 9 9 and 2 6 6 9 5 contain two pairs of duplicated OMP genes that are essentially identical to each other, and yet differ significantly between the strains (7). This high intrastrain identity coupled with significant interstrain diversity strongly suggests that DNA up­ take from surrounding cells and homologous recom­ bination between the duplicated loci keeps the paralo­ gous genes essentially identical while allowing the orthologous genes in independent strains to diverge (7), perhaps providing a mechanism for host defense evasion or determination of host specificity. Signifi­ cantly, these duplicated gene pairs were also found, at the same relative chromosomal location, in other H. pylori strains examined, although the N-terminal region had been deleted in a few cases (7, 5 3 ) . H. pylori contains homologs of all genes neces­ sary for 2-keto-3-deoxyoctulosonic acid (KDO)-lipid A biosynthesis, a common structure found in the lipo­ polysaccharide (LPS) of the majority of gram-negative bacteria. Synthesis of the LPS core requires the se­ quential addition of sugar moieties, carried out by glycosyltransferases. While H. pylori 2 6 6 9 5 and J 9 9 have seven putative glycosyltransferase genes in com­ mon, they contain one and two unique genes, respec­ tively, that have been identified as glycosyltransfer­ ases that may be involved in the specific addition of LPS core sugars (21). The O-antigen chain of the LPS of H. pylori is composed of Lewis acids (Le and L e ) , which are identical to those found on host tissues and have been implicated in colonization and persistence of H. pylori and may also play a role in autoimmunity ( 1 2 , 1 3 ) . Both H. pylori genomes encode two 2 million molecular weight) that are sulfated and heavily glycosylated (carbohy­ drate > 8 0 % ) and represent a major component of mucus. These glycoproteins serve as host mucosal de­ fenses by preventing colonization of microbes, as well as by protecting the epithelial cells from injury. Mucin contains a wide array of structurally distinct carbohy­ drate side chains, such as sulfated or nonsulfated sugar moieties, Lewis a, b, X , and/or Y moieties, and sialic acid residues. Because of its lifestyle as a mucosal pathogen, H. pylori has evolved a number of mecha­ nisms to overcome the nonspecific host defenses due to mucin (Fig. 3). First, H. pylori has the ability to bind to gastric and salivary mucin, and optimal bind­ ing requires that the mucin be sulfated and/or sialylated (141, 142, 2 1 3 , 2 2 9 , 2 5 7 , 2 9 4 , 3 4 0 , 3 4 5 , 350). Sulfated galactose or sulfated Lewis a within salivary mucin was the likely antigen bound by H. pylori (350), although sialic acids and Lewis b are also possi­ ble antigens (104, 2 1 3 , 2 3 6 ) . Mucin binding is ele­ vated by exposure of H. pylori to low pH in vitro, and by the presence of divalent cations (especially nickel and zinc), and is mediated most likely by elec­ trostatic interactions (236, 3 5 0 ) . Gastric mucin blocks H. pylori from adhering to the gastric epithelial cells, presumably by competing with the cell surface for H. pylori adhesins (104, 142, 294, 340). Desulfation of mucin by the anti-ulcer compound sulglycotide results in loss of binding of H. pylori to mucin (141, 257), and sialidase treatment of mucin likewise de­ creases H. pylori binding to gastric mucin and hemag­ glutination to red blood cells ( 1 4 1 , 1 4 2 , 2 1 3 , 3 4 0 ) . An H. pylori protein that binds mucin has been recently identified as the 16-kDa neutrophil-activating protein (Nap), a protein that binds pleiotropically to sulfated oligosaccharides such as sulfo-Lewis a, sulfogalactose, and sulfo-N-acetylglucosamine found on mucin (229, 327). Nap also binds to Lewis X and sialylated glycoconjugates in vitro (229, 327) and causes en­ hanced adhesion to human neutrophils by an un­ known mechanism (102). Second, H. pylori possesses several mucinase ac­ tivities (proteins not yet identified): one desulfates mucin and the other proteolytically cleaves the proteinaceous component of mucin ( 2 9 9 , 3 0 0 , 304). Deg-

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Chemotactic movement toward mucin

Binding to mucin (low pH; high Zn, Ni)

Binding to mucin receptor on host cell mediated by H. pylori iLPS

Mucinases (Urease required)

Reduction of mucin M>ligomers by thioredoxin

Desulfation

Proteolysis

A

Host

Tighter adherence to gastric epithelial cells

Tighter adherence to gastric epithelial cells

Proteinaceous monomer of mucin Sialic acid Sulfate

Depletion of mucincontaining granules in gastric epithelial cells

Mucin oligomer

Nap/Mucin Receptor o

Mucin-containing granule

Figure 3. Interactions of H. pylori with mucin. H. pylori chemotactically moves from a region of low concentration of mucin to a higher concentration and then binds to it. There may be competition between Nap binding to mucin (left pathway) and LPS interacting with the host mucin receptor, which would block the ability of mucin to bind its own receptor (right pathway). H. pylori mucinase activity degrades mucin either by removing sulfate residues (desulfation) or by cleaving the proteinaceous component of mucin (proteolysis). This allows H. pylori access to receptors on the host cell, leading to tighter adherence. Following adherence, mucin-containing granules are depleted, so that mucin release is perturbed. This leads to decreased mucin concentration in the mucous layer and would allow additional H. pylori to bind to gastric epithelial cells. There are very likely other H. pylori adhesins that interact with mucin, since mucin has a diverse repertoire of glycoconjugate moieties.

CHAPTER 34 • ADHERENCE AND COLONIZATION

radation of mucin allows H. pylori to migrate to and adhere to the gastric epithelial cell surface more quickly. In another study, urease of H. pylori was suggested to disrupt the carbonate-bicarbonate buffer of the stomach through the production of ammonium from urea, thereby leading to breakdown of gastric mucin (293). This was suggested based on the finding that filtrates of H. pylori passaged only once in vitro from gastric biopsies failed to degrade mucin unless urea was present. Whether urease can more directly degrade mucin has not been reported. Third, H. pylori displays chemotactic activity to­ ward mucin (111; T. L. Testerman, D. J . McGee, and H. L. T. Mobley, unpublished observations), which requires a two-component signal transduction system CheA/Y (111). Fourth, the H. pylori thioredoxin was shown to reduce mucin such that oligomers of mucin are poten­ tially converted to monomers (359). Reduction of mucin to monomeric form may destroy the ability of mucin to interact with the mucin receptor. Thus, H. pylori may chemotactically move toward mucin, bind mucin, and degrade it. This would decrease the viscos­ ity of the mucous gel and allow H. pylori to facilitate migration to the epithelial cell surface, an event that prevents clearance by peristalsis. Fifth, H. pylori may occasionally enter the mucin-containing compartment of goblet cells at areas of intestinal metaplasia (a precursor of gastric carcinoma) (117). This could explain the clinical ob­ servations that, upon adherence of H. pylori to the gastric epithelium, depletion of the mucin-containing mucous granules occurs (26). The mechanism of de­ pletion of mucin secretion by the glandular epithelium has been further explored in two studies. A study of human biopsies showed that H. pylori infection sig­ nificantly lowers the amount of mucin contained in cells from antral biopsies, but little effect is seen in cells from gastric body biopsies (170). Rats fed 0 . 0 1 % ammonia for 4 weeks develop a similar pattern of mucin depletion in antral cells, suggesting that the ammonia produced by H. pylori urease may be re­ sponsible for effects on mucin (170). Micots and col­ leagues used a mucin-secreting cell line to further study the effects of H. pylori on mucus secretion (215). They found that baseline mucin biosynthesis was not impaired over a 24-h period; however, secre­ tion of mucin was impaired at 2 4 h. Secretion by H. pylori-'miected cells was more strongly inhibited fol­ lowing stimulation by forskolin and ionophore A23187, agents that raise intracellular cAMP levels. These studies indicate that H. pylori perturbs the re­ lease of mucin, which would facilitate H. pylori ad­ herence to the gastric epithelial cell layer and may contribute to ulcer formation in the antrum. All of

389

these interactions of H. pylori with mucin may also explain the clinical observations that H. pylori is found both free in the mucous layer and attached to gastric epithelial cells and strongly suggest that more research is needed to understand the H. pylori genes involved in these mucin interactions. Also, given the potential for H. pylori LPS to interact with host cell glycoconjugates of the same structure (homotypic in­ teractions) (92, 3 2 4 ) , a future area of examination should be determination of whether LPS is involved in adherence of H. pylori to mucin as both LPS and mucin contain Lewis X . Hemagglutination To investigate the interaction of H. pylori with human cells, hemagglutinating activity of various spe­ cies of red blood cells (RBCs) by H. pylori has been extensively studied. On the basis of studies from nu­ merous laboratories ( 1 0 , 1 7 , 4 7 , 4 8 , 5 1 , 9 5 , 1 0 0 , 1 4 7 , 149, 1 5 1 , 160, 1 6 1 , 1 8 4 - 1 8 6 , 2 1 6 , 2 1 8 , 2 2 8 , 2 4 5 , 269, 3 2 5 ) , H. pylori exhibits a broad spectrum of hemagglutination. This activity depends on the H. py­ lori strains used, how long they have been passaged in vitro, how the strains are grown (e.g., broth-grown cells lack sialic acid-dependent hemagglutination ac­ tivity in contrast with agar-grown bacteria), and the species of red blood cells used in the hemagglutination study. Both cell surface-associated and soluble hemag­ glutinins exist in H. pylori. Hemagglutinin binding to human RBCs is mediated by at least three specific interactions. Two of the interactions are mediated by distinct sialic acid linkages to carbohydrate side chains of host glycoconjugates, either a 2 , 3 - or a 2 , 6 specific for strongly hemagglutinating strains. These interactions are inhibited by pretreatment of the erythrocytes with neuraminidase or by fetuin or mucin, glycoproteins rich in sialylated oligosaccha­ rides, but not by pretreatment with asialofetuin or asialomucin. A third interaction is observed for weaker hemagglutinating strains, and this interaction is sialic acid independent. A protein of 59 kDa has been suggested to be involved in this interaction, but its receptor specificity is unknown (147). In general, hemagglutination activity is inhibited by preincuba­ tion of H. pylori at 56°C. This heat treatment does not necessarily alter adherence, however. In vivo, H. pylori is unlikely to interact with human RBCs. Thus, the physiological role(s) of the hemagglutinins has been addressed more carefully using gastric epithelial cells and human neutrophils and monocytes. Sialic acid-dependent hemagglutinat­ ing H. pylori strains appear to resist phagocytosis by human neutrophils and monocytes, in contrast with sialic acid-independent hemagglutinating strains (10,

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TESTERMAN ET AL.

47, 4 8 , 5 1 ) . These results suggest a correlation be­ tween possession of sialic acid-dependent lectins and protection from phagocytic killing. However, these studies need to be repeated using freshly isolated H. pylori strains and mutants bearing isogenic gene dis­ ruptions in candidate genes, such as the gene encoding the putative sialic acid lectin (see below), hpaA, to address what genes are responsible for adhesion and whether they contribute to the virulence of H. pylori. Several studies have attempted to correlate hem­ agglutination with interactions of H. pylori strains to KatoIII, AGS, M K N 4 5 , or primary gastric epithelial cell lines or to nongastric cell lines. It was found that adherence to some cell lines and primary gastric cells from various animals was sialic acid and heparan sul­ fate dependent (47, 9 8 , 174). Adherence is inhibited by preincubation of host cells with neuraminidase or by preincubation of H. pylori with the sialic acid-con­ taining proteins fetuin or mucin. Contradictory evi­ dence for a correlation of hemagglutination and ad­ herence to gastric cells has been widely documented (46, 4 7 , 5 6 , 1 0 8 , 174, 2 4 5 , 2 6 8 ) , although the strain selected and how long it is passaged for each of these studies play a critical role in the type of data obtained (145, 368). Data from various studies suggest H. py­ lori adherence levels to various cell types as follows: primary gastric cells > HEp-2 (human larynx carci­ noma) > AGS > M K N 4 5 cells > KatoIII (174, 234).

that additional genes are necessary for transport, as­ sembly, or regulation of hemagglutination expression in H. pylori. Additionally, HpaA has also been shown to be an inner membrane lipoprotein in E. coli ex­ pressing hpaA (246), rather than the expected outer membrane location (101). HpaA has also been ob­ served as a component of the extracellular flagellar sheath (162, 195). An isogenic hpaA mutant of H. pylori still retains sialic acid-dependent hemagglutina­ tion activity and adheres normally to five human gas­ tric carcinoma cell lines and to fixed human gastric tissues (162, 2 4 6 ) , suggesting that other sialic acid lectins exist and that adherence to epithelial cells is multifactorial. Although these latter studies ques­ tioned the relevance of HpaA in H. pylori attachment and virulence, experiments using human neutrophils, monocytes, primary human gastric epithelial cells, and animal studies have not been reported with the hpaA mutant. Simon and colleagues have discovered that 3'-sialyllactose does not inhibit adherence of highly passaged isolates of H. pylori (294), in contrast with lowly passaged isolates, suggesting that highpassage isolates, such as the ones used by O'Toole and colleagues (246), may have lost the expression of certain sialic acid adhesins on the cell surface. Interest­ ingly, the genome sequence of H. pylori predicts an additional HpaA ortholog (HP0492, 3 0 % identical to HpaA at the amino acid level), which may explain some of the perplexing findings (332).

Sialic Acid-Binding Adhesins

Evidence for the existence of other sialic acid ad­ hesins in H. pylori was provided in several studies. Five candidate sialic acid adhesins of H. pylori of mo­ lecular mass of 64, 6 3 , 5 6 , 2 5 , and 2 0 kDa have been described, but their further characterization has not been reported (147, 184, 185). It is unclear whether the 20-kDa protein may be the same as the 20-kDa conserved antigen reported by Doig and colleagues (75). Additional evidence for H. pylori binding to sia­ lic acid-containing glycoconjugates comes from transgenic mice lacking parietal cells (321). These mice were developed to mimic the presumed in vivo situation; following H. pylori adherence to the gastric epithelium, parietal cell function rapidly deteriorates (128). In the parietal cell-negative transgenic mice, epithelial multipotent stem cells bearing sialic acidcontaining glycoconjugates proliferate; H. pylori spe­ cifically attaches to these glycoconjugates in vitro and in vivo (321). This finding confirms the early study of Bode and colleagues (26), who observed up-regulation of sialic acid-containing glycoconjugates follow­ ing H. pylori adherence to gastric tissue. Other inves­ tigators studying normal versus gastric carcinoma gastric tissue have observed elevated sialyltransferase activity (254), leading to increased expression of sialy-

From the data summarized above on hemaggluti­ nation and mucins, there is strong evidence for sialic acid adhesins in H. pylori. Likewise, sialic acid-con­ taining glycoconjugates have been shown to exist on the surface of gastric epithelial cells and on human neutrophils by staining with sialic acid lectins or by conducting binding assays; these sialic acid-contain­ ing glycoconjugates are up-regulated upon contact of H. pylori with the host cell ( 2 6 , 2 1 7 , 321). Confirma­ tion of the hypothesis for sialic acid adhesins of H. pylori came from Evans and colleagues, who cloned and characterized the first sialic acid adhesin, HpaA (100, 101). The gene encoding the sialic acid adhesin HpaA, hpaA (HP0410), has been cloned, sequenced, and expressed in E. coli (101). The purified protein ( ~ 2 9 kDa) binds to sialoconjugates mainly in an a2,3-specific manner and can be detected on Western blots with antiserum directed against HpaA (100, 101). Antibodies against the putative HpaA sialic acid-binding motif, KRTIQK, inhibit H. pylori sialic acid-dependent hemagglutination, and immunogold labeling shows that HpaA is surface-exposed and is functional (101). However, in E. coli expressing HpaA, no hemagglutination is observed, suggesting

CHAPTER 34 • ADHERENCE AND COLONIZATION

lated glycoconjugates in gastric adenocarcinoma; this is rarely observed in normal gastric mucosa (8, 196, 197, 276). Similarly, drastically elevated sialic acids in the gastric juice of duodenal ulcer patients were observed as compared to normal healthy controls (251, 348), while expression of fucose-rich (Lewis bpositive) glycoproteins is down-regulated in gastric ulcer patients (27). These results prompt the interest­ ing hypothesis that H. pylori directly alters host cell receptor expression and that there is a temporal expression of host cell receptors following H. pylori binding. However, few follow-up experiments have been conducted. Taken together, the sialic acid adherence studies support two conclusions: (i) H. pylori binds multiple types of sialic acids in vitro and in vivo and (ii) H. pylori adherence to normal gastric epithelium, per­ haps first through glycolipids or through the Lewis b antigens (see below), causes the up-regulation of the expression of sialic acid containing glycoconjugates, which allow tighter binding of H. pylori. Lewis b-Binding Adhesins An early study by Bode and colleagues (26) sug­ gested the presence of fucose-containing glycopro­ teins on the surface of gastric epithelial cells. Subse­ quently, several reports have confirmed that stationary-phase H. pylori can bind to fucosylated glycoconjugates containing Lewis b structures on the surface of gastric epithelial cells within human gastric biopsies (28, 104). Monoclonal antibodies to Lewis b or soluble Lewis b antigens inhibit the binding of H. pylori to fixed gastric tissue (28, 2 6 6 ) . In these latter two studies, no evidence for sialic acid-depen­ dent adhesion was obtained. Further support for H. pylori using Lewis b as a receptor comes from transgenic mouse studies. H. pylori binds in a cell line­ age-specific manner to the gastric epithelial cells and to the gastric pit in the mucosa of transgenic mice expressing the human a l , 3 / 4 fucosyltransferase gene, in contrast with nontransgenic littermates (103). This enzyme adds a fucose residue to the Lewis b precursor H type 1 antigen. Soluble Lewis b antigens inhibit binding of H. pylori to gastric tissue from these transgenic mice (103). Guruge and colleagues (128) extended this study and found that simultaneous coinoculation of eight fresh clinical H. pylori isolates resulted in similar colonization levels of both transgenic and nontransgenic littermates, with only one strain predominating (128). H. pylori stayed in the mucous layer in nontransgenic mice, whereas in transgenic animals, H. pylori was found in the mu­ cous layer and adherent to the surface of mucus-se­ creting gastric epithelial cells, resulting in severe

391

chronic gastritis, parietal cell loss, and production of autoantibodies to Lewis X antigen (found in H. pylori LPS and on parietal cells) (128). Thus, attachment of H. pylori to the gastric epithelial cell surface alters the disease outcome. The fresh H. pylori clinical isolates bound in vitro only to mouse gastric biopsies obtained from the Lewis b transgenic mice, and binding did not depend on presence of flagella (128). Further stud­ ies with this transgenic mouse model demonstrated that parietal cell ablation results in amplification of gastric epithelial multipotent stem cells (321). H. py­ lori attachment to these cells resulted in enhanced cel­ lular and humoral immune responses. The first H. pylori adhesin responsible for this Lewis b-binding was identified as BabA, which is a member of the large family of paralogous outer mem­ brane proteins (155). The presence of the babA gene in H. pylori correlates with duodenal ulcer and adeno­ carcinoma and use of this gene marker concomitant with certain vac A and cagA subtypes further strength­ ens this correlation (118). Presence of babA also cor­ relates well with the ability of H. pylori to bind to Lewis b antigens in vitro (118); babA-deficient clinical isolates always fail to bind Lewis b. The above studies have contributed much to our understanding of H. pylori adherence to Lewis b re­ ceptors. However, there are inconsistencies: (i) not all H. pylori strains bind Lewis b antigens (see below), (ii) Lewis b antigens are widely distributed on epithe­ lial cell types to which H. pylori does not interact and thus does not explain the specific tissue tropism of H. pylori, and (iii) Lewis antigen expression in the host can vary. Thus, host and bacterial strain heterogeneity may play a role in determining bacterium-host cell interactions, and H. pylori can clearly interact with multiple receptors as recently confirmed in vivo using transgenic mice (321). Recent studies have questioned whether Lewis b serves as the major receptor for H. pylori adherence. Su and colleagues (316) demonstrated that Lewis b antigen-independent adherence to gastric cell lines re­ quires de novo protein synthesis in both host and bac­ terium. In contrast, Lewis b-dependent adherence oc­ curred to fixed gastric tissue (316), as was originally reported (104). In a second study, using primary gas­ tric cells isolated from gastric biopsies, Clyne and Drumm showed evidence of Lewis a and b on the host cell surface of 18 of 19 samples tested, but three different H. pylori strains bound gastric cells indepen­ dent of Lewis a or b using quantitative flow cytometry (58). Preincubation of these cells with monoclonal an­ tibodies to Lewis a or b from two different commer­ cial sources did not block binding of H. pylori to these cells. Similar results were obtained using the KatoIII gastric cell line. In other studies, presence of Lewis

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antigens on gastric cells could not be correlated with Lewis expression on H. pylori LPS ( 1 3 7 , 3 2 4 ) . Finally, only 4 of 32 H. pylori isolates from infected children expressed the Lewis b binding ability in an in vitro filter binding assay, suggesting that BabA is not ex­ pressed in the majority of H. pylori isolates from chil­ dren (36). BabA is also not expressed in about 5 0 % of H. pylori fresh clinical isolates (118), probably due to phase variation of the babA gene via slipped-strand mispairing (155, 3 3 2 ) . Notably, there are also rare babA-positi\e strains that also fail to bind Lewis b (118). All H. pylori strains examined to date (n = 49) synthesize a neuraminidase, and 2 0 % of strains pro­ duce fucosidase (83), which may cleave host cell sialic acid or fucose residues, respectively, from glycoconju­ gates. This could result in unmasking of other sugar moieties to which putative H. pylori adhesins could bind and could explain the discrepancies in H. pylori adherence characteristics. However, there is no or­ tholog of either enzyme in the sequenced genome of H. pylori strain 2 6 6 9 5 , suggesting that presence of these enzymes is artifactual (143), that H. pylori con­ tains nonhomologous genes, or that only some strains possess these enzymes. A 61-kDa protein from H. pylori was shown to interact with the H type 2 antigen (the blood group O antigen) and may explain the finding that patients with blood group O are more susceptible to peptic ulcer disease (4). This protein also could bind Lewis a and Lewis b antigens, yet is distinct from BabA. Taken together, these observations suggest that (i) H. pylori binds to Lewis b in vitro and in vivo, but this is not an absolute requirement for adherence; (ii) adhesins independent of BabA may interact with Lewis b; and (iii) the host cell surface can change its sugar repertoire in response to H. pylori adherence. Sulfate and Lipid-Binding Adhesins H. pylori has been shown to interact with a num­ ber of sulfated and lipidated compounds in vitro, in­ cluding heparan sulfate, phosphatidylethanolamine (PE), lactosylceramide, galactosyl- and lactosylceramide sulfate (collectively known as sulfatides), ganglioside G M 3 , gangliotriaosyl ceramide (Gg3), and gangliotetraosylceramide (Gg4) ( 1 3 , 1 9 , 4 9 , 5 0 , 1 2 3 , 1 2 4 , 144, 167, 1 9 1 - 1 9 3 , 2 7 5 , 302). H. pylori also binds to gastric mucin, a sulfated protein that is heavily gly­ cosylated (229, 2 5 7 , 340). In the study by Kamisago and colleagues, it was observed that adherence of H. pylori to KatoIII gastric epithelial cells is inhibited by sulfated glycoconjugates (167). Lewis b, sialic acid, and G M 3 were not involved in this process, but sul­ fatides were, since a monoclonal antibody to sulfa­

tides markedly reduced H. pylori adherence to KatoIII cells (167). Additionally, bovine milk diluted up to 100-fold inhibits binding of H. pylori to sulfatide by 5 0 % (130). Adherence of H. pylori to human cells correlates well with the amount of PE present in lipids extracted from these host cells ( 8 4 , 1 2 3 ) . PE, however, does not explain the specific tissue tropism of H. pylori since PE is ubiquitously present in cell membranes through­ out the human body. The H. pylori adhesin for Gg3, Gg4, and PE was shown to be related to exoenzyme S of Pseudomonas aeruginosa, since monoclonal anti­ bodies to exoenzyme S block the interaction between H. pylori and these lipids in vitro (124, 1 9 1 , 193). N-terminal sequence analysis of the purified 63-kDa protein from H. pylori was determined and, based on the complete genome sequence (332), the protein is now known to be catalase, a protein not homologous with exoenzyme S. This result is surprising since bac­ terial catalases are typically cytosolic, not surface ex­ posed. H. pylori also has a sphingomyelinase, which hydrolyzes sphingomyelin and PE and has hemolytic activity (40). Perhaps after H. pylori interacts with PE, it releases sphingomyelinase to hydrolyze PE re­ ceptors, thereby gaining deeper access to the specific cell surface molecules found in the gastric mucosa. Laminin-Binding Adhesins H. pylori has been shown in a number of studies to bind to laminin, a basement membrane sialylated glycoprotein (75, 3 3 4 , 3 4 1 , 342). A sialic acid glycoconjugate inhibits binding of H. pylori to laminin by 7 0 % and neuraminidase pretreatment of laminin re­ duces binding by 5 0 % . Heat or protease treatment of H. pylori markedly reduced binding, suggesting that one or more H. pylori proteins are involved in laminin binding (334, 3 4 1 , 343). Indeed, Doig and colleagues (75, 76) purified a 19.6-kDa laminin-binding protein resembling bacterioferritin from multiple H. pylori strains, and others identified a 25-kDa laminin-bind­ ing protein distinct from HpaA that binds in an a2,3 sialic acid-dependent fashion (343). Evidence from several other laboratories suggests that H. pylori LPS is also involved in laminin binding (223, 2 5 5 , 3 0 3 , 342). Specifically, a phosphorylated oligosaccharide in the core of H. pylori LPS (in some but not all strains) may be involved in initial binding (342). Re­ markable diversity of laminin-binding was observed among distinct H. pylori strains (334). These data taken together suggest that LPS me­ diates initial binding of H. pylori to laminin, followed by subsequent tighter binding to laminin by one or more sialic acid lectins, of which the 19.6-kDa and 25kDa proteins participate. H. pylori binding to laminin

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393

may take place in vivo after damage to the epithelial cell layer has exposed the basement membrane.

pylori is primed for adhering to the gastric epithelial cell once the organism migrates there.

Role of LPS in Adherence

Role of Outer Membrane Proteins in Adherence

H. pylori LPS can express antigens that molecularly mimic the Lewis antigens (a, b, X , and Y) found on the surface of gastric epithelial cell glycoconjugates (324). Several studies support the role of LPS as an adherence factor for H. pylori. A monoclonal anti­ body that inhibits H. pylori adherence to gastric epi­ thelial cells by up to 7 5 % was shown to target the LPS possibly through the Lewis X portion (244). In another study, the core LPS oligosaccharide bound to laminin (342). The O antigen was not involved in binding to laminin, since rough variants (lacking O antigen) still bound laminin. In recent studies, the O antigen LPS side chain, which contains Lewis X anti­ gens and molecularly mimics human cell surface gly­ coconjugates, was shown to bind to the gastric epithe­ lium in a Lewis X-dependent fashion, and the receptor may be Lewis X itself on the host surface via homotypic interactions (92, 2 4 4 , 3 2 4 ) . Finally, H. pylori LPS interferes with the interaction of gastric mucin with the 97-kDa mucin receptor (256). All of these studies are complicated by the observation that H. pylori LPS content varies from one strain to the next and undergoes phase and antigenic variation within a single strain (15, 16, 2 2 2 ) , leading to variable expression of Lewis antigens on H. pylori LPS (262). Despite LPS variation, it is clear that H. pylori LPS plays in important role in adherence and future re­ search should focus on construction of defined LPS mutants to investigate this further. Role of Heat Shock Proteins in Adherence Heat shock proteins of H. pylori have surpris­ ingly been shown by numerous laboratories to be sur­ face exposed (150, 3 6 5 , 366) and involved in adher­ ence ( 1 5 0 , 1 5 1 , 3 6 4 - 3 6 7 ) . For example, the intensity of Hsp60 on the H. pylori surface, which is variable among strains, correlates with the degree of adherence (365). Monoclonal or polyclonal antibodies to Hsp60 or Hsp70 block adherence of H. pylori to MKN45 cells, primary gastric epithelial cells, and in an in vitro thin layer chromatography assay (151, 3 6 4 , 3 6 7 ) . However, not all strains of H. pylori could be blocked in adherence by anti-Hsp60 (367). The host receptor for Hsp60 and Hsp70 was found to be sulfatides (150, 151). The Hsp-sulfatide interaction was dependent on a stress induction (exposure to pH 2.5 or 42°C for 5 min). Thus, in the stomach lumen where the pH is 1 to 2, expression of Hsp may be induced so that H.

Cotranscribed genes for two outer membrane proteins, AlpA and AlpB, are necessary for Lewis b antigen-independent (and BabA2-independent) ad­ herence of H. pylori to KatoIII gastric epithelial cells and gastric tissue (239, 2 4 0 ) . The host receptor for AlpA/B is unknown. HopZ also mediates adherence, since a hopZ isogenic mutant has reduced adherence (253). However, adherence was only qualitatively de­ scribed and the host receptor is unknown. AlpA, AlpB, BabA, HopZ, and Hops A - E all be­ long to a paralogous family of 32 outer membrane proteins (OMPs), 8 of which undergo phase variation by alteration of the number of dinucleotide repeats by slip-strand mispairing (7, 77, 97, 155, 2 4 0 , 2 5 3 , 332). That four of these proteins (HopZ, AlpA, AlpB, BabA) are involved in adherence suggests that the en­ tire protein family may have roles in adherence and that the diverse repertoire and phase variation of OMPs allow fine specificity of H. pylori adherence to specific host cell glycoconjugates under different conditions in vivo. This hypothesis may be very diffi­ cult to test directly in H. pylori. Rather, an E. coli model that carries one or more H. pylori OMPs could be useful to determine the role and receptor specificity of various OMPs. Role of Flagellar Secretion Apparatus in Adherence An isogenic nonmotile mutant in the flagellar se­ cretion apparatus component FliQ results in 3 0 % re­ duced adherence to AGS cells (112), presumably be­ cause the mutation decreases release of a protein involved in adherence that would normally be trans­ ported through the apparatus. Since another flagellar, nonmotile mutant (in the flhB gene) adhered similarly to wild type, flagella per se are probably not necessary for adherence (112). However, an open question that still needs to be addressed is whether the flagellar sheath, which contains proteins, including the adhesin HpaA, is involved in adherence. Some of the best stud­ ied adhesin-receptor interactions are shown in Fig. 4 .

ADHERENCE ANTAGONISTS Adherence of H. pylori to the gastric epithelium is a crucial initial step in colonization, as nonadhering bacteria would be washed away during peristalsis-me­ diated flushing of the stomach. Therapeutic ap­ proaches to inhibit adherence may thus prevent colo-

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Figure 4. Interaction of H. pylori adhesins with host cell receptors. For simplicity, only the best studied H. pylori adhesinreceptor interactions are shown. The in vivo situation is likely much more complex, as described in the text, in that there may be dynamic and temporal expression of adhesins and receptors, and during any given time, H. pylori may interact with only a subset of the cellular receptors. (A) Interaction of H. pylori adhesins to gastric epithelial cell receptors. (B) Interaction of H. pylori adhesins to the basement membrane protein laminin. H. pylori has evolved multiple mechanisms to interact with host laminin.

nization and disease through eradication of H. pylori. Many adherence antagonists have been tested, and some promising candidate adherence antagonists are briefly summarized (Table 2 ) . First, administration of 3'-sialyllactose to persis­ tently colonized rhesus monkeys cures or decreases H. pylori colonization in some monkeys (225). 3'-sialyllactose also significantly reduces gastric bacterial load in H. pylori-iniected humans (D. Zopf, P. M. Simon, M . Hurley, E. McGuire, and S. Roth, unpub­ lished observation). These data were obtained follow­

ing promising in vitro studies showing that 3'-sialyllactose inhibits adherence of low-passage isolates of H. pylori to epithelial cells (294). The 3'-sialyllactose presumably binds to and saturates H. pylori sialic acid adhesins, such as HpaA, and prevents binding of these adhesins to host cell sialic acid receptors. This adher­ ence antagonist is safe, is nonimmunogenic, and can potentially be used in combination with other thera­ peutic agents. Second, the anti-ulcer compound sofalcone inhibits H. pylori adhesion to mucin in a dosedependent fashion (319), probably by inhibiting the

CHAPTER 34 • ADHERENCE AND COLONIZATION

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Table 2. H. pylori adherence antagonists Antagonist 3'-sialyllactose Sofalcone Sucralfate, sulglycotide

Ecabet sodium Rebamipide Co-magaldrox (Maalox) Omeprazole L. salivarius Milk

Bile acids Fucoidan

Mechanism of adherence antagonism Inhibits sialic acid-mediated adherence Inhibits H. pylori adherence to mucin by inhibiting chemotaxis toward mucin Blocks interaction of H. pylori LPS with laminin and gangliosides; decreases H. pylori mucinase activity by desulfating mucin; blocks interaction of LPS with host mucin receptor Increases stability of mucin to pepsin degradation; inhibits H. pylori surface urease activity Unclear Interferes with IL-8 secretion; reduces H. pylori surface expression of Hsp60 Unclear Protobiotic agent against H. pylori Inhibits Lewis b and sulfatide-mediated adherence; prevents host cell vacuolation; possibly saturates H. pylori mucin-binding proteins Unclear Inhibits Lewis b and sulfatide-mediated adherence

chemotaxis of H. pylori toward mucin (370). Third, the anti-ulcer compounds sucralfate and sulglycotide inhibit adherence by blocking the interaction of H. pylori LPS with laminin, G M 3 ganglioside, and lactosylceramide at the gastric epithelium surface (255, 2 5 8 , 301, 3 0 5 , 309). These compounds also decrease H. pylori mucinase activity by desulfating mucin (300, 304), and they also block the interaction of H. pylori LPS to the 97-kDa mucin receptor by 9 0 % (256). These activities lead to increased availability of mucin to bind normally to the mucin receptor on the gastric epithelium surface. Fourth, pretreatment of H. pylori with the anti-ulcer compound ecabet sodium inhibits adhesion of H. pylori to gastric epithelial cell lines by increasing stability of mucin to pepsin degra­ dation and also inhibits surface urease activity (133, 172, 2 9 1 ) . Fifth, pretreatment of gastric epithelial cells lines MKN-28 and M K N 4 5 with the anti-ulcer compound rebamipide, or derivatives thereof, inhibits adhesion of H. pylori by about 5 0 % (131). Sixth, pre­ treatment of M K N 4 5 gastric epithelial cells with the commonly used antacid co-magaldrox (Maalox) in­ hibits adherence of H. pylori by 7 5 % and specifically interferes with IL-8 secretion from the epithelial cells, while reducing surface expression of H. pylori Hsp60 (168). Seventh, the proton pump inhibitor omepra­ zole inhibits adherence of H. pylori extracts to human neutrophils (320). Eighth, addition of Lactobacillus salivarius, indigenous flora of the mouth, to MKN45 and KatoIII gastric epithelial cells decreases H. pylori adherence by up to 9 0 % , suggesting that lactobacilli could be used as a probiotic agent against H. pylori (166). Lactobacilli are also colonization antagonists

Reference(s) 225, 294 319, 370 255, 256, 258, 300, 301, 304, 305, 309 131, 172, 291 133 168 320 1, 166 59, 130, 315 205 290

in a gnotobiotic murine model (1, 166). Ninth, milk could serve as a potential adherence antagonist by blocking adherence of H. pylori to sulfatide and Lewis b and by preventing cell vacuolation (130). Indeed, human milk inhibits adherence of H. pylori to KatoIII cells by 50 to 7 0 % (59). The inhibitory fraction of milk was shown not to be secretory IgA (59), but rather K-casein, in a fucose-dependent fashion (315). There is also a casein-independent fraction of human milk that inhibits H. pylori adherence, which could perhaps be the mucin found in milk (59). Tenth, bile acids, such as chenodeoxycholic acid, are effective in­ hibitors of adherence of certain H. pylori strains by mechanisms that are unclear (205). Finally, fucoidan, a sulfated a l , 3 fucan, inhibits both Lewis b and sul­ fatide-mediated adherence of H. pylori to MKN28 and KatoIII gastric epithelial cells (291). Seven to 10 fucoidan-binding proteins of H. pylori were detected but not identified. Adherence antagonists are already being tested for efficacy in combination with antibiotic therapy regimens, such as omeprazole with amoxicillin. An­ other use for adherence antagonists is dissection of molecular mechanisms of adherence. For example, several adhesin-receptor interactions can be blocked so that still additional interactions may be explored (132).

DOES INVASION I N T O H O S T CELLS OCCUR? A long-standing question in H. pylori research is whether the bacterium can invade gastric epithelial

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cells and, if so, whether the organisms can persist as viable organisms intracellularly. The answers to these questions, however, are still controversial. The vast majority of researchers agree that invasion is rare (26, 30). However, rare invaders could still be important for intracellular persistence, resistance to antimicro­ bial therapy, and protection from the human immune response. H. pylori is occasionally localized within endocytic vacuoles in gastric epithelial cells, on the basis of studies using human gastric biopsy specimens or various cell lines (26, 2 3 5 , 2 7 4 , 3 3 3 , 3 6 1 , 362). Inva­ sion into parietal and chief cells was also observed in patient gastric biopsies in one study (362). Addition­ ally, H. pylori can invade the lamina propria and gain access to the basement membrane (10), where laminin is present. Using a nongastric epithelial cell line (HEp2) and a gentamicin protection assay, Evans and col­ leagues observed that invasion of H. pylori was rapid and dependent on the sialic acid adhesin HpaA (99). However, little replication of H. pylori occurred after 24 h, and numerous coccoid forms were observed, suggesting the H. pylori may have died following entry. Indeed, using the same cells and experimental protocol for assessing invasion, Megraud and col­ leagues failed to recover viable intracellular organisms (211). More recent experiments suggest that the gen­ tamicin protection assay is not useful for accurately assessing invasion of H. pylori because the organism finds extracellular pockets that protect it from the an­ tibiotic, leading to falsely elevated invasion levels (62). HEp-2 cells are also probably a poor model to assess invasion and adherence of H. pylori because it is a nongastric cell line, and since only some strains of H. pylori adhere to these cells (174). An early elegant study by Bode and colleagues (26) demonstrated internalization of H. pylori into the cytoplasm of mucus-secreting duodenal epithelial cells. This occurred in 1 0 % of the duodenal ulcer pa­ tient biopsies examined. This study was subsequently confirmed by Noach and colleagues (235), who used human gastric biopsies in their study and found six samples that were highly suggestive of invasion. H. pylori has also been found inside the mucin compart­ ment of goblet cells in gastric biopsies (117). H. pylori has a predilection for association with intercellular tight junctions ( 2 6 , 1 3 4 , 3 3 0 ) . Initial attachment of H. pylori to the cell surface may weaken tight junctions between adjacent host cells ( 2 6 , 2 3 5 ) . This weakening may allow deeper penetration of H. pylori between cells, with subsequent internalization ( 2 6 , 2 3 5 ) . How­ ever, that coccoid forms of the organism were ob­ served in some of these transmission electron mi­ crographs suggests that the invaders may have been nonviable. Evidence for invasion in the studies men­

tioned above were by electron microscopy or by gen­ tamicin protection assays and thus "invasion arti­ facts" cannot unequivocally be ruled out. In contrast, other investigators were unable to show evidence for invasion in gastric biopsies (134, 330) or in cell lines (62, 263). Whether internalized H. pylori is viable was still not clear until more recently, when invasion of H. pylori was confirmed by immunohistochemical stain­ ing for bacterial Hsp60, which was found intracellu­ larly in gastric biopsy specimens (96). Treatment of patients with standard antimicrobial triple therapy for 2 weeks resulted in clearance of the bacteria by day 14, but 5 of the 10 patients in the study relapsed by one month posttherapy, providing the first sugges­ tive evidence for antimicrobial therapy failure due to persistent (and thus, viable) intracellular H. pylori (96). If H. pylori lives in an intracellular environment, then poorly penetrating antibiotics, such as the widely used amoxicillin, would fail and may explain occa­ sional clinical failures of antimicrobial regimens. Using a tissue culture-based system, H. pylori was shown to invade AGS cells using a gentamicin protection assay (317). Type I (cag ) strains invaded better than type II strains (cag mutant). Invasion was blocked by cytochalasin D, which inhibits actin poly­ merization, or by a tyrosine phosphatase. Interest­ ingly, a 125- to 130-kDa protein that is almost cer­ tainly CagA, based on more recent studies (18, 64, 238, 2 8 3 , 313), was tyrosine phosphorylated. Inva­ sion was markedly enhanced by transfection of AGS cells with the 81 integrin gene, suggesting that integrins might serve as receptors for H. pylori in this system (317). A highly sensitive method of assessing invasion of 18 clinical isolates of H. pylori into HEp-2 cells was recently reported by Wilkinson and colleagues (355). In this study, acridine orange was used to stain microbes; intracellular viable microbes fluoresce green, whereas nonviable organisms fluoresce red. Fluorescence of extracellular microbes is quenched by a counterstain. This study showed H. pylori invasion frequencies that exceeded those for Shigella flexneri and paralleled those of Yersinia enterocolitica (355). Despite being mostly coccoid in shape, intracellular H. pylori remained viable for at least 6 h after entry, and no significant correlation was observed between invasion frequencies and clinical state from which the H. pylori was isolated (355). It will be of great interest to apply this sensitive method to more relevant cell lines such as KatoIII gastric epithelial cells and to fresh gastric biopsies. One potential problem with assessing H. pylori invasion is that VacA may contribute to cell lysis, leading to disruption of tissue culture monolay+

CHAPTER 34 • ADHERENCE AND COLONIZATION

ers. Thus use of a vacA mutant of H. pylori may help investigate invasion mechanisms more closely (355). Except for possibly HpaA (in a nongastric cell model), H. pylori invasins have not yet been identified in any study and specific genes need to be disrupted to lend stronger support for the hypothesis that H. pylori can occasionally invade host cells.

INTERACTIONS OF H. PYLORI W I T H PHAGOCYTIC CELLS H. pylori adheres to and enters human mono­ cytes and neutrophils, phagocytic cells that infiltrate the gastric mucosa. Adherence to neutrophils leads to up-regulation of the integrin CD l i b , independent of the H. pylori genes cagA, vacA, and cagE (129). Entry of H. pylori into human neutrophils (9, 2 6 ) , mono­ cytes, and macrophages (5,9) is opsonin independent, suggesting that H. pylori adhesins are responsible for entry. Whether these phagocytosed organisms remain viable or are killed depends on the presence of the cag PAI. Type I strains have a delay in phagocytosis followed by homotypic phagosome fusion into "megasomes," whereas type II strains are rapidly phagocy­ tosed and killed (5). The megasomes contained viable organisms (5). The reason for survival of type I strains inside phagosomes is unclear, but perhaps catalase and superoxide dismutase produced by the bacterium detoxify reactive oxygen intermediates inside of phagosomes (241, 3 1 2 ) . Also of importance is whether strains possess sialic acid hemagglutinins. Those H. pylori strains containing sialic acid hemag­ glutinins are more resistant to phagocytosis by human neutrophils and monocytes, in contrast to those lack­ ing sialic acid hemagglutinins (9, 4 6 , 4 8 ) . However, H. pylori adherence, which can be clearly distin­ guished from phagocytosis, does require sialic acids on the surface of human monocytes (51). Therefore, sialic acid hemagglutinating strains adhere better yet are more resistant to bactericidal killing and phagocy­ tosis compared with nonhemagglutinating strains. Heparan sulfate significantly enhances phagocytosis of H. pylori by human neutrophils (46, 4 9 ) . H. pylori activates monocytes by both LPS-dependent and -independent mechanisms, resulting in the production of IL-1 and TNF-a and in expression of major histocompatibility (MHC) antigens and the IL-2 receptor (199). Monocytes could thus be crucial for ingestion of H. pylori and presentation of bacterial antigens on the cell surface in the context of MHC antigens for eventual humoral or cell-mediated im­ mune response. The role of specific bacterial genes in the interaction of H. pylori with phagocytic cells is a largely unexplored area of research.

397

Summary of Adherence Assimilation of all these data supports a model whereby H. pylori first encounters gastric mucin and degrades it. Next, H. pylori induces Hsp expression during their transient exposure to acid in vivo, allow­ ing binding to sulfatides. Upon neutralization of acid by urease and movement of H. pylori to the gastric epithelial cell surface (pH neutral) via motility, the Hsps may be down-regulated and H. pylori then can bind to phosphatidylethanolamine. Following cleav­ age of PE by sphingomyelinase, H. pylori adheres to Lewis b and up-regulates sialic acid-containing glyco­ conjugates to which H. pylori subsequently binds. After damage to the gastric epithelium, H. pylori ad­ heres to the basement membrane protein laminin. Hence, H. pylori is a very sticky bacterium that has evolved numerous adhesins to bind to host cell sur­ faces. Temporal regulation of adhesin and host cell receptor expression is probably very important in vivo, but has not been well studied. The real challenge is to temporally dissect these adhesins and receptors to test the ideas in this model and determine which are important in vivo.

COLONIZATION Host Factors Even before the discovery of H. pylori, research­ ers had found significant correlations between certain genetic markers and prevalence of ulcer disease (Table 3). Similar studies have been performed more re­ cently, and there are early indications that some alleles of host genes may predispose one to become colonized in the first place, while others are associated with sub­ sequent development of ulcers or cancer. This section will focus primarily on factors believed to increase

Table 3. Factors influencing colonization by H. pylori Factors that enhance colonization High-salt diet Low antioxidant levels Pregnancy Ethnicity (Chinese, Indian, Polynesian) Thl response Gastrin Urease Motility/chemotaxis GGT Phospholipase

Factors that inhibit colonization Lactobacilli Vitamin C intake Ethnicity (Malay, European) Th2 response Gastric mucus Urease inhibitors

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susceptibility to infection, rather than disease out­ come; however, the present state of knowledge scarcely allows differentiation between susceptibility and prognosis. Numerous epidemiological studies have exam­ ined a range of populations and environmental factors to determine what constitutes a risk factor for devel­ opment of chronic H. pylori infection and subsequent pathologies. Although the evidence is circumstantial, it appears that the H. pylori infection rate varies due to both exposure level and intrinsic susceptibility to colonization. Studies have revealed that nursing home patients, endoscopists, and individuals consuming food from street vendors may be more likely to be­ come infected with H. pylori than the general popula­ tion (24, 122, 2 0 7 , 2 6 4 ) . These studies point toward a higher risk of exposure to the organism. In other studies, however, it is more difficult to imagine why the exposure rates should differ. For instance, com­ parisons of H. pylori-specific IgG, indicating an estab­ lished infection, and IgM, suggesting recent exposure, reveal that women are more than twice as likely to become infected with H. pylori while pregnant than nonpregnant controls (181). Since pregnancy is known to result in many immunological changes, it is possible that host immune status could render the host less able to combat the initial infection with H. pylori. Although numerous studies have shown that acquisition often occurs during childhood (93, 201), at least one study has also demonstrated a high rate of spontaneous clearance among children before age 11 (331). Thus, it is possible that children are both more likely to be exposed to H. pylori and more likely to clear the infection as their immune systems mature, whereas adults are less likely to clear an established infection but are infrequently exposed to the or­ ganism. Many investigators have accumulated evidence that factors specific to an individual host influence colonization by H. pylori. Studies of twins revealed that monozygotic twins were 8 1 % concordant for in­ fection, compared with 6 3 % concordance in dizygotic twins (200). That is, 8 1 % of identical twins were either both infected or neither infected. There were few cases in which one twin was infected and the other twin was not. This difference was maintained regard­ less of whether the twins were reared together or apart. In a multiethnic Malaysian city, a study of 1,060 consecutive endoscopy patients revealed that, overall, the prevalence of H. pylori infection mea­ sured by rapid urease tests of biopsies was 4 9 % ; how­ ever, only 1 6 . 4 % of ethnic Malay patients were in­ fected, whereas 4 8 . 5 % of Chinese and 6 1 . 8 % of Indian patients were H. pylori positive (121). Another study in a multiethnic region also revealed differences

in H. pylori positivity related to ethnicity. Even when socioeconomic status was accounted for, Europeans were significantly less likely to be seropositive than Maori or Pacific Islanders (113). Neither research group could rule out dietary factors or cultural prac­ tices that may increase risk of infection; however, Campbell and colleagues noted that Maori and Pacific Islander populations interact more frequently with Europeans than with each other (34). This group also found that strains isolated from Europeans were al­ most three times more likely to express a biologically inactive VacA cytotoxin than strains isolated from Polynesians. This could mean that VacA confers an advantage to H. pylori only in certain hosts, while it is detrimental for the bacterium in others. Specific host characteristics could therefore influence the ability of H. pylori to colonize in the first place, and subse­ quently exert adaptive pressure leading to measurable differences in the colonizing strain. A single encounter with a given strain of H. pylori may not be sufficient for lifelong infection. Ethnic cor­ relations among inhabitants of the same geographic region merit further investigation to determine whether identifiable host markers predispose certain individuals to infection. Polynesians are known to ex­ press the Lewis b antigen at a lower frequency than Europeans, but this has not yet been correlated to risk of infection or strain type likely to be present (138, 139). Data regarding the identities or mechanisms of such host factors influencing H. pylori growth are sparse, due to the inherent difficulties in conducting controlled studies. Nonetheless, one study performed in rhesus monkeys has yielded some interesting data regarding host specificity, and apparent changes in selective pressure that occur as colonization pro­ gresses. A group of four monkeys that had been previ­ ously cured of their natural H. pylori infections was inoculated with a mixture of seven fresh clinical iso­ lates (81). By 10 months postinfection, randomly am­ plified polymorphic DNA (RAPD) fingerprinting re­ vealed that clinical isolate J I 66 predominated in all four animals, even though this strain had been de­ tected in only one animal at the earliest timepoint. Two other strains were found early in infection in all four animals, but both had nearly reached undetecta­ ble levels by 10 months. Although only a small num­ ber of animals were used, this study strongly suggests that either the gastric environment changes as infec­ tion becomes established, and that this change affects the relative survival of H. pylori strains, or that strains differ in their relative competence for infection and persistence. It is possible that, had only one strain been used as an inoculum, the monkeys would have been only transiently infected. Thus far, little is known about specific host fac-

CHAPTER 34 • ADHERENCE AND COLONIZATION

tors that influence H. pylori infection. The HLA types that appear to be correlated with susceptibility to helicobacter infection are HLA DR and HLA D Q * 0 3 0 1 . HLA DQA1*0301 and HLA DQ5 are frequently as­ sociated with more severe disease but may not predis­ pose an individual to becoming infected in the first place. HLA-DQA1*0102 appears to confer partial protection to H. pylori-related atrophic gastritis and intestinal-type gastric adenocarcinoma (21). The identification of additional host cell markers relevant to H. pylori may provide important clues for under­ standing H. pylori infection and developing treatment strategies. Immune Factors The quest for a fully protective H. pylori vaccine has been frustrating, but there is ample evidence that innate and elicited immunity serves to modify and/or partially control H. pylori infection (120, 163, 213). One study revealed a difference between infections of germ-free athymic mice and their euthymic counter­ parts (169). Although both groups of mice were per­ sistently infected, the athymic mice had colony counts approximately eightfold higher than euthymic mice from week four until the conclusion of the study. Bac­ terial counts increased rapidly in euthymic germ-free mice during the first week postinfection but subse­ quently declined until reaching a stable level by week three. In contrast, the bacterial counts in the athymic germ-free group continued to increase until at least week four. Interestingly, the germ-free euthymic group consistently had less inflammation than the athymic group, suggesting that infiltration of inflam­ matory cells alone is insufficient to reduce H. pylori numbers. Although the authors did not measure anti­ body titers or other markers of immune response, H. pylori numbers declined after week one, when anti­ body titers are expected to be rising. Since H. pylori infection induces a polarized T h l response, characterized by increased IFN-y, it was hy­ pothesized that directing the immune system toward a Th2-type response might reduce infection. An initial investigation of I L - 4 m i c e , which exhibit a strong T h l response, infected with H. felis showed increased colonization and inflammation, supporting this hy­ pothesis (221). Chen and colleagues (41) did not find similar increases in colonization or immune response when they compared H. pylori-'miected I L - 4 mice to wild-type mice. Nonetheless, convincing evidence for the role of the Th2 response in control of H. pylori infection is provided by a study utilizing adoptive transfer of splenocytes (221). Mice injected with an H. pylori-specitic Th2-cell line showed dramatic re­ ductions in bacterial burden. Mice injected with _ / _

_ / _

399

splenocytes from immunized mice also harbored fewer bacteria, while no difference was noted when splenocytes were transferred from mice that had been infected with H. pylori but not previously immunized. Reductions in bacterial burden correlated signifi­ cantly with elevated serum IgGl levels (a marker of the Th2 response) in the recipient mice. Gastrin is a polypeptide hormone produced by G cells in the antrum of the stomach that stimulates secretion of gastric acid (Fig. 1). H. pylori-positive individuals frequently have elevated gastrin levels, both fasting and following a meal, and often have increased acid output in response to gastrin (33). Both gastrin levels and acid secretion return to normal fol­ lowing eradication of H. pylori ( 9 4 , 1 2 7 ) . Gastrin has been shown to augment growth of H. pylori and may aid in colonization. When grown in brain-heart infu­ sion or Brucella broth medium supplemented with 1 0 % fetal calf serum, H. pylori showed a dose-depen­ dent response to gastrin, with a decreased lag phase and higher final bacterial density, while the gastric peptides somastatin and epidermal growth factor had no effect (53). Gastrin had no effect on growth of Campylobacter jejuni or E. coli. Experiments with I-labeled gastrin demonstrated that gastrin is spe­ cifically bound and taken up by H. pylori (53). The structurally similar peptides pentagastrin and cholecystokinin octapeptide were able to compete with ra­ diolabeled gastrin for uptake but did not similarly stimulate H. pylori growth. Uptake of radiolabeled gastrin, as measured by autoradiography, was inhib­ ited at 4°C, suggesting an energy-dependent internali­ zation mechanism. Several mechanisms were initially proposed to explain H. py/on-mediated increases in gastrin output. H. pylori might reduce secretion of the G-cell inhibitor somastatin, which is produced by nearby D cells; H. pylori products could directly stim­ ulate G-cell activity (353); or H. pylori could mediate effects on gastrin indirectly via other host cells. Re­ duced D-cell numbers, somatostatin concentrations, and response to cholecystokinin seem to support the first hypothesis ( 3 3 , 1 7 7 ) ; however, other experimen­ tal data indicate that the last hypothesis is correct. The proinflammatory cytokines IL-1B and TNF-a produced by monocytic cells have been shown to in­ crease gastrin secretion by endocrine cells and by an­ tral G cells (353). Thus, there are two interactions between H. py­ lori and gastrin: (i) H. pylori induces elevated gastrin release, and (ii) H. pylori uses gastrin as a growth factor. H. pylori proteins involved in this response have not yet been identified. Nor is it known what advantage is conferred by H. pylori's uptake and growth response to gastrin. Given the specificity of the response, the simple use of gastrin for nutritional 125

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purposes seems unlikely. The utility of using gastrin as a homing signal directing H. pylori toward the gastric mucosa makes intuitive sense; however, it is less clear whether the host side effects of H. pylori colonization are also beneficial to the bacteria. As one of the host's primary innate defenses, an increase in acid secretion following bacterial insult and mucosal inflammation m a y be beneficial. Moreover, a link has previously been shown between the immune system and gastrin production. Following immunization, reexposure of animals to the immunogen caused an increase in gas­ trin secretion (326). H. pylori, however, has adapted to cope with the resulting acid load, and perhaps gas­ trin secretion somehow aids in the perpetuation of H. pylori colonization. Dietary, Drug, and Environmental Factors Following colonization, H. pylori is envisioned to reside in a protected niche, safe from stomach acid and many components of the immune system. But how accurate is this picture? Some elements of the immune system are able to cross the mucosal epithe­ lium. Antibodies are secreted, and macrophage-like cells may be able to traverse the gastrointestinal epi­ thelium (349). The gastric mucous layer effectively prevents back-diffusion of hydrochloric acid but does allow diffusion of other solutes, including some over 30 kDa (73, 354). Thus, H. pylori could be exposed to many elements of the host diet. Dietary factors may have a larger influence during and immediately fol­ lowing infection, as H. pylori struggles to colonize the gastric mucosa. Dietary factors Excessive salt intake is a dietary factor common to Japanese and to many Americans. A high-salt diet has previously been correlated with gastritis and in­ creased cancer risk but may in fact influence H. pylori colonization as well (336). Mice fed 7 . 5 % salt prior to and following experimental infection with H. py­ lori SSI became colonized at a higher level than mice fed a normal diet (110). Salt alone causes atrophy of parietal cells and increases the rate of N-methyl-N'nitro-N-nitrosoguanidine-induced tumor formation in rodents (352). Perhaps these changes produce a more favorable environment for H. pylori replication. Furthermore, gastric cell proliferation rates correlate with salt intake only when the subject is infected with H. pylori (72, 357), which could make the high Sart­ re, pylori combination particularly carcinogenic. Dietary vitamin C (ascorbic acid), on the other hand, is epidemiologically linked to lower rates of gas­ tric carcinoma (31, 2 9 5 ) . There is evidence that lower

risks may be partially attributable to scavenging of reactive oxygen radicals in the stomach by ascorbate (80), but more specific effects of vitamin C on H. py­ lori may also play a role. Researchers have shown inhibition of H. pylori, both in vitro and in vivo by vitamin C (373). Vitamin E, another antioxidant, had no effect on growth, even at high concentrations. Fur­ thermore, this inhibitory effect appears to be specific for H. pylori and the closely related Campylobacter species, as various E. coli strains, Salmonella, and Vibrio species were unaffected. Vitamin C is bacteri­ cidal for helicobacter in vitro, but only partially inhib­ itory in vivo. Gerbils dosed orally with 10 mg of vita­ min C daily following H. pylori infection had 100fold fewer CFU per stomach than control mice. The attainable concentrations of vitamin C near the mu­ cosal epithelium are not known, and it is possible that in vivo levels are not high enough for full suppression or that H. pylori is otherwise partially protected in its gastric niche. Additionally, H. pylori may inhibit the secretion of ascorbic acid from the circulation, lowering concentrations in the colonized stomach (280). Vitamin C inhibits urease activity in vitro (231); however, Zhang and colleagues (373) report that growth of urease-negative mutants is similarly inhibited by vitamin C in vitro, suggesting urease-independent mechanisms of growth inhibition. Given the accepted importance of urease for colonization, it is difficult to differentiate between urease-dependent and -independent effects of vitamin C. In related research, another antioxidant, astaxanthin, was found to reduce both the bacterial load and gastric inflammation in H. pylori-iniected mice (25). Astaxanthin, a carotenoid present in high levels in the algae Haematococcus pluvialis, has previously been studied as an anti-cancer agent (45, 323). Astax­ anthin may mediate both chemoprotection and ame­ lioration of H. pylori infection via its effects on the immune system. Dietary astaxanthin resulted in in­ creased phytohemagglutinin-induce ed lymphocyte proliferation and increased cytotoxic activity (45). Additionally, astaxanthin suppresses interferon-y (IFN-y) production in vitro (165) and increases IL-4 production in vivo, suggesting that astaxanthin pro­ motes a shift from a predominantly T h l response to a mixed T h l / T h 2 response (25). Since IFN-y activates phagocytic cells, which can result in tissue damage, suppression of IFN-y may decrease tissue pathology. Thus, astaxanthin may protect the mucosal surface both by decreasing phagocyte activation and via its intrinsic antioxidant activity. Reports that certain Chinese and Italian popula­ tions consuming large amounts of garlic are at de­ creased risk for gastric cancer have prompted investi­ gations of the antimicrobial effects of garlic ( 3 2 , 3 7 2 ) .

CHAPTER 34 • ADHERENCE AND COLONIZATION

Garlic tablets or extracts have demonstrated in vitro growth inhibitory activity against H. pylori in several studies (38, 5 5 , 1 6 4 , 2 4 2 , 2 9 6 ) . In two studies, a syn­ ergistic effect between omeprazole and garlic was ob­ served (38, 164). Unfortunately, human trials have not yet demonstrated amelioration of H. pylori infec­ tion (126). Other dietary components have been tested and discarded as potential risk factors for helicobacter in­ fection. Some early data suggested a role for vitamin E and beta-carotene in prevention of gastric cancer (78, 176); however, a 5-year clinical trial involving male smokers with atrophic gastritis failed to show any protective effect of alpha-tocopherol (vitamin E) or beta-carotene supplementation (347). Likewise, a study of polyunsaturated fatty acid intake showed no decrease in colonization or inflammation. The au­ thors believe that earlier correlations were spurious, and possibly due to confounding factors such as afflu­ ence, social class, or smoking (82). Probiotics Lactobacilli can also influence levels of coloniza­ tion by helicobacter. Although gastric acid is famous for its ability to kill ingested organisms, the stomach routinely contains live organisms in the mucus and associated with the gastric wall. Sampling of gastric juice, both fasting and postprandial, routinely pro­ duced Viridans streptococci, Neisseria spp., Candida spp., and Lactobacillus spp. (311). One possible rea­ son for the failure of most H. pylori strains to colonize mice may be the higher level of Lactobacillus coloni­ zation observed in mice. Isogai et al. hypothesized that increasing the amount of time H. pylori is able to contact the gastric mucosa may give H. pylori a better chance at competing with the normal flora of the mouse (156). They were able to transiently infect con­ ventional mice by temporarily clamping the duode­ num for 30 min, thus preventing the H. pylori inocu­ lum from passing through the stomach. They noted, however, that the numbers of lactobacilli in the stom­ ach fell following surgical treatment and H. pylori inoculation. Subsequently, Lactobacillus numbers rose as H. pylori numbers fell. In a more controlled study of the effects of lactobacilli, Kabir et al. infected gnotobiotic BALB/c mice in the presence or absence of L. salivarius, a species frequently found in the human stomach and oral cavity (166). Germ-free animals could be infected by all H. pylori strains tested in the study, including NCTC 11637, which had failed to infect conventional animals in previous studies. Fur­ thermore, the infection was sustained at about 1 0 CFU per g of tissue for 9 weeks. When the germ-free animals were infected with L. salivarius one week 5

401

prior to H. pylori inoculation, H. pylori was unable to colonize. Likewise, infecting previously H. pyloricolonized mice with L. salivarius reduced both the level of H. pylori colonization and the anti-H. pylori IgG titers. In contrast, two other organisms found in the mouse stomach, Enterococcus faecalis and Staph­ ylococcus aureus, were unable to prevent H. pylori colonization. This suggests that the inhibitory effects of lactobacilli are due to more than simple competi­ tion. Several investigators have observed that Lacto­ bacillus spp. inhibit adherence of H. pylori in vitro (60, 166), but there is evidence of other inhibitory mechanisms used by at least some lactobacillus strains. A whey-based culture supernatant of Lacto­ bacillus acidophilus (johnsonii) L a i administered daily for 2 weeks was able to decrease urease activity, as measured by C-urease breath test ( C - U B T ) , in H. pylori-infected human volunteers (214). Four weeks after cessation of therapy, C - U B T values re­ mained significantly below pretreatment levels. In vitro studies performed using the same supernatants and those from a related strain, L. acidophilus La 10, showed that strain L a i was able to inhibit H. pylori growth in vitro, whereas strain La 10 could not. The two strains produce similar amounts of lactic acid, implying that lactic acid was not responsible for the effect. In a similar study involving mice, the Servin group examined the effects of human-derived L. aci­ dophilus strain LB on H. pylori adhering to cultured mucus-secreting cells and on mice infected with H. felis. Culture supernatants from strain LB caused a significantly greater decrease in H. pylori viability than did a similar supernatant from L. acidophilus strain GC (60). The activity of both supernatants could be partially abrogated by heating, suggesting a heat-labile antagonist. Lactic acid alone, at a similar concentration and pH to that found in supernatants, had no effect on H. pylori viability. Thus, H. felis is presumably resistant to products produced by naturally occurring lactobacilli in the conventional BALB/c mouse stomach but is sensitive to L. acidophi­ lus LB. Although the in vivo studies were not per­ formed with H. pylori, the experiments demonstrate that addition of a new Lactobacillus strain to the ex­ isting flora can alter the state of the Helicobacter in­ fection and may reduce gastric pathology. Lactobacilli are known to produce hydrogen peroxide and bacteriocins, which may be responsible for some of the H. pylori inhibition (159, 344). The above data in con­ junction with mouse data suggest that consumption of L. acidophilus or dairy products fermented with these organisms may prevent colonization from oc­ curring or reduce symptoms in previously infected in­ dividuals. A common theme appears to be emerging from 13

13

13

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TESTERMAN ET AL.

research on dietary influences on H. pylori infection. Agents, which in themselves promote tissue damage or cellular proliferation, appear to increase the risk of helicobacter infection and eventual cancerous lesions. Tissue damage may give H. pylori an advantage as it establishes colonization of the gastric mucosa. Dietary supplements and alteration may at least form part of a rational treatment regimen, in combi­ nation with drug therapy. Dietary interventions may be less expensive than drugs; this is useful for develop­ ing countries or other populations lacking access to advanced health care. Extracts of commonly con­ sumed plants, such as garlic, or bacteria may also prove safer and less likely to cause side effects, an important consideration when treating children and pregnant women. Lack of correlation between epide­ miological evidence and in vitro and in vivo data war­ rants caution when investigating dietary supplements. Correlations based on epidemiology may be spurious, and confounding host factors may limit in vivo effi­ cacy. Therefore, carefully designed human and/or ani­ mal studies are required. Non-antibiotic drugs Bismuth, which has antidiarrheal, antibacterial, and anti-inflammatory properties (109), has been used to treat gastrointestinal disorders for at least 300 years (180, 2 4 9 ) , yet its mechanism of action is only now being uncovered. Bismuth-containing com­ pounds appear to have several activities relevant to inhibition of H. pylori. Colloidal bismuth subcitrate inhibits activity of phospholipases 2 and C (PLA2, PLA-C) derived from H. pylori lysates or culture supernatants (247, 2 4 8 ) . There are conflicting reports regarding whether bismuth is competing with calcium ions for binding to PLA2 (247, 2 4 8 ) . It has been sug­ gested that PLA activity damages the hydrophobic barrier that protects the gastric mucosa against dam­ age by acid (79). Inhibition of phospholipases may help preserve the integrity of the gastric mucosa, thereby interfering with H. pylori colonization by re­ stricting access to the gastric epithelium. Bismuth subsalicylate also appears to scavenge reactive oxygen species. Damage to normal human gastric tissue removed by biopsy was assessed follow­ ing exposure to ethanol, hydrochloric acid, or sodium hydroxide (22). Increases in superoxide production, hydroxyl radical production, and DNA fragmenta­ tion were measured following exposure to each of the above agents. Bismuth subsalicylate measurably re­ duced these forms of damage in a dose-dependent manner. Although these experiments did not involve helicobacter, other lines of evidence support the no­ tion that H. pylori infection results in increased reac­

tive oxygen species within tissues. Both increased re­ active oxygen species and increased DNA damage have been detected in H. pylori-infected patients (23, 7 1 , 204). This tissue damage was initially thought to be due to activation of neutrophils; however, Obst and colleagues (237) demonstrated that DNA damage induced by H. pylori extracts can be inhibited by var­ ious radical scavengers. The antioxidant activity of bismuth has been shown to interfere with at least one H. pylori enzyme, alcohol dehydrogenase (270). The relevance of alco­ hol dehydrogenase inhibition in terms of colonization ability is uncertain; however, several hypotheses have been proposed. Acetaldehyde, produced by the action of alcohol dehydrogenase on ethanol, is toxic to mu­ cosal cells (232) and inhibits proliferation of gastric cell lines in vitro (206). Thus, acetaldehyde may im­ pair the ability of the mucosal layer to repair damage. Alternatively, it has been proposed that the reverse reaction may occur under microaerobic conditions, resulting in ethanol production and energy produc­ tion (278). In support of the notion that fermentation may be an important means of energy production, a potent alcohol dehydrogenase-inhibitor, 4-methylpyrazole, also inhibited growth of H. pylori in culture (277). Reduction in damage may make the gastric epi­ thelium more resistant to colonization. Therefore, antioxidant activities of bismuth or other compounds may comprise one facet of the complex set of interac­ tions leading to clearance of H. pylori or long-term colonization. Bismuth may have other direct inhibitory effects on H. pylori. Investigators have reported disruptions in the H. pylori cell wall, loss of motility, and de­ creased viability following treatment with various bis­ muth compounds (314, 360). Cytotoxicity associated with cell wall disturbances was also inhibited by diva­ lent cations (314). Thus, displacement of required di­ valent cations may be a common mechanism of bis­ muth activity. The anti-ulcer drug, ecabet, has two reported ac­ tivities that may interfere with H. pylori colonization. First, ecabet has a high affinity for gastric mucus and decreases degradation of mucous glycoproteins by pepsin (172). Second, ecabet inhibits urease activity (158). In experiments with jack bean urease, ecabetmediated urease inhibition was shown to be irreversi­ ble (157). The authors of this study suggest that per­ manent inhibition is due to specific binding and denaturation of urease. H. pylori is able to survive in buffer at pH 3 if 10 mM urea is present. Addition of ecabet to the buffered urea results in decreased viability, sim­ ilar to survival seen in the absence of urea (158). No evidence has been presented to address the question of whether ecabet is taken up by H. pylori, where it

CHAPTER 34 • ADHERENCE AND COLONIZATION

could suppress activity of intracellular urease. None­ theless, several clinical studies have demonstrated im­ proved clearance of H. pylori infection when ecabet is given in combination with antibiotics, suggesting that the drug's anti-urease activity is relevant to infec­ tion (243, 2 8 7 , 292). Improvement of mucin stability by ecabet may therefore force H. pylori to spend longer in the lumen of the stomach, where it is ex­ posed to the lowest pH values, while ecabet simul­ taneously deprives H. pylori of its ability to neutralize acid via urease activity. Bacterial Factors Since H. pylori has no known animal reservoir and is not believed to replicate outside the host, all naturally occurring strains of H. pylori are presumed able to infect some subset of the population. The search for genes that are not required for viability but do influence colonization is progressing rapidly, with the assistance of two fully sequenced H. pylori ge­ nomes. One strategy is to examine properties that are common to all H. pylori strains but absent in other bacteria. Genome sequences also facilitate the search for homologs of genes known to be critical to other pathogens. Some genes may influence pathogenesis, without having an effect on colonization. For exam­ ple, the vacuolating cytotoxin VacA (see chapter 9) has profound effects on host cells, but insertional dele­ tion of the vacA gene does not reduce colonization (87). This section summarizes current knowledge on genes that may influence gastric colonization by H. pylori. Urease was the first colonization factor identified in H. pylori, and continuing experimentation has un­ covered new roles for this enzyme (see chapter 16). Urease was initially thought to be important solely for neutralization of gastric acid, yet it is required for colonization even in achlorhydric animals (88). Research has uncovered potential roles for urease in nitrogen assimilation and provision of energy for fla­ gellar rotation (356, 371). Several drugs used to com­ bat H. pylori infection, including bismuth, sucralfate, and sulglycotide, exert at least some of their effects through inhibition of urease activity. The first urease mutant of H. pylori was gener­ ated by mutation with nitrosoguanidine (85). This mutant was unable to colonize gnotobiotic piglets. Due to the undefined nature of the original mutants, questions remained regarding possible second site mu­ tations. The Eaton group later reported the use of an insertional deletion urease mutant and repeated the colonization study, with similar findings (88). Other groups found similar results with urease mutants tested in normochlorhydric nude mice, ferrets, and

403

cynomolgus monkeys (12, 3 2 2 , 335). The insertional deletion mutant persisted at low numbers for at least 5 days in some piglets rendered achlorhydric by treat­ ment with omeprezole. Fewer than 100 CFU per g of tissue were recovered, however, from two of four animals at 5 days postinfection. In contrast, eradica­ tion therapy with the urease inhibitor flurofamide failed to eradicate H. mustelae infection in ferrets, even when given in combination with amoxycillin (260). These data suggest that acid neutralization is one important function of urease, but that urease also plays other roles during the infection process. Further­ more, urease may not be absolutely required once in­ fection is established. H. pylori has mechanisms other than urease for protection against acid. These include arginase, an ali­ phatic amidase, and amino acid deamidases (210, 212, 298). The rocF gene encoding arginase protects H. pylori against acid in the presence of arginine by converting it to ornithine and urea (210). The rocF mutant is 1,000 times more sensitive to acid than the wild-type strain in the absence of urea. One rocF mu­ tant examined had a 2-log reduction in the ability to colonize mice, suggesting that arginase could be an important bacterial factor in vivo. y-Glutamyl transpeptidase (GGT) is constitutively expressed by all strains of H. pylori but has only been found in a few other bacteria (44). This enzyme catalyzes the transfer of glutamyl groups between compounds such as glutathione and amino acids. In mammalian cells, G G T influences glutathione metab­ olism. Its role in bacteria is unknown, but G G T has been hypothesized to play a role in the transport of amino acids. A ggt isogenic mutant of the mouse-viru­ lent SSI H. pylori strain was convincingly shown to be unable to colonize mice (44). Additionally, the mutant strain was unable to infect in the presence of wildtype H. pylori. Further studies on the H. pylori ggt mutant have not yet been performed, however, and an E. coli ggt mutant was found to be sensitive to hydrogen peroxide and chlorine compounds, and suppression of G G T activity resulted in leakage of glutathione from the bacterial cells (43, 2 2 7 ) . Al­ though the gene is constitutively expressed, the ggt mutant strain demonstrated no obvious deficiencies when grown in vitro. Elucidation of the role played by H. pylori GGT in vivo may provide clues regarding the relevant stresses faced by H. pylori during coloni­ zation. Phospholipases are believed to play roles in the virulence of several organisms, including Clostridium perfringens, Listeria monocytogenes, and P. aerugi­ nosa. H. pylori has PLA1, PLA2, and PLC activities (247). As mentioned above, the anti-ulcer drug bis­ muth inhibits phospholipase activity. T o test the im-

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TESTERMAN ET AL.

portance of phospholipase activity in vivo, Dorrell and coworkers (79) constructed a mutation in pldA, the only H. pylori phospholipase gene identified to date. The resulting mutant H. pylori strain had no detectable PLA2 activity, no zone of clearing on egg yolk agar, and markedly reduced hemolytic activity. This mutant was not detected in mice at 2 or 8 weeks following infection, suggesting an inability to colo­ nize. Nonetheless, infected mice generated H. pylorispecific antibody titers. Although titers were signifi­ cantly lower than those elicited by wild-type H. py­ lori, this may indicate that the pldA mutant is able to persist within the mouse for a short period. PLA2 lev­ els and lysolecithin, the product of cellular phospho­ lipid hydrolysis, have been found to be higher in H. py/on'-infected patients than in uninfected controls (182). This indicates that PLA2 could contribute to cellular damage in vivo and thus may play an active role in pathogenesis. Iron is required for growth of nearly all patho­ gens. H. pylori actively acquires iron from the host and may, in fact, contribute to anemia in some pa­ tients ( 5 2 , 2 5 2 ) . Human transferrin is the protein used to sequester iron in the systemic circulation, while lactoferrin is used in the mucosa, including tears, saliva, and milk (70). Other bacterial species are known to utilize transferrin and lactoferrin as iron sources, and specificity for the human or bovine proteins is be­ lieved to contribute to species specificity of Neisseria gonorrhoeae and Pasteurella haemolytica, respec­ tively ( 2 8 1 , 2 8 2 ) . H. pylori produces a 70-kDa outer membrane protein, Lbp, which specifically binds human lactoferrin (74). Bovine lactoferrin partially inhibits interaction with human lactoferrin; however, bovine lactoferrin cannot be effectively used by H. pylori for growth in iron-deficient medium (153). Lactoferrin may be a particularly important source of iron for bacteria residing in the gastric mucosa, as it retains binding capabilities even at pH extremes (153). Serum antibodies specific for H. pylori Lbp were found in patients infected with H. pylori but not in control patients, suggesting that this protein is expressed by H. pylori in vivo (74). Other bacterial colonization factors have not yet been precisely identified. Van Doom and colleagues (346) found that a spontaneous mutation within strain SPM326 affected colonization in BALB/c and C57B1/6 mice. The wild-type strain induced strong IL8 production in KATOIII cells, whereas the sponta­ neous mutant did not induce IL-8. The wild-type strain, however, was unable to colonize BALB/c mice, whereas the IL-8 mutant strain could infect these ani­ mals. Both strains were able to colonize C57B1/6 mice, but the wild-type stain colonized at a significantly lower level, and with a further decrease in bacterial

load by 12 weeks postinfection. Wild-type strain SPM326 is able to persistently colonize other mouse strains (119), suggesting that host factors play a role as well. The site of the spontaneous mutation is not known; however, mutations in certain genes in the cag pathogenicity island influence IL-8 production (3, 39, 3 3 9 ) . The authors speculate that the wild-type SPM326 strain may be more susceptible to host factors than strain S S I , which readily infects both BALB/c and C57B1/6 mice. Perhaps the mutant defi­ cient in IL-8 induction elicits a weaker immune re­ sponse than the parent strain. In three of seven SPM326-infected C57B1/6 mice, increases were noted in the number of T cells in the mucosa, and increased MHC class II antigen expression was not confined to small cells located between the gastric glands, as it was in control mice (346). The mechanism of these colonization and immunological effects will need to be elucidated to determine whether similar gene dis­ ruptions influence H. pylori colonization in humans. One significant impediment to studying bacterial colonization factors is the lack of data regarding the natural route of transmission and the physiological state of the organism at the time of infection. Since H. pylori is capable of converting to a coccoid mor­ phology, and this conversion takes place after pro­ longed in vitro culture, it is possible that coccoid or­ ganisms are responsible for naturally acquired infections. The literature contains conflicting reports regarding the capacity of coccoid H. pylori to infect animals. Cellini and colleagues were able to demon­ strate colonization of BALB/c mice by culture, histol­ ogy, and serology following administration of coccoid organisms (37). A similar attempt to infect gnotobi­ otic piglets with coccoid H. pylori failed to produce any evidence of even transient infection (86). Neither group attempted to assess viability of coccoid organ­ isms using any measure other than the formation of colonies. Therefore, one cannot infer what proportion of each inoculum was dormant or truly dead. Far more research on the conversion to—and possible re­ version from—the coccoid state is needed to resolve the question of whether coccoid H. pylori is infec­ tious. Motility and Chemotaxis To avoid being continually bathed in acid, and eventually flushed from the stomach entirely, H. py­ lori must be motile. H. pylori flagella are composed of flagellin, encoded by flaA and flaB ( 1 7 9 , 1 8 8 , 3 1 8 ) , and are surrounded by a membranous sheath contain­ ing LPS and protein (116, 195). Aflagellate H. pylori mutants are unable to colonize (89,90) but still gener­ ate an immune response, indicating survival at least

CHAPTER 34 • ADHERENCE AND COLONIZATION

for a short time (91). Repeated in vivo passage of H. pylori resulted in greater colonization efficiency (203). Further investigation revealed that bacteria from later passages expressed more flaA mRNA and flagellin protein than did earlier passage isolates. The flagella of H. pylori possess unique bulb structures at their tips, called terminal bulbs. The function of the terminal bulbs and the genes control­ ling their development are not yet known. These structures may aid in adherence or in propelling the bacteria through the viscous mucous layer. Indeed, viscosity has been shown to stimulate chemotaxis (369). The panorama of bismuth's antihelicobacter activities may include reduction in motility. Inhibition of urease activity with various inhibitors abolished the motility and the chemotactic response in a viscous solution, in spite of the presence of flagella and the ability to move through a nonviscous solution (226, 369). The energy source for flagellar movement is de­ rived from the proton motive force; hydrolysis of urea may generate the additional energy required for movement through a viscous environment (371). The ability to swim may not be sufficient to main­ tain the H. pylori population in the stomach. Some evidence has arisen indicating that the capacity for directed motility, or chemotaxis, may be crucial. Analysis of the H. pylori genome revealed genes ho­ mologous to those encoding the known chemotaxis regulators, CheA and CheY. The H. pylori homologs were designated cheYl and cheAY2. Mutations in these genes, which abolished chemotaxis, also ren­ dered the mutants unable to colonize mice, although significant antibody responses were detected (111).

CONCLUSIONS We are only beginning to understand the com­ plex mechanisms used by H. pylori to establish infec­ tion and to maintain colonization in the same host over a period of many years. Penetration of the mu­ cous layer likely involves motility, phospholipase ac­ tivity, and mucinase activity. H. py/on'-mediated re­ duction of mucus synthesis or secretion may also assist the bacterium in gaining access to the epithelium and persisting at this location. Adherence allows the bacteria to anchor themselves to the epithelial layer, but bacteria that remain attached to epithelial cells will eventually be swept away as these cells die and are exfoliated. Thus, a proportion of the H. pylori population exists in the nonadherent state. H. pylori must also contend with antibodies and phagocytic cells as the host mounts an immune response. Colonization by H. pylori has only been con­ firmed in the gastric mucosa. Limitation to this site

405

could be due to competition by other organisms in the mouth and small intestine. Other Helicobacter spp. have been isolated from blood and deep tissues, but H. pylori has not. Perhaps the exquisite sensitivity of H. pylori to complement-mediated lysis (261, 265) renders it incapable of causing extragastric disease; however, circumstantial evidence suggests that H. py­ lori may be present in the liver (233, 259). Preventing the establishment of long-term coloni­ zation, or even curing an established H. pylori infec­ tion, may be achieved either by killing all organisms or by antagonizing one or more of the persistence mechanisms and suppressing growth until the combi­ nation of host immune response, exfoliation, and movement of gastric contents purge the last bacterium from the stomach. Data in humans on prevention of colonization or spontaneous cure are sparse; how­ ever, there is some evidence for both phenomena (29, 115, 198, 2 0 2 , 3 2 8 , 372). Widespread treatment of H. pylori infection with multidrug regimens is prob­ lematic even in industrialized nations. Thus, until an effective vaccine is developed to prevent H. pylori in­ fection, improved sanitation and public education may be the most practical means of limiting infection. At present, dissecting the individual contribu­ tions of the factors discussed in this chapter is exceed­ ingly difficult. The development of new research tools, such as the completed genome sequence and superior in vivo and in vitro models will no doubt facilitate future studies of H. pylori adherence and colonization.

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CHAPTER 34 • ADHERENCE AND COLONIZATION

361. 362.

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368.

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Helicobacter pylori: Physiology and Genetics Edited by H . L. T. Mobley, G. L. Mendz, and S. L. Hazell © 2 0 0 1 ASM Press, Washington, D.C.

Chapter 35

Lipopolysaccharide Lewis Antigens BEN J . APPELMELK AND CHRISTINA M. J . E.

As determined by serological techniques, the O-anti­ gen of lipopolysaccharide (LPS) of more than 8 0 % of Helicobacter pylori strains tested worldwide express Lewis blood group antigens ( 3 6 , 6 0 , 7 1 ) . This percent­ age possibly represents an underestimation; it was demonstrated that some H. pylori strains do not react with anti-Lewis x (Le ) monoclonal antibodies (MAbs) while structurally they were shown to express Le (39). Thus, Lewis antigen expression in H. pylori is highly conserved. This restricted diversity in O-anti­ gen structure is striking, and the question arises whether H. pylori Lewis antigens play a role in patho­ genesis. An analogous situation is found in Neisseria gonorrhoeae, where conserved LPS O-antigen epi­ topes directly interact with the host via ligand-lectin binding (35). x

x

There are additional reasons why H. pylori LPS Lewis antigens are thought to play a role in pathogen­ esis beyond merely providing length to the LPS (al­ though length itself already contributes to virulence) (7). (i) H. pylori LPS displays phase variation, defined as the high frequency of reversible change of LPS phe­ notype (2, 5, 6 8 , 69). In other bacteria (Neisseria spp. and Haemophilus influenzae), phase variation of LPS is crucial to virulence (37, 65). (ii) H. pylori LPS dis­ plays molecular mimicry with the host (4). Gastric human epithelial cells also express L e ^ blood group antigens. The expression by microorganisms of sur­ face structures similar to those found in the host is called molecular mimicry. Examples of other patho­ gens displaying molecular mimicry are Campylo­ bacter jejuni and Neisseria spp. (33). The role of mim­ icry in pathogenesis can be twofold, (a) H. pylori mimicry is pathogenic. Infection might break toler­ ance to the shared epitopes and induce autoanti­ bodies. Bound antibodies may induce tissue damage, for instance, by fixing complement, (b) Molecular mimicry might provide immune escape by preventing

VANDENBROUCKE-GRAULS

the formation of antibodies directed to the epitopes shared by self and microorganism; the lack of re­ sponse to a surface-located antigen might contribute to persistence of infection, (iii) H. pylori Lewis anti­ gens might interact with host lectins. Several host lec­ tins are known to interact with host Lewis antigens (22, 42); the same lectins may interact with H. pylori Lewis antigens. Such interaction may have biological consequences such as bacterial adhesion, coloniza­ tion, and cytokine induction. In this chapter, we will discuss phase variation of H. pylori LPS, including LPS biosynthesis and ge­ netics; the biological significance of Lewis antigen mimicry; and the role of Lewis antigens in interactions of H. pylori with host lectins.

PHASE VARIATION EST H. PYLORI LPS The structures of LPS isolated from a variety of H. pylori strains have been determined chemically. The overall architecture of H. pylori LPS is similar to that of LPS of other gram-negative pathogens. The lipid A moiety is connected to the oligosaccharide core region that in turn is connected to the O-antigen (or Lewis antigen). In many strains, the O-antigen con­ sists of L e and/or Le (Table 1), but other blood group antigens (H type 1, L e , L e , nonfucosylated polylactosamine [=i-antigen], sialyl Lewis x, blood group A) have also been found (10, 1 1 , 4 6 , 47, 4 9 ) . Strains expressing H type 2 have not been identified. Often, strains express more than one Lewis antigen (Table 2 ) . For example, strain NCTC 1 1 6 3 7 (ATCC 43504) expresses polymeric L e with n up to 8 or 9 that is substituted terminally in nonstoichiometric amounts with Le or H type 1. x

y

a

b

x

y

Ben J . Appelmelk and Christina M. J . E. Vandenbroucke-Grauls • Department of Medical Microbiology, Vrije Universiteit Medical School, van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands.

419

420

APPELMELK AND VANDENBROUCKE-GRAULS

Table 1. Structures of Lewis blood group antigens and glycosyltransferases required for biosynthesis Structure

Antigen

Fucal—2Galpi—3GlcNAc" Fucal—2Galpi—4GlcNAc ' Galfil—4GlcNAc 3 i

Glycosyltransferases required

H type 1 H type 2 Lewis x (Le )

a2FucT, f33GalT a2FucT, fi4GalT a3FucT, f34GalT

Lewis y (Le )

a2FucT, a3FucT, fi4GalT

Lewis a (Le )

a4FucT, (33GalT

Lewis b (Le )

a2FucT, a4FucT, 03GalT

x

T Fucal Fucal-»2Galf31—4GlcNAc 3

y

T Fucal

a

Galf31—3GlcNAc 4 t Fucal Fucal—2Galf31—3GlcNAc 4

b

T Fucal -3(Gair31-.4GlcNAcf31-.) NANA2—3 Galfil—4GlcNAc 3 n

i-antigen Sialyl Lewis x (sLe )

f34GalT a3FucT, p4GalT, sialylT

Blood group k

a2FucT, pGalT, a3GalNAcT

x

T Fucal Fucal—Gaipi—3GlcNAc 3 t GalNAcal

c

" Abbreviations used: Gal, D-galactose; Fuc, L-fucose; GlcNAc, N-acetyl-D-glucosamine; NANA, N-acetylneuraminic ( = sia­ lic) acid; a 2 / 3 / 4 F u c T , a273/4 fucosyltransferase; B3/4GaiT, 0 3 / 4 galactosyltransferase; GalNAcT, N-acetyl-D-galactosaminyltransferase; GlcNAcT, N-acetyl-D-glucosaminyltransferase. For biosynthesis of all blood group antigens shown, an active B3-GIcNAcT is also required. Structural chemistry has failed to detect the presence of H type 2 epitopes in H. pylori LPS. H. pylori and H. mustelae both express blood group A type 1. b

c

Phase Variation

Table 2. LPS phase variants of strains NCTC 11637 and P466 Phase variant lb lc 2b 3a

Serotype

Equivalent strain(s)"

Le'' Le , (Le ) , Le ".. .(Le ) , H type 1 Le% (Le ) Nontypeable i-Ag, H type 1 Le . .(Le ) , H type 1 Le , (Le )„, i-Ag sLe , L e . . .(Le )

M019, 0 6 P466 NCTC 11637* UA948

y

x

fc

n

1

x

n

x

n

Di.r' K4.1 K5.1

e

y

J223 NCTC 11637

x

n

mif

y

sP466*

x

x

y

x

n

Equivalent strains are strains with an O-antigen structure identical to that of a particular N C T C 1 1 6 3 7 LPS phase variant. * L e , (Le )„ designates a strong expression of both Le" and L e (variant l c , strain P466); L e . . .(Le )„ designates a weak expression of L e and a strong expression of L e (variant 2b, strain N C T C 1 1 6 3 7 ) . Variant 2b represents the predominant serotype of strain N C T C 1 1 6 3 7 . D l . l was isolated as an H type 1-negative variant from a K4.1-like variant. "K4.1 was isolated from N C T C 1 1 6 3 7 as an i-Ag-positive phase variant, K5.1 was isolated from K4.1 as an Le -positive variant and expresses a serotype identical to that of strain N C T C 1 1 6 3 7 . ^Variants l b through H l l are derived from N C T C 1 1 6 3 7 . * T h e sLe -negative variant of P 4 6 6 expresses L e , (Le )„.

a

y

x

y

y

x

y

x

c

d

x

x

y

x

Phase variation is defined as the random switch­ ing of LPS phenotype at frequencies that are much higher (sometimes > 1 % ) than classical mutation rates. This process results in reversible loss and gain of certain LPS epitopes and results in a bacterial popu­ lation that is heterogeneous with regard to LPS expression. Phase variation contributes to virulence by generating heterogeneity; certain environmental or host pressures select those bacteria that express the best adapted phenotype. An example is LPS sialylation in Neisseria spp. While nonsialylated bacteria are adherent and invasive, they are sensitive to the lytic action of serum; in contrast, sialylated bacteria adhere less well but are more resistant to serum (65). Phase variation allows outgrowth of nonsialylated bacteria during adhesion or invasion and of bacteria express­ ing sialylated LPS upon contact with serum. Phase variation can be detected by colony-blot­ ting with MAbs specific for LPS (5). An example is given in Fig. 1 where an H. pylori strain was probed

CHAPTER 35 • LIPOPOLYSACCHARIDE LEWIS ANTIGENS

421

sectors observed. By colony-blotting, many LPS phase variants were isolated from a single strain (NCTC 11637) (see Table 2 ) . Subsequently, variants were serotyped in en­ zyme-linked immunosorbent assay and subjected to sodium dodecyl sulfate-polyacrylamide gel electro­ phoresis (SDS-PAGE) and immunoblotting (Fig. 2, Table 2 and 3 ) . The frequency of phase variation is in the range of 0.5 to 1 % , but the frequency of switch­ ing on is not necessarily the same as switching off: the switch frequency of NCTC 1 1 6 3 7 to variant l b is in the 0.5 to 1% range, but the switch-back fre­ quency to parent phenotype is only 0 . 0 7 % (5). Phase variation is not restricted to laboratory strains; it also occurs in other strains including clinical isolates. Figure 1. LPS phase variation in H. pylori LPS. H. pylori strain B1.5 was colony-blotted with an anti-H type 1 MAb and immunostained.

with a MAb specific for H type 1. Three types of colo­ nies are present: first, those that are completely reac­ tive (dark colonies); the bacteria forming this colony originate from a single bacterial cell expressing H type 1, with no switching off to the H type 1-negative phe­ notype occurring during multiplication. Likewise, nonreactive colonies originate from a bacterial cell with a switched-off phenotype. Colonies with a dark sector originate from a cell with a switched-off pheno­ type that switched on during multiplication (often on more than one independent event per colony); clonal outgrowth of a switched-on variant gives rise to the

3 4 5

Molecular Mechanisms of LPS Phase Variation The sequencing of the genome of two H. pylori strains has identified many LPS-related genes, includ­ ing several glycosyltransferases potentially involved in phase variation (1, 12, 5 9 , 6 2 ) . Two similar but not identical a3-fucosyltransferse (a3-fucT) genes have been identified both in strain 26695 (HP0379 and HP0651) and in strain J 9 9 (JHP1002 and JHP0596). Functional studies with the cloned and ex­ pressed gene products show that both FucT enzymes encoded by these two genes are able to form L e from lactosamine acceptors (31, 4 4 ) . However, insertional mutagenesis studies have shown that they differ in fine-specificity (2). The HP0379-encoded a3-FucT has a preference for internal GlcNAc residues (i.e., not located at the nonreducing terminus) and yields x

3 4 5 6

Figure 2. Characterization of LPS phase variants by SDS-PAGE visualized by silver stain (a); immunoblot with anti-Le MAb (b); and anti-Le MAb (c). Lane 1, NCTC 11637; lane 2, variant lb; lane 3, variant lc; lane 4, variant 2b; lane 5, variant 3a. The low molecular weight fraction in (a) represents the core-lipid A moiety. x

y

422

APPELMELK AND VANDENBROUCKE-GRAULS

Table 3 . Reactivity of monoclonal antibodies with H. pylori strains in relation to ct3-fucosyltransferase gene C-tract length Specificity of monoclonal antibody used (MAb code) Strain

Mono-Le (6H3)"

NCTC 11637 K5.1 2b K4.1 lc lb 4187E 4187E-K0651 4187E-K0379 4187E-K0379/651

Poly-Le (54.1F6A)"

x

x

+++ ++ +++ +++ +++ +++ +++ -

C

6

a3-fucT C-tract length

+ + +++ + +++ -

i-antigen

H type 1

—

+++ ++ +++ +++ + ++ +++ + +++

+++ + +++

d

d d

H type 2

Ley

+

+ + + + +++ +++ + + +" + +++ +

Not done Not done

+ +

Not done

-

e

HP0651

HP0379

C9 (off) C9 (off) C9 (off) C9 (off) CIO (on) C9 (off) CIO (on) ND ND ND

CIO (on) CIO (on) CIO (on) C l l (off) CIO (on) CIO (on) CIO (on) ND ND ND

MAbs 6 H 3 and 5 4 . 1 F 6 A recognize monomeric and polymeric L e , respectively. * Strains K 5 . 1 , 2b, K 4 . 1 , l b , and l c are LPS phase variants of strain N C T C 1 1 6 3 7 . " - , OD < 0.3; + , 0.3 < O D < 1.3; + + , 1.3 < O D < 2.3; + + + , O D > 2.3. In titration this MAb reacted 128-fold more with 4 1 8 7 E - K 0 6 5 1 than with strain 4 1 8 7 E and 64-fold less with 4 1 8 7 E - K 0 3 7 9 than with the 4 1 8 7 E parent. ' In titration, MAb Hp 151 (anti-Le'') reacted equally well with strains 4 1 8 7 E and 4 1 8 7 E - K 0 3 7 9 . 0

x

4 9 2

4 9 2

4 9 2

4 i ) 2

d

polymeric L e , while HP0651-encoded a3FucT has a preference for terminal GlcNAc residues and forms mono/oligomeric L e . The HP0379-encoded a3-FucT can also function as an a4-FucT and can therefore also form Le " (3, 5 7 ) . HP0093/94 (JHP0086) is an al-fucT; the gene product is required for biosynthesis of both L e and H type 1 (see below) (3, 67, 6 9 ) . H type 2 epitopes do not occur in H. pylori LPS, and knocking out both a3-fucT genes in a strain that expresses L e ^ yields LPS that expresses i-antigen but no H type 2 (2). Thus, a3-fucosylation precedes a2-fucosylation. This was confirmed in enzyme assays with cloned a2FucT that forms L e from synthetic L e but not H type 2 from GalBl—•4GlcNAc (67). In contrast, this enzyme is able to form H type 1 with a GalBl—>3GlcNac ac­ ceptor. Sequencing of a 2 - and both ct3-fuel genes re­ vealed that they all carry long poly-C stretches close to the 5 ' end of the gene. C-tracts are also present in LPS genes of Neisseria spp. and are a well-character­ ized cause of LPS phase variation (37). On replication, DNA slippage (slipped-strand mispairing) in C-tracts may give rise to daughter DNA that is either one C shorter or longer; this can occur at very high ( 1 % ) frequencies. The result is a high-frequency, reversible frameshifting. The consequence is a rapid on-off switching of enzyme activity. When a C-tract is pres­ ent in the parent strain that to leads to a full-length, active gene product in the C + 1 or C — 1 daughters, the frameshifting will lead either to the production of nonsense polypeptides that have no or little enzyme activity or, due to the occurrence of early stop codons, to a truncated inactive gene product. The molecular basis of phase variation in H. pylori was determined x

x

37

5

y

y

x

by sequencing the C-tracts in the a3-fucT genes of the parent strain (NCTC 11637) and in the phase variants (Table 3) (2). In the N C T C 1 1 6 3 7 HP0651 is "off" due to the presence of a C9 tract; HP0379 is "on" in this strain (CIO). Phase variation from L e to i-Ag and back to L e x

x

In the phase variant expressing i-Ag plus H type 1 (variant K4.1), both HP0651 (C9) and HP0379 ( C l l ) are off; this explains the lack of L e and the biosynthesis of nonfucosylated polylactosamine ( = i antigen) in strain K4.1. In addition, K4.1 expresses H type I due to the presence of an active a2FucT. In strain K5.1, the Le -positive switch-back variant isolated from K4.1, HP0379 is " o n " again (CIO). Thus, phase variation from L e to i-Ag and back to L e can be understood at the molecular level through reversible length changes in the C-tract of a3-fucT gene HP0379, that is, from CIO to C l l and back to CIO. A HP0379/HP0651-double knockout of strain 4 1 8 7 E ( 4 1 8 7 E - K 0 3 7 9 / 6 5 1 ) expresses a serotype identical to that of strain K4.1 (i.e., i-antigen and H type 1). Clinical isolate J233 expresses H type 1 plus i-Ag both as determined by structural chemistry (47) and by serology, and in that strain also both ct3-fucT genes are off. We conclude that LPS serotype is deter­ mined by the on-off status of a3-fucT. x

x

x

x

Phase variation from L e to L e plus L e x

x

y

While strain N C T C 1 1 6 3 7 expresses polymeric L e , H type 1, and a little L e , phase variant l c strongly expresses both L e and Le . C-tract analysis shows that both HP0379 and HP0651 are "on" in x

y

x

y

CHAPTER 35 • LIPOPOLYSACCHARIDE LEWIS ANTIGENS

strain l c . Knockout studies in strain 4 1 8 7 E also showthat the presence of an intact HP0651 is associated with a stronger L e expression and with reactivity with Mab 6H3 that recognizes monomeric L e . We conclude that HP0651 FucT preferentially fucosylates GlcNAc at the nonreducing terminus, thus forming an efficient acceptor for a2-FucT to form Le . In con­ trast, HP0379 a3-FucT would prefer internal GlcNAc, thus forming polymeric L e from the inside out, a structure that is evidently a less efficient accep­ tor. Consequently, as compared to variant l c , less Le is formed in the parent strain. y

x

y

x

y

Phase variation from L e to L e x

back to allow the stronger interaction with AAA. This second mechanism may therefore compensate for + 1 frameshifting due to C-tracts. These two mechanisms operate in the genome strain 2 6 6 9 5 . While this strain expresses Le (46), its al-fucT gene is frameshifted ( + 1 ) due to the C-tract (62) and theoretically would yield an inactive a2FucT. However, presence of the translational - 1 frameshift cassette AAAAAAG causes a - 1 shift in the reading frame, an active en­ zyme to be formed and L e synthesis to take place. The mechanism of — 1 slippage has been well investi­ gated for the dnaX gene of Escherichia coli (29, 63). y

y

Other phase variants

y

Variant H l l expresses L e , Le , but no H type 1; hence, phase variation has to take place in the gene coding for B3-GalT (3). Variant D l . l expresses a truncated LPS and does not react with any anti-Lewis MAb. This variant arose through phase variation from K4.1 through subsequent loss of the elongating GlcNacT (5). An sLe -expressing variant of P466 was isolated and characterized (46); neuB (HP0178), a gene required for biosynthesis of sialyl-Le , contains a C6-tract in strains 26695 and J 9 9 . x

Variant l b has a truncated LPS (Fig. 2) that strongly expresses L e (Table 2); this serotype is simi­ lar to that of strains M O 19 and 0 6 . Enzymatic analy­ sis showed that this variant lacks GlcNAcT activity (5). The serotype of this strain can be explained by the following model. Likely there are two GlcNAcT enzymes, one that recognizes the core and adds the first GlcNAc and a second one that recognizes Gal and thus is responsible for chain elongation. Likely, the lack of GlcNAcT activity in variant l b signifies lack of the second, elongating enzyme. Thus, first the core plus a single GlcNAc is formed in this variant. HP0379 is "on" in variant l b , so that terminal L e is formed; a2-FucT then forms Le . Although GlcNAcT genes have been identified in other species (13), they do not show significant homology with H. pylori open reading frames. y

x

y

Phase variation forming L e

a

Variant 3a expresses polymeric L e plus Le (3). Hence, compared to NCTC 11637, this variant has lost both Le and H type 1. This can be explained by phase variation in al-fucT, and indeed insertional inactivation of this gene in NCTC 11637 yields a mu­ tant with a serotype indistinguishable from that of strain 3a (3). The al-fucT gene also contains a Ctract and hence phase variation occurs along the lines sketched above for a aih-fucT. However, a second mechanism for phase variation was observed in the al-fucT gene, namely a sequence (AAAAAAG) that allows mRNA slippage at the translational level (69). The result of this slippage is a - 1 frameshift. The mechanisms involved are as follows: there are two anticodons for lysine, UUU and CUU. However, from the whole genome sequence it is known that H. pylori codes only for a t R N A with the UUU anticodon while t R N A with the CUU anticodon is missing. Hence, when AAG is encountered in the mRNA of alfucT, the loaded t R N A (UUU) slips one base x

y

Lys

Lys

Lys

423

a

y

x

x

Biological Role of LPS Phase Variation Is phase variation relevant in vivo? We isolated 30

E

//. py/on-specific Tcell

4 o

Activation and proliferation of H. py/o/7-specific T cells Figure 2.

ing of H. pylori to epithelial M H C class II molecules induces apoptosis of epithelial cells (31). However, the role of direct bacterium-epithelium contact-me­ diated apoptosis in atrophic corpus gastritis may be questioned, since colonization usually takes place in the antrum and H. pylori is not invasive, but apoptosis occurs deep in the mucosa.

3

C3

Continued.

The loss of glands in the mucosa, that is, the de­ velopment of gastric atrophy, may thus be caused by an H. py/on'-induced autoimmune T-cell attack against epithelial and parietal cells rather than through autoantibodies. In analogy with EAIG, C D 4 T cells, and not B cells or autoantibodies, are the criti­ cal mediators in Helicobacter-associated gastric pa+

438

BERGMAN ET AL.

thology (21, 5 4 , 6 0 , 7 0 , 71). Consequently, antigas­ tric autoantibodies may only serve as a marker for ongoing antigastric autoimmunity mainly carried out by autoreactive T cells. REFERENCES 1. Alderuccio, F., P. A. Gleeson, S. P. Berzins, M. Martin, I. R. van Driel, and B. H. Toh. 1997. Expression of the gastric H/KATPase alpha-subunit in the thymus may explain the dominant role of the beta-subunit in the pathogenesis of autoimmune gastritis. Autoimmunity 25:167-175. 2. Alderuccio, F., and B. H. Toh. 1998. Spontaneous autoim­ mune gastritis in C3H/He mice: a new mouse model for gastric autoimmunity. Am. J. Pathol. 153:1311-1318. 3. Alderuccio, F., B. H. Toh, P. A. Gleeson, and I. R. van Driel. 1995. A novel method for isolating mononuclear cells from the stomachs of mice with experimental autoimmune gastritis. Autoimmunity 21:215-221. 4. Alderuccio, F., B. H. Toh, S. S. Tan, P. A. Gleeson, and I. R. van Driel. 1993. An autoimmune disease with multiple molec­ ular targets abrogated by the transgenic expression of a single autoantigen in the thymus. /. Exp. Med. 178:419-426. 5. Appelmelk, B. J., G. Faller, D. Claeys, T. Kirchner, and C. M. J. E Vandenbroucke-Grauls. 1998. Bugs on trial: the case of Helicobacter pylori and autoimmunity. Immunol. Today 19: 296-299. 6. Archimandritis, A., S. Sougioultzis, P. G. Foukas, M. Tzivras, P. Davaris, and H. M. Moutsopoulos. 2000. Expression of HLA-DR, costimulatory molecules B7-1, B7-2, intercellular adhesion molecule-1 (ICAM-1) and Fas ligand (FasL) on gas­ tric epithelial cells in Helicobacter pylori gastritis; influence of H. pylori eradication. Clin. Exp. Immunol. 119:464-471. 7. Ardill, J. E., C. J. Larkin, D. Fillmore, and K. D. Buchanan. 1998. Autoantibodies to gastrin in patients with pernicious anaemia: a novel antibody. Q. J. Med. 91:739-742. 8. Bamford, K. B., X. Fan, S. E. Crowe, J. F. Leary, W. K. Gourley, G. K. Luthra, E. G. Brooks, D. Y. Graham, V. E. Reyes, and P. B. Ernst. 1998. Lymphocytes in the human gastric mu­ cosa during Helicobacter pylori have a T helper cell 1 pheno­ type. Gastroenterology 114:482-492. 9. Barrett, S. P., P. A. Gleeson, H. de Silva, B. H. Toh, and I. R. van Driel. 1996. Interferon-gamma is required during the initiation of an organ-specific autoimmune disease. Eur. J. Im­ munol. 26:1652-1655. 10. Barrett, S. P., A. Riordon, B. H. Toh, P. A. Gleeson, and I. R. van Driel. 2000. Homing and adhesion molecules in autoim­ mune gastritis. /. Leukoc. Biol. 67:169-173. 11. Barrett, S. P., B. H. Toh, F. Alderuccio, I. R. van Driel, and P. A. Gleeson. 1995. Organ-specific autoimmunity induced by adult thymectomy and cyclophosphamide-induced lymphope­ nia. Eur. J. Immunol. 25:238-244. 12. Burman, P., O. Kampe, W. Kraaz, L. Loof, A. Smolka, A. Karlsson, and A. Karlsson-Parra. 1992. A study of autoim­ mune gastritis in the postpartum period and at a 5-year followup. Gastroenterology 103:934-942. 13. Callaghan, J. M., M. A. Khan, F. Alderuccio, I. R. van Driel, P. A. Gleeson, and B. H. Toh. 1993. Alpha and beta subunits of the gastric H + /K( + )-ATPase are concordantly targeted by parietal cell autoantibodies associated with autoimmune gas­ tritis. Autoimmunity 16:289-295. 14. Cappell, M. S., and A. Garcia. 1998. Gastric and duodenal ulcers during pregnancy. Gastroenterol. Clin. North Am. 27: 169-195.

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CHAPTER 36 • GASTRIC AUTOIMMUNITY

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Helicobacter pylori: Physiology and Genetics Edited by H . L. T. Mobley, G. L. Mendz, and S. L. Hazell © 2 0 0 1 ASM Press, Washington, D.C.

Chapter 37

Vaccines JACQUES PAPPO, STEVEN CZINN, AND JOHN NEDRUD

T H E I M M U N E RESPONSE AGAINST HELICOBACTER PYLORI: IMPLICATIONS F O R VACCINATION

Gastric Mucosal Immune Response to Infection

uli that profoundly restructure gastric cell composi­ tion and signaling. Early events in H. pylori binding to gastric epithelial cells are mediated by interactions of epithelial cell glycoconjugate and integrin receptors with their cognate H. pylori ligands (83, 148, 149, 152). Adhesion induces translocation of H. pylori protein antigens into epithelial cells by type IV secre­ tion (125) and the focal reorganization of cytoskeletal proteins into membrane pedestals (44). Tyrosine phosphorylation of host proteins (45) and activation of N F - K B transcription factor (90) then promote the production of inflammatory cytokines and chemokines. Accordingly, gastric biopsies from infected sub­ jects exhibit elevated levels of interleukin-lB (IL-1B), IL-6, IL-8, IL-12, tumor necrosis factor alpha (TNFct), growth-related oncogene, monocyte chemotactic protein-1, macrophage inflammatory protein-1 alpha, and regulated-upon-activation T expressed and secreted (RANTES) chemokines (reviewed in refer­ ence 81). The chemical gradients created by these mol­ ecules finely tune the expression of cell adhesion receptor-ligand pairs and favor leukocyte recruitment, accumulation, and activation.

In the absence of H. pylori antigenic stimulation, the stomach appears as a relatively quiescent organ, with little evidence of immunologic activity. Even after oral immunization, which supports the traffick­ ing and migration into mucosal organs of antigenspecific T cells and IgA B cells originating in gut-asso­ ciated lymphoid tissues (GALT), the uninfected stom­ ach is segregated from the continuous entry of lym­ phocytes into mucosal sites ( 1 1 2 , 1 2 8 ) . These findings indicate a paucity of local gastric cytokines and chemokines involved in guiding integrin expression, leuko­ cyte homing, and influx in the absence of H. pyloridriven inflammation. On the other hand, there is sub­ stantial evidence that H. pylori delivers powerful stim­

It is clear that infection activates immune mecha­ nisms that ordinarily lie dormant in gastric tissue. H. pylori upregulates expression of the CD l i b / C D 18 in­ tegrin and its receptor, intercellular-adhesion molecule-1 (ICAM-1; CD54) used for leukocyte transmi­ gration into inflammatory sites ( 2 5 , 7 1 , 73). H. pylori can also increase gastric epithelial cell expression of CD80 and CD86 (164) required for T cell costimulation and upregulates class II major histocompatibility complex (MHC) in vivo (47, 159). Importantly, the class II M H C heterodimer may itself function as a receptor for H. pylori (52). Thus, H. pylori reprograms the stomach to function as a target end organ for leukocyte homing and amplifies the immunologic

The immune response to H. pylori is remarkably di­ verse. Evidence from human and animal studies has shown that the immune system expends substantial energy in response to H. pylori. Yet, the infection is commonly lifelong, and the immune response acti­ vated against this organism does not effect clearance or prevent reinfection after successful antimicrobial treatment (131). The immune functions in cases of spontaneous "clearance" in pediatric populations (92, 109) or in animal models (37) are unknown. However, mathematical (16) and animal (38) infec­ tion models describe a central role for the host re­ sponse in the regulation of H. pylori load throughout the course of the infection, and thus, predict that shifts in the host response can affect the dynamics of this host-microbial equilibrium.

Jacques Pappo • Department of Immunology, Infection Discovery, AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, MA 02451. Steven Czinn • Division of Gastroenterology, Case Western Reserve University, Cleveland, OH 44106. John Nedrud • Department of Pathology, Case Western Reserve University, Cleveland, OH 44106.

441

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PAPPO ET AL.

machinery to buttress local T-cell activation during infection. These observations also raise the possibility that vaccine-specific effectors may accumulate and undergo local activation in the infected stomach. B-cell recruitment and local proliferation The compartments where H. pylori antigens are scanned by the immune system are not completely understood, but these antigens must permeate both mucosal and peripheral lymphoid organs, as evi­ denced by the development of heterogeneous serum IgG and mucosal IgA antibody responses during infec­ tion (24, 9 5 , 1 1 4 ) . H. pylori-infected gastric tissue harbors increased numbers of H. pylori-specific anti­ body-secreting cells (113). The polymeric Ig receptor, involved in transport of IgA dimers into the lumen, is substantially upregulated in infected gastric epithe­ lium and permits concomitant secretion of elevated levels of anti-H. pylori secretory IgA (2, 5 9 , 84). The gastric B cells and their clonal progeny commonly as­ semble into mucosal follicles (147), but these B cells appear unable to convert gastric epithelial cells into the specialized M cell lineage (93) found elsewhere at mucosal surfaces overlying lymphoid aggregates in GALT (127). The chemokine B lymphocyte chemoattractant and its receptor, C X C R 5 , direct B-cell move­ ment and follicle formation (54). This receptor-ligand pair localizes to B-cell aggregates in H. pylori-induced mucosa-associated lymphoid tissue (MALT) lym­ phoma and gastric lymphoma (115) and thus appears to be involved in shaping H. pylori follicular reac­ tions. The size of the B-cell repertoire for H. pylori is not known, but substantially diverse antibody speci­ ficities are used during infection (95), including speci­ ficities directed against epithelial and Ig self-epitopes (67, 155). Commitment and activation of specialized gastric T cells H. pylori infection leads to the progressive accu­ mulation of T cells in the proximal and distal stom­ ach. These T-cell infiltrates are frequently dominated by the C D 4 T helper (Th) cell phenotype (7) and may organize into discrete parafollicular T-cell regions re­ sembling the GALT architecture (59). Gastric resident T cells exhibit upregulated IL-2 receptor expression (82) and recognize H. pylori peptide ligands using var­ ious T-cell receptor (TCR) VB chains (35), although only a relatively small (ca. 2 0 % ) proportion of gastric T-cell clones derived from infected subjects is able to proliferate to H. pylori antigens in a class II MHCrestricted fashion (32). The infected gastric tissue con­ stitutes an enriched source of effector T cells, with +

frequencies of H. pylori-specific mucosal T cells in the range of 1 to 1 0 % compared with a frequency of < 0 . 0 5 % antigen-specific T cells in the peripheral lymphocyte pool (33, 35). Functionally, these gastric T cells display Th-cell function for B-cell proliferation and IgA production (32, 3 3 ) , but also appear to me­ diate CD95 (APO-l/Fas) interactions resulting in apoptosis of B lymphocytes and epithelial target cells (33, 135). Another response to H. pylori infection is charac­ terized by the emergence of gastric intraepithelial lym­ phocytes (glEL) (74). The vast majority of murine glEL derived from infected tissue are T C R o.B CD4 , exhibit a memory/activation phenotype, and display ubiquitous signaling/adhesion molecules CD2 and CD28. These glEL are well equipped for transendothelial migration, as evidenced by the expression of C D l l a , CD44, and lymphocyte-Peyer's patch adhe­ sion molecule 1 (LPAM-1) and LPAM-2 (79). The homing of a B C D 4 into the stomach has been estab­ lished in vivo, and gradual acquisition of CD103, which mediates adhesive interactions with gastric epi­ thelial cell E-cadherin, marks the conversion of a B C D 4 T-cell emigrants into resident glEL postin­ fection (80). +

+

h l

+

+

+

+

Programming by H. pylori for Host Persistence during Natural Infection The type and magnitude of immune responses are governed by cytokines derived from functionally polarized Th-cell subsets. The T h l subset drives cellmediated immunity and inflammatory responses through the production of interferon-y (IFN-y), IL-2, IL-12, IL-18, lymphotoxin a, and TNF-ct, while Th2 cells are associated preferentially with mucosal IgA induction and secrete IL-4, IL-5, IL-6, IL-10, IL-13, and transforming growth factor B. Little is known about the sorting of H. pylori peptides and the strength of T C R signaling throughout the course of the infection. However, there is increasing evidence that H. pylori biases Th-cell differentiation and that the ensuing polarization toward a Thl-type response contributes to H. pylori gastric disease. Live H. pylori differentially stimulates secretion of IL-12 (70), a cytokine produced by antigen-pre­ senting cells (APCs) involved in Thl-cell generation via upregulation of IFN-y production (108). Popula­ tions of gastric T cells analyzed from H. pylori-in­ fected biopsies show an overabundance of IFN-y cells and IFN-y-secreting cells relative to IL-4-producing cells ( 7 , 1 0 5 ) , a profile consistent with a T h l phe­ notype. Further, IFN-y appears to predominate in the stomach even in the absence of H. pylori gastritis (88), raising the possibility that the gastric microenviron+

CHAPTER 37 • VACCINES

merit itself may select for Thl-cell development. The skewing of the Th response is also observed in the clonal progeny of gastric C D 4 T cells isolated from infected stomach tissue, since a majority of the H. py/on'-specific T-cell clones established from infected subjects display a T h l type ( 3 3 , 1 4 6 ) . Recent evidence points to the ability of chemokines and their receptors to control the selective migration of polarized Th sub­ sets (126). The C-C chemokine RANTES, which is upregulated during H. pylori infection (142), me­ diates transendothelial migration of T h l but not Th2 cells (89). Therefore, proinflammatory Thl-type cells predominate in the infected stomach not only because T h l cells are expanded by their encounters with H. pylori, but also because the repertoire of chemokines may exclude Th2-cell influx. +

Immune evasion strategies employed by H. pylori To explain its persistence in human populations, H. pylori has been suggested either to tolerize the host from mounting a protective immune response or to interfere with immune responses that would other­ wise result in its elimination (15). While H. pylori carriage does not induce peripheral tolerance (60), several studies indicate the ability of H. pylori to downregulate T-cell proliferation (97) and IL-15 tran­ scription (106) and to restrict cognate interactions for T-cell activation through perturbation of endocytosis and antigen processing (122). Recent findings show that the survival of H. pylori may also be linked to its capacity to negatively select T cells via induction of apoptosis (156). It is precisely because H. pylori polarizes the type and magnitude of the host response that immunization may be viewed as a means to de­ viate a Thl-dominant response resulting in infection and gastritis to another immunologic state capable of interfering with H. pylori persistence and disease.

PREVENTIVE AND THERAPEUTIC VACCINES Rationale for Vaccination The global H. pylori burden is estimated at 1 0 organisms (23). The rising rate of antimicrobial-resis­ tant strains (65, 116), the emergence of multidrugresistant strains (30), and the development of adverse events to treatment (20) represent important causes of primary treatment failures and pose formidable challenges for the successful treatment of this infec­ tion. Vaccination to prevent infection or to treat an already established infection has surfaced as an in­ creasingly viable approach for the clinical manage­ ment of H. pylori infection. Vaccination should prove 1 6

443

efficacious against drug-resistant strains and may well limit the development of drug-resistant H. pylori. As shown in experimental models (see "Experimental Models of Immunization and Vaccine Efficacy," below), immunization also protects against reinfec­ tion and can interfere with transmission. Although the prevalence of H. pylori infection in the developed world appears to be declining, estimates from a mathematical model indicate that the disappearance of H. pylori in the United States will take in excess of a century (137). Indeed, a recent study designed to evaluate the impact of a vaccine on the cost of H. pylori disease argues for a very favorable cost-benefit ratio (136). However, a salient and compelling argu­ ment for vaccination lies in its ability to modulate the host immune response programmed by infection, and thus, in the potential to avert the development of longterm inflammatory sequelae. There is now direct evi­ dence that the proinflammatory cytokine macrophage migration inhibitory factor inactivates transcription of the p53 tumor suppressor (77) and that immune deviation can lessen the severity of chronic gastritis and limit the evolution of gastric atrophy (56). Fur­ thermore, recent studies have suggested that polymor­ phisms in the IL-1B gene cluster, controlling the mag­ nitude of the host immune and inflammatory responses, may explain clinical outcomes of H. pylori gastroduodenal disease (46). Therefore, vaccination against H. pylori is currently poised as a singularly advantageous strategy for infection management with the potential to prevent the development of gastric adenocarcinoma. Feasibility of Vaccination against H. pylori The first studies on oral immunization with H. pylori antigens (27) were stimulated by observations that IgA responses can protect against mucosal infec­ tions and that H. pylori is a noninvasive pathogen that remains at the mucosal surface. The existence of a surrogate H. pylori model based on murine Helico­ bacter felis infection (100) enabled the rapid expan­ sion of immunization studies for the control of helico­ bacter infection (Table 1). Observations of immune protection from challenge in orally vaccinated mice were generated in short order ( 1 8 , 1 9 , 28). In the pig­ let H. pylori model available at that time, mucosal or parenteral vaccinations were found to diminish the infection density, but systemic vaccination resulted in a more severe neutrophilic and lymphocytic gastritis than that of unimmunized animals (41). Although the nature of the protective immune response was not understood, subsequent experiments using the murine H. felis model firmly established the principle of oral vaccination in the context of a mucosal adjuvant for

444

PAPPO ET AL.

Table 1. Animal models of helicobacter vaccine immunity Animal model Mouse H. felis

Chronic active gastritis Atrophy, metaplasia, and gastric cancer in mutant and transgenic strains Influx and GALT-like assembly of CD4 T cells and IgA B cells Quantitation of infection density by histology and urea hydrolysis assays H. felis lacks cag pathogenicity island Chronic active gastritis and progressive atrophy Long-term persistent infection Gastric erosion and ulcer in some models Host genetic background controls extent of gastric disease and infection density Infection with cagA or cagA strains Quantitation of infection density by bacterial culture Chronic gastritis, multifocal atrophic gastritis, ulcer Natural infection Experimental reinfection Prominent lymphofollicular gastritis Natural infection Experimental infection variable Chronic gastritis, erosion, ulcer +

Mouse H. pylori

Cat H. pylori

Germ-free piglet H. pylori Rhesus monkey H. pylori

Selected references

Mucosal immunization —• long-term (~1 yr) protection from challenge Therapeutic immunization "cures" infection Passive antibody administration protects Protection —> "postimmunization gastritis"

19, 28, 57, 118, 157

Mucosal immunization prevents infection, attenuates established infection, and protects against reinfection Parenteral immunization protects Nontoxic adjuvants and salmonella vectors efficacious Immune protection is antibodyindependent—requires CD4 T cells and class II MHC functions

50, 68, 110, 111, 129

Prophylactic immunization with MDP adjuvant ineffective; promotes mucosal damage Therapeutic mucosal immunization cures 30% of naturally infected ferrets Mucosal immunization protects against challenge

8, 26, 58, 162

Parenteral and mucosal immunizations protect Parenteral immunization —• severe gastritis Mucosal immunization prevents natural transmission Variable effects on H. pylori burden by oral, parenteral, or combined routes of prophylactic immunization Oral therapeutic immunization not protective

41, 99

+

+

Ferret H. mustelae

Vaccination findings

Salient features

-

Chronic active gastritis High incidence of natural infection Persistent experimental infection with some strains

prevention ( 5 3 , 1 0 4 , 1 1 8 , 1 2 8 ) and treatment ( 2 1 , 3 6 ) of the infection. The seminal observations of interfer­ ence with helicobacter infection by immunization rep­ resent the scaffold for human vaccine development aimed at control of H. pylori disease on a global scale. Experimental Models of Immunization and Vaccine Efficacy The recognition that H. felis lacks the cag patho­ genicity island and that it must signal the host in an epithelial pedestal-independent fashion (141) acceler­ ated the development of murine H. pylori models and widened the search of predictive models of human infection and vaccination outcome. In recent years, experimental models of H. pylori vaccine immunity

+

9, 55

39, 102, 103, 145

have been described in mice, felines, germ-free piglets, and nonhuman primates (Table 1). The utilization of murine models, in particular, has confirmed the pro­ tective effect of vaccination against H. pylori infection with adapted cagA or cagA mutant strains of varying colonization densities (96, 1 1 0 , 1 3 0 ) . Not only can immunization protect against challenge with H. py­ lori, but it may also eradicate or substantially dimin­ ish the extent of an established infection and confer protection against rechallenge (61). In ferrets, oral im­ munization can result in cure of natural Helicobacter mustelae infection in approximately one-third of the treated animals (26). Recent findings in gnotobiotic piglets (43) and in cats (9) have provided further cues on the ability of vaccination to limit H. pylori coloni­ zation. Sequential oral immunizations in rhesus mon+

CHAPTER 37 • VACCINES

keys appear to prevent the natural transmission of H. pylori in about 3 0 % of vaccinated animals (39). However, vaccination of rhesus monkey hosts has lit­ tle (102) or no effect (145) in preventing colonization upon deliberate challenge, and treatment of a chronic infection by immunization in this animal model re­ mains unproven (103). A survey of outcomes from vaccine studies in ex­ perimental H. pylori models reveals a gradient of vac­ cine efficacy from murine to nonhuman primates. This therapeutic gradient may reflect the degree of genetic adaptation of H. pylori strains for coloniza­ tion of murine hosts not naturally susceptible to infec­ tion ( 1 0 1 , 1 1 0 ) when compared with H. pylori strains naturally fit for long-term survival in susceptible rhe­ sus monkey hosts (38). On the other hand, vaccina­ tion outcomes ranging from complete immune protec­ tion in murine models (63, 110) to minimal vaccine efficacy in rhesus monkeys (103) could simply be rea­ soned on the basis of our limited knowledge of mu­ cosal immunization of nonhuman primates. Thus, while the proposition of immune intervention for con­ trol of H. pylori infection is supported by the available evidence, to what extent, or indeed which animal model of H. pylori infection is predictive of vaccina­ tion outcomes in humans is presently unknown. A human challenge model (66), while limited to an acute infection, may now enable preventive vaccination trials to be conducted in a human population. What Is Meant by Immune Protection? Effect on Infection Density and Gastritis Important features of animal models of vaccine efficacy are the endpoints that are measured and how these endpoints relate to human bacterial burden and disease. It is now recognized that the vaccination out­ come must be interpreted carefully and that "immune protection" is not necessarily synonymous with the abstract notion of "sterilizing immunity," i.e., abso­ lute blockade or eradication of the infection. For ex­ perimental models involving H. felis, complete pre­ vention or cure of the infection has been judged by quantitation of the bacterial population by using his­ tological methods, urea hydrolysis assays, or both. However, both of these methods appear unable to reliably detect low levels of infection (below 1 0 to 1 0 organisms/g). Indeed, antimicrobial treatment of immunized "protected" mice effects resolution of the underlying gastritis driven by residual organisms (49). In H. pylori models, a common readout of the vacci­ nation outcome is determination of bacterial burden by quantitative culture of hosts challenged with a sin­ gle H. pylori strain within weeks postvaccination. While bacterial culture is considered to be a reprodu­ 4

5

445

cible "gold standard" to measure the presence of via­ ble organisms, it is apparent that characteristics of the host genetic background and the H. pylori challenge strain can specify for a wide range of infection density (29, 3 8 , 1 0 1 , 1 1 0 , 1 5 4 ) . Therefore, the determination of vaccine efficacy in murine models might be more instructive if examined in host strains of various ge­ netic backgrounds challenged with different H. pylori strains. Further, the observation of transient experi­ mental infections (29, 37) raises the possibility that "immune protection" in certain models may represent the accelerated rate of clearance of an otherwise tran­ sient infection. Vaccination can provide a complete barrier to infection in some murine models with modest coloni­ zation efficiency (110, 140), but immune protection when the infection burden is comparable to that of humans (6) ordinarily involves the attenuation of in­ fection density by 1 to 3 log CFU/g of stomach (50, 96, 111, 129). Because the H. pylori density is associ­ ated with the extent of gastritis and epithelial injury (6), the findings of immune protection also point to vaccination as a means to downregulate H. pylorimediated inflammation and support the notion that a histological readout of gastritis should be investi­ gated in vaccine studies. This is particularly germane in view of observations of increased leukocytic influx upon immunization against H. mustelae (162) and H. pylori (41, 103). In murine H. felis models, this phenomenon of "postimmunization gastritis" has been described months postvaccination and is charac­ terized by increased corpus infiltration of IgA B cells and C D 4 and C D 8 T-cell populations (49, 118) of unknown antigen specificity. On the other hand, re­ cent findings from vaccination against H. pylori in nonhuman primates (39) and humans (119) suggest that oral immunization does not power the develop­ ment of gastritis. Further studies are required to fully understand the relationships of animal model, anti­ gen-adjuvant combination, and in particular, vaccine efficacy on the onset of gastritis postimmunization. +

+

+

Targeted Sites for Immune Induction and Antigen Recognition MALT is largely represented by the organized lymphoid tissue in the oropharyngeal, intestinal, and genital tracts. These structures contain discrete cyto­ kine microenvironments that direct mucosal T-cell activation and IgA B-cell differentiation upon appro­ priate antigenic stimulation. Early studies on immunization suggested that immune protection re­ quires the genesis of effectors from GALT (18, 19, 28). In more recent studies, it has been further shown that vaccine delivery to oral/buccal, nasal, Peyer's

446

PAPPO ET AL.

patch, and rectal tissues confers immune protection from infection (40, 9 6 ) . During mucosal immuniza­ tion, IgA B cells and C D 4 T cells are recruited and expanded in the gastric mucosa (49, 6 3 , 1 2 8 ) . Gastric localization of vaccine-specific leukocytes requires the upregulation of homing receptor-ligand pairs signaled by challenge with live organisms or by the presence of an underlying infection (see "Gastric Mucosal Im­ mune Response to Infection," above). However, it has not been conclusively established that the gastric resi­ dent leukocytes generated by vaccination are MALT emigrants. Indeed, recent phenotypic studies of gastric a 4 T C R a B cells after oral immunization point to the peripheral lymphoid pool as a contributor of gas­ tric vaccine effectors (129) and support findings of parenteral immunization, especially at subcutaneous sites that target the lymph nodes draining the stom­ ach, as an additional route that promotes immune protection (69) and accretion of IgA cells and T cells in gastric tissue (50). +

+

+

+

+

Antigens, Adjuvants, and Delivery Systems for H. pylori Vaccines The initial vaccine studies employed bacterial ly­ sates delivered orally with cholera toxin (CT) as a prototype mucosal adjuvant (reviewed in references 11, 5 1 ) . However, the complex nature of whole-cell vaccines and their potential for eliciting undesirable immune reactions have favored the use of purified recombinant antigens for vaccine development. Sev­ eral approaches have guided the identification of H. pylori vaccine antigens, including in silico prediction from genomic analyses, comparison of antigenic pat­ terns using proteomics or libraries of H. pylori geno­ mic DNA, and theoretical and experimental analyses of putative virulence factors. Genomic analyses have revealed five paralogous gene families of outer membrane proteins represented in both sequenced H. pylori strains. These gene fami­ lies comprise from 3 to 33 members, display a C-ter­ minal hydrophobic motif, and consist of potential vaccine candidates with porin function and adhesive properties (3). The genomics approach has also re­ vealed that candidate vaccine proteins may be suscep­ tible to antigenic diversification, since expression of these antigens may be switched on and off by a slipped-strand mispairing repair mechanism, as found in the fucosyltransferase genes encoding the enzyme required for Lewis X and Y side chain addition on lipopolysaccharide (5). By proteomic analyses, the screening of sera from infected subjects has resulted in the identification of about 30 immunodominant H. pylori antigens, including the neutrophil-activating protein HP-NAP, flagellar and heat shock proteins,

the urease B subunit, and elongation factors (95). Screening of sera from vaccinated mice against an H. pylori expression library has likewise identified ure­ ase, the heat shock protein HspB, putative membrane proteins, and the lipoprotein Lpp20 (75). In vitro as­ says with antibody-secreting cells (113) or with T-cell clones (32) derived from infected subjects have further identified membrane proteins, the hemagglutinin HpaA, and CagA and VacA as potential candidates. Of these, the enzymes urease and catalase, the UreB subunit of urease, the heat shock proteins HspA and HspB, the leukocyte-activating HP-NAP, the lipopro­ tein Lpp20, and the CagA and VacA antigens have been shown to be protective in infection models (21, 5 3 , 9 1 , 104, 1 1 0 , 1 1 8 , 1 2 8 , 130, 1 3 9 ) . Virtually all of the vaccine studies reporting effi­ cacy have shown a strict adjuvant requirement. The mucosal adjuvants CT and the closely related E. coli heat-labile toxin (LT) are A B molecules with superior capacity for driving Th2 differentiation and IgA pro­ duction when delivered to MALT (107). Multiple im­ munizations in the absence of C T adjuvant, even at high antigen doses, generate appreciable levels of serum IgG and mucosal IgA antibody but fail to me­ diate immune protection (161). The toxicity of CT and LT molecules in humans has stimulated the inves­ tigation of alternative adjuvants and delivery systems for H. pylori vaccination. Dissection of the A B toxin adjuvant activity has shown that genetic detoxifica­ tion of LT via site-directed replacement of serine to lysine at position 63 is consistent with vaccine efficacy (61, 111), while immunization with the recombinant CTB or LTB subunits is not (13, 160). Immunization with the orally active adjuvant muramyl dipeptide has no measurable effect on antibody induction or H. mustelae infection, but instead appears to lead to mu­ cosal damage (162). Oral delivery of antigen encapsu­ lated in poly (D-L-lactide-coglycolide) microspheres, aimed at augmenting M cell-dependent uptake (48), enhances the anti-H. pylori antibody responses (94), but the value of this strategy in protection against infection has not been established. The construction of additional mutant L T molecules (reviewed in refer­ ence 132), as well as the design of novel CTA-based fusion protein adjuvants (1) and nanoparticulate de­ livery systems with IL-12-dependent adjuvant activity (144), should further encourage work into the search for clinically viable mucosal adjuvants for H. pylori vaccines. Several adjuvants exhibit efficacy in parenteral immunization protocols. Alum, complete Freund's adjuvant, and incomplete Freund's adjuvant effect im­ mune protection of varying magnitude when delivered subcutaneously or intraperitoneally with H. pylori antigens ( 4 1 , 5 0 , 6 4 , 1 6 3 ) , although repeated intra5

5

CHAPTER 37 • VACCINES

muscular injections of a conventional alum-based vac­ cine appear ineffective (102). Immunization by the subcutaneous route with the saponin adjuvant QS21 is protective against challenge, and highly efficacious when used therapeutically in a murine model (68, 69). Recent studies have shown subcutaneous immuniza­ tion with the LT adjuvant, or with combinations of LT and LTB, to have equivalent protective activity to oral vaccination with LT (160). An alternative approach to the use of mucosal adjuvants for recombinant subunit vaccines involves the use of live vectors. A replication-defective adeno­ virus vector delivered by intramuscular injection re­ duces the extent of H. felis infection (85). Injection of poliovirus replicons encoding the urease B subunit generates specific IgG2a antibody and IFN-y re­ sponses (124), but its effect on protection is currently unknown. Oral or intranasal immunization with a live attenuated Salmonella enterica serovar Typhimurium-vectored vaccine is also efficacious in controlling the extent of H. pylori colonization (22, 62). Whether employing mucosal, parenteral, or com­ bined immunization routes, vaccine studies accompa­ nied by a histopathology readout of the target end organ should reveal whether a particular antigen-ad­ juvant pair has the instrinsic ability to further drive the proinflammatory T h l response committed during infection. Studies using C D 4 T cells and mucosal APC (70, 72) have the potential to guide the selection of antigens, adjuvants, and delivery systems as a func­ tion of their effects on Th-cell differentiation and may ultimately aid in the rational design of H. pylori vac­ cines. +

MECHANISMS OF I M M U N E P R O T E C T I O N B Cells and Antibody Induction In murine H. pylori infection models, the gastric B-cell pool is in the order of 1 to 2 X 1 0 IgA B cells/ stomach (29); this population of IgA cells harbors a very high frequency (ca. 1 0 % ) of specific antibodycontaining cells that cluster in gastric mucosa after immunization (96). Local salivary gland immunity ap­ pears to contribute substantially to the overall IgA antibody level during oral immunization (143), and administration of high concentrations of IgA mono­ clonal antibody can protect from challenge (28). In humans, IgA antibody may delay the onset of H. py­ lori infection in some populations (153), and its fine specificity can be shown to differ after immunization and during spontaneous clearance (14). On the other hand, IgA deficiency does not increase the susceptibil­ ity to infection (17), and B-cell deficiency in |xMT 6

+

447

mice has little effect on the severity of the infection (12) and, in fact, may be compatible with spontaneous clearance (134). While a frequent outcome of protec­ tive immunization against H. pylori is the generation of elevated serum IgG and mucosal IgA antibody re­ sponses (19, 2 8 , 104, 128), careful analysis reveals that the relationship between IgA antibody level and the extent of immune protection is discordant (96, 161). Indeed, recent studies in B-cell-deficient u.MT mice now indicate that both prophylactic and thera­ peutic vaccinations mediate immune protection in a B-cell- and antibody-independent fashion (14, 5 0 , 151). Gastric T Cells and the Th-Cell Theorem If effector mucosal IgA responses are not central to immune protection, what then mediates H. pylori vaccine immunity? The evidence has suggested, and more recently established, the involvement of T cells in vaccine-directed protection. Approximately 1 0 C D 4 T cells/stomach are generated during experi­ mental infection of mice (29), and this C D 4 popula­ tion is substantially expanded during protective vacci­ nation ( 4 9 , 9 6 ) . The mucosal adjuvant CT, frequently employed in protective vaccination trials, requires C D 4 T cells for demonstrable adjuvant activity (76). Direct evidence from studies in M H C gene knockout mice shows that T cells regulate the infection burden (129) and that vaccine efficacy is strictly dependent on the genesis of activated C D 4 T cells (129) and governed by intact class II M H C function (50, 129). Furthermore, adoptive transfer of Th cells from vacci­ nated donors into unimmunized recipient mice re­ duces the severity of the infection (64, 87, 121) (Fig. 1). The frequency of antigen-specific T cells has not been estimated, but the homing into gastric tissue of about 2 X 1 0 T C R a B C D 4 cells after adoptive transfer is sufficient to confer immune protection (87). Interestingly, adoptive transfer of Th-cell effec­ tors circumvents CD4 deficiency and protects from challenge, but adoptive transfer into class II M H C gene knockout mice does not, even though transferred C D 4 T cells can be shown to populate the stomach (Fig. 1). These observations suggest that C D 4 T-cell effectors must experience interactions between the TCR and the MHC to result in persistence and expres­ sion of their Th-cell program. 6

+

+

+

+

4

+

+

+

+

The polarization of Th-cell effectors during anti­ genic stimulation constitutes an important determi­ nant of immune protection. In vaccine studies using CT adjuvant, sequential oral immunizations give rise to a progressive increase in IL-4 and downregulation of IFN-y production (138). Oral immunization may also increase IFN-y expression (22, 6 3 ) , but immune

448

PAPPO ET AL.

Adoptive Transfer

Mouse strain

Infection density Protection

Frequency of af?CD4 +

o C 5 7 B L / 6 wild-type

62

2.08

C 5 7 B L / 6 wild-type

724

0.19

p=0.0003

C 5 7 B L / 6 TacfBR-[KO]A p N 5 MHC Class I H -

245

1.42

p=0.50

C57BL/6-Cd4""'N7 CD4'-

490

0.09

p=0.0014

b

Figure 1. Vaccination with H. pylori. Groups of C57BL/6 wild-type donor mice were immunized intranasally with 100 |xg H. pylori lysate antigen and 10 (xg CT adjuvant, or with CT adjuvant alone. H. pylori immunization protected from infection relative to treatment with CT alone (2.9 X 10 vs. 2.4 X 10 CFU/biopsy, respectively; p < 0.0001 by Wilcoxon rank sum analysis). Spleen Th-cell effectors were isolated from protected donor mice and expanded in vitro with H. pylori lysate, IL2, and IL-4. H. py/on'-specific CD4 T cells were then magnetically sorted and adoptively transferred (10 ) by intravenous injection into groups of C57BL/6, MHC class I I ~ , or CD4"'~ recipient mice. The mice were subsequently challenged with H. pylori, and immune protection was assessed 2 weeks later by quantitative bacterial culture. Micrographs show representative cross sections (7 |xm) of stomach from the indicated strains. T cells were stained with anti-TCRfi and anti-CD4, and the mean frequency per mm of stomach was derived from serial sections. The circles enclose TCRc

TNFa, IFNY,IL1,IL6,1L8

1

Mutations in Key Genes Recruitment of Inflammatory Cells

Direct Effects of H. pylori

Environmental and Genetic Co-factors

Impaired

Inhibition of cell proliferation

- • DNA Repair

Reactive Oxygen Species

Compensatory proliferation of stem cell pool

Damage to Epithelial Cells

Figure 2. Molecular pathways linking H. pylori and gastric carcinogenesis.

Inflammation-Related Mechanisms of H. pyloriAssociated Gastric Carcinogenesis H. pylori infection of the gastric mucosa induces an inflammatory response by the host that consists of infiltration of the mucosa by polymorphonuclear leukocytes as well as by macrophages and T and B lymphocytes. Both H. pylori and cytokines induced during infection can stimulate the recruitment and ac­ tivation of inflammatory cells. When activated, in­ flammatory cells produce chemical mediators that in­ clude reactive oxygen species (ROS). Reactive oxygen intermediates in turn can up-regulate interleukin-8 (IL-8), further promoting the inflammatory response stimulus (143). Intermediate ROS are partly responsi­ ble for an increased oxidative stress status of gastric epithelial cells, which may be potentiated by de­ creased antioxidant levels associated with H. pylori infection, as reflected by lower concentrations of vita­ min C in the gastric juice during H. pylori infection (23). ROS can induce DNA damage with the accumu­ lation of DNA mutations, contributing to the patho­ genesis of gastric cancer (54). In addition, epithelial cell turnover is affected by the inflammatory response to H. pylori. This notion is supported by studies de­ scribing an increase in both epithelial cell prolifera­ tion and programmed cell death (apoptosis) in re­ sponse to H. pylori infection. Apoptosis is a regulated process of cell death that is triggered by H. pylori as well as by various inflammatory mediators, including

tumor necrosis factor (TNF) and interferon-gamma (IFN-y). Exposure to H. pylori-activated peripheral blood mononuclear cells (PBMCs), but not H. pylori itself, induced Fas antigen expression in RGM-1 gas­ tric epithelial cells, indicating a Fas-regulatory role for inflammatory cytokines in this system. When exposed to Fas ligand, RGM-1 cells treated with PBMC-conditioned medium underwent massive and rapid cell death associated with increased proliferation (69). All these changes can contribute to clonal expansion of epithelial cells that suffered mutational events and to the development of gastric cancer (25, 3 2 , 3 3 , 142).

Direct Toxic Effects of H. pylori on Gastric Epithelial Cells Studies in vivo and in vitro have demonstrated a number of toxic effects of H. pylori on gastric epithe­ lial cells that may trigger cellular apoptosis and com­ pensatory proliferation in vivo, providing a link to the effect of direct toxicity by bacterial products and carcinogenesis (173). Exposure of human gastric epi­ thelial cells to high concentrations of H. pylori super­ natant caused lethal cell injury and increased sensitiv­ ity of AGS cells to injury by superoxide (148). H. pylori can weaken the mucous component of the gas­ tric mucosal barrier and impair the secretory function of mucous cells. Lipase, phospholipase A2, and pro­ tease of H. pylori were shown to cause a rapid degra­ dation of mucus glycoprotein polymer to glycopeptides, resulting in gradual loss of mucus viscosity and

CHAPTER 40 • GASTRIC CANCER

487

increased permeability of mucus to H (135, 147). These enzymes were also shown to inhibit mucus se­ cretion from a mucus-secreting human cell line, result­ ing in decreased cytoprotection (108). Further, phos­ pholipase A2 of H. pylori has been shown to damage epithelial cell membranes by disrupting the protective phospholipid layer at the apical surface of mucous cells (12, 106, 119, 170). The H. pylori cytotoxin VacA induces the forma­ tion of vacuoles related to the late endosomal/lysosomal compartment in primary gastric epithelial cells in culture (59, 148). A cellular protein, VIP54, was recently described by its ability to bind H. pylori VacA (27). VIP54 is a 54-kDa protein with a cellular distri­ bution similar to that of intermediate filaments and might be involved in interactions between intermedi­ ate filaments and late endosomal compartments (27).

vated expression of Fas antigen in mucosal cells con­ current with the presence of Fas ligand-expressing lymphocytes. Furthermore, H. pylori stimulates apoptosis of gastric epithelial cells in vitro in associa­ tion with the enhanced expression of Fas receptor (76, 132) and activation of caspase-3 (85). Additionally, H. pylori induces apoptosis in the gastric epithelium, through up-regulation of proapoptotic Bax and down-regulation of antiapoptotic Bcl-2 proteins. This phenomenon was observed in H. py/on'-infected pa­ tients with duodenal ulcer and in Kato III gastric can­ cer cells, indicating a direct apoptotic effect of H. py­ lori on mucosal cells (89). Recent studies in the Mongolian gerbil model of H. pylori infection have shown that in H. py/on'-infected gerbils antral apoptosis is seen early during infection and is fol­ lowed later by increased cell proliferation (123), which correlates well with human studies (75).

H. pylori Affects the Cellular ApoptosisProliferation Balance

An antiproliferative activity of H. pylori was shown to affect the proliferation of various mamma­ lian cell lines (U937, Jurkat, AGS, Kato III, HEP-2, and P388D1), and this effect was associated with di­ minished protein synthesis. The responsible H. pylori factor might be a protein of 100 kDa (PIP, for prolifer­ ation-inhibiting protein) (87, 149). Ammonia, which is a cytotoxic factor generated by H. pylori, is in­ volved in gastric mucosal injury and inhibited the pro­ liferation of HGC-27 cells in a dose-dependent man­ ner. Flow-cytometric analysis showed S-phase accumulation of HGC-27 cells, suggesting that am­ monia inhibits the growth of gastric cells in S phase. This mechanism could make a significant contribu­ tion to the pathogenesis of H. pylori-associated gastric mucosal atrophy, a known risk factor of gastric can­ cer (105). In summary, H. pylori bacterial products as well as inflammation-related products have been shown to perturb cell proliferation and increase apoptosis in gastric epithelial cells. In vivo, these effects result in increased cellular turnover and compensatory prolif­ eration of surviving epithelial cells ( 3 8 , 1 2 2 , 1 3 8 ) . Ac­ cordingly, H. pylori eradication results in decreased cell proliferation and apoptosis (68). The increased cell turnover resulting from H. pylori infection liter­ ally exposes the epithelium to a greater risk of expan­ sion of cells that have incurred mutations, in this man­ ner promoting cancer development.

+

The maintenance of gastric mucosal integrity de­ pends on the balance between cell loss due to pro­ grammed cell death (apoptosis) and cell proliferation ( 1 2 2 , 1 6 7 ) . In the uninfected stomach, apoptotic cells are rare and superficial, but during H. pylori infec­ tion, apoptotic cells are more numerous and located throughout the depth of gastric glands (110). The apoptotic index is higher in specimens from patients with H. pylori gastritis than in noninflamed controls, and apoptosis decreases following H. pylori eradica­ tion and resolution of gastritis (77). H. pylori strains carrying the cag pathogenicity island in general ex­ press CagA. Infection with CagA-positive strains re­ sulted in increased gastric cell proliferation as com­ pared to CagA-negative strains, because CagApositive strains induced a lesser degree of apoptosis. This finding might explain the increased risk for gas­ tric carcinoma that has been reported in some studies to be associated with infection by CagA-positive H. pylori strains (130, 149). The precise mechanism of increased apoptosis with H. pylori infection is not known, but a number of studies have begun to provide evidence for potential mechanisms. H. pylori lipopolysaccharide (LPS) may be a virulence factor responsible for the induction of gastric epithelial cell apoptosis. LPS caused gastric mucosal responses typical of acute gastritis and marked epithelial apoptosis in rats (125). Inoculation of the H. pylori Sydney strain (SSI) in C57BL/6 mice induced caspase-3 activation followed by DNA frag­ mentation, which are hallmarks of apoptosis (155). Normal gastric and small bowel tissues express low levels of Fas antigen and nondetectable levels of Fas ligand. Consequent to H. pylori infection, there is ele­

H. pylori Causes Imbalance of Transduction Pathways and Affects Gene Transcription in Gastric Epithelial Cells Attachment of H. pylori to gastric cells results in pedestal formation and cytoskeleton rearrangement similar to that described for enteropathogenic Esche-

488

ASAKA ET AL.

richia coli. H. pylori cell adherence was shown to in­ duce tyrosine phosphorylation of two proteins of 145 and 105 kDa in gastric epithelial cells (139, 140). There is now evidence that the 145-kDa protein corre­ sponds to H. pylori CagA (3, 152). CagA-positive H. pylori strains translocate the bacterial protein CagA into gastric epithelial cells by a type IV secretion sys­ tem, encoded by the cag pathogenicity island. CagA is tyrosine phosphorylated and induces changes in the tyrosine phosphorylated state of distinct cellular pro­ teins (118). H. pylori infection is associated with the induc­ tion of several cytokines, including IL-8 (25, 105, 175). N F - K B regulates a variety of genes involved in cell growth and immune response and is a transcrip­ tional regulator of IL-8 production. N F - K B activation after bacterial infection may be an important defense mechanism or part of the response of gastrointestinal epithelial cells to infection. Infection with H. pylori was shown to activate N F - K B directly in gastric epi­ thelial cells in vitro and in vivo and to induce nuclear translocation of both N F - K B p50/p65 heterodimers and p50 homodimers. Nuclear translocation of N F KB is followed by increased IL-8 mRNA and protein levels, consistent with N F - K B up-regulation of IL-8 gene transcription (81). Activation of IL-8 through N F - K B appears to occur through a sphingomyelin-ceramide pathway (104). Infection of AGS cells with an H. pylori Cag-positive strain rapidly induced a dosedependent activation of extracellular signal-regulated kinases (ERK), p38, and c-Jun N-terminal kinase (JNK) MAP kinases (82). Another study showed that exposure of gastric epithelial cells to H. pylori induces activation of the transcription factors c-fos, c-jun, and AP-1. This ef­ fect appears to occur through activation of the ERK/ MAP kinase cascade, resulting in Elk-1 phosphoryla­ tion and increased c-fos transcription (107). Since MAP kinases regulate cell proliferation, differentia­ tion programmed death, stress, and inflammatory re­ sponses, activation of gastric epithelial cell MAP ki­ nases by H. pylori may play a role in inducing gastroduodenal inflammation and carcinoma (82). H. pylori infection can alter the expression of many other epithelial cell genes. Increased expression of amphiregulin and heparin-binding epidermal growth factor­ like growth has been shown to be mediated by an H. pylori factor greater than 12 kDa in size (131). The availability of high-throughput screening using microarray gene expression analysis is expected to pro­ vide an abundance of information at a fast pace. H. pylori Infection, Oxidative Stress, and the Accumulation of Genetic Mutations Oxidative injury has been implicated in various diseases associated with chronic inflammation, such

as H. pylori infection. Apoptosis and oxidative stress are closely interrelated and may play a determinant role in the evolution of chronic gastritis to gastric car­ cinogenesis. Exposure of gastric epithelial cells to H. pylori resulted in the generation of ROS (116). More­ over, addition of either TNF-a or IFN-y for 2 4 h re­ sulted in enhanced ROS production in response to bacteria or H 0 . DNA 8-hydroxydeoxyguanosine (80HdG) is a sensitive marker for oxidative DNA damage. Concentrations of 8HdG were detected at significantly higher frequency in chronic atrophic gas­ tritis, in the presence of severe disease activity, intes­ tinal metaplasia, and H. pylori infection (39). After eradication of H. pylori, 80HdG contents were signif­ icantly decreased (55). Patients infected with H. pylori expressed more inducible nitric oxide synthase (iNOS), and higher levels of iNOS were caused by infection with CagA-positive H. pylori strains (101). Increased levels of iNOS and cyclooxygenase (COX2) were demonstrated in H. pylori-associated gastritis (44). Both nitric oxide and COX-2 products have been shown to have mutagenic potential, possibly linking these molecular alterations seen with chronic gastritis with increased risk of gastric carcinoma de­ velopment (54, 126). Although many studies using surrogate markers for biological mutations have been done, much less is known about the specific gene mutations that occur critically in the early stages of gastric carcinogenesis. Mutation of the TP53 gene has been described in a small number of cases of premalignant gastric mucosa with intestinal metaplasia (73, 141). K-ras mutations were found in 1 4 . 4 % of all baseline biopsies from atrophic gastritis patients, and an association was found between the presence of K-ras mutations in baseline biopsies and the progression of preneoplastic lesions. Among those patients with atrophic gastritis without metaplasia, 1 9 . 4 % (6 of 25) contained K-ras mutations, suggesting that mutation of this gene is an early event in the etiology of gastric carcinoma (49). 2

2

H. py/on-Associated Impairment in DNA Mismatch Repair Mutation surveillance and repair are carried out by the DNA repair system. DNA mismatch repair (MMR) corrects mutations that occur during cell rep­ lication (88). With increased cell turnover of the gas­ tric mucosa during active H. pylori infection, an in­ creased load of mutations may occur as a consequence of the infection and other environmental risk factors. Under these conditions, a situation where DNA repair might be overwhelmed may develop. Microsatellite instability (MSI) is a marker of mutations that develop subsequent to deficient DNA M M R activity. The

CHAPTER 40 • GASTRIC CANCER

DNA M M R genes hMSH3 and hMSH6 and growth factor receptors and transforming growth factor B-RII are frequently mutated in MSI-positive gastric cancers (16, 1 1 1 , 174). The expression of the M M R genes h M L H l and rarely hMSH2 is usually abolished in MSI-positive gastric cancers (41, 5 6 , 99). MSI can result in mutations in the coding regions of other critical genes involved in regulation of cellu­ lar proliferation and differentiation. Recent studies found that patients with MSI-positive tumors showed a significantly higher frequency of previous H. pylori infection (171), and patients with MSI-positive tu­ mors were more likely to have active H. pylori infec­ tion (100). Using a coculture in vitro system, gastric cancer cell lines exposed to H. pylori showed de­ creased levels of both MutS (hMSH2 and hMSH6) and MutL ( h M L H l , hPMS2, and hPMSl) DNA M M R proteins (84). Additionally, H. pylori caused MSI in longer-term cocultures. These data suggest that deficient M M R caused by H. pylori underlies MSI in the gastric epithelium, providing a mechanism of mutation accumulation in the gastric mucosa dur­ ing early stages of H. pylori-associated gastric carci­ nogenesis. H. pylori-Associated Changes in Epithelial Adhesion Molecules Proteins involved in epithelial adhesion are essen­ tial for maintenance of tissue structure and have a prognostic importance in gastric cancer. E-cadherin is essential for maintaining cell adhesion, as well as for differentiation, and it is thought to act as a sup­ pressor of epithelial tumor cell invasiveness and me­ tastasis. A study by Terres et al. reported that H. py­ lori infection was significantly associated with downregulation of E-cadherin (158). However, Blok et al. found no significant differences in E-cadherin expres­ sion between H. pylori-positive and H. pylori-nega­ tive early gastric carcinoma patients (15).

DEVELOPMENT OF GASTRIC CANCER IN H. PYLORZ-INFECTED ANIMALS Gastric carcinogenesis is multifactorial and some environmental factors are believed to be involved in this process (19), including excessive intake of salt (21), N-nitroso compounds in foods, and low con­ sumption of fresh fruits and vegetables. H. pylori has been shown to be associated with an increased risk of both intestinal type and diffuse type gastric cancers (97), which correspond, respectively, with well-differ­ entiated and poorly differentiated types in the Japa­ nese classification (6, 3 4 , 57, 7 0 , 97, 115, 121).

489

Recently, evidence that H. pylori infection may induce gastric adenocarcinomas in animal models has accumulated ( 6 2 , 6 7 , 1 4 5 , 1 5 3 , 1 5 9 , 1 6 9 ) . Mongolian gerbils with long-term infection with H. pylori devel­ oped gastric cancer, with treatment with low-dose chemical carcinogens, N-methyl-N-nitrosourea (MNU) or MNNG, or without them. The Mongolian gerbil model resembles the human situation in its suscepti­ bility and response to H. pylori infection (63), since the bacterium can induce chronic active gastritis, gas­ tric ulceration, and duodenal ulceration. Therefore, the Mongolian gerbil model has an advantage for in­ vestigating gastric carcinogenesis developed as a result of H. pylori infection in humans. In this section the development of gastric cancer in Mongolian gerbil models with H. pylori infection and the relevance of this animal model will be discussed. Development of Animal Models of H. pylori Infection The IARC/WHO conclusions were based pre­ dominantly on several epidemiologic studies in hu­ mans. To generate direct evidence of the causal rela­ tionship between H. pylori infection and occurrence of gastric cancer, we need to conduct clinical interven­ tion studies in which eradication of H. pylori can re­ duce the occurrence of gastric cancer in humans. Al­ ternatively, we can observe the development of gastric cancer in animal models of H. pylori infection. In the first decade after the discovery of this or­ ganism, an inoculation of H. pylori was reported to induce gastritis in beagle dogs, Japanese monkeys, miniature gnotobiotic pigs, mice, and Mongolian ger­ bils (63, 79, 80, 9 0 , 128, 146, 168). These animal models indicated that H. pylori infection could induce histologic gastritis, which was characterized by nu­ merous infiltrations of inflammatory cells, epithelial erosion, and degeneration. Fujioka et al. infected Japanese monkeys with H. pylori and then followed the animals for a prolonged period, detecting the oc­ currence of gastric mucosal atrophy 1.5 years after inoculation (45). However, these animals are too big to use for cancer experiments, and there are still no reports on the development of gastric cancer in these animal models. Although a mouse model resembling human H. pylori chronic gastritis is available, a specialized H. pylori strain, SSI, or Helicobacter felis was used, with gastric carcinoma not being detected (98). Hirayama et al. (63) reported first in 1996 that H. pylori could induce gastritis, gastric ulceration, and intestinal metaplasia during long-term infection in Mongolian gerbils models. In this model, H. pylori could colonize the stomach and induce gastritis 12 weeks after inocu-

490

ASAKA ET AL.

lation and induce gastric ulceration at 2 4 weeks and intestinal metaplasia at 2 4 to 48 weeks. These histo­ logic characteristics, infiltration of numerous neutro­ phils and lymphocytes, with defects in the gastric mu­ cosal tissue reaching the muscular layer, and occurrence of intestinal metaplasia resembled that of human H. pylori infection. After this report, Mongo­ lian gerbil models began to be used in experiments in the study of gastric carcinogenesis by H. pylori infec­ tion in Japan. In 1998, three papers on the development of gas­ tric cancer using Mongolian gerbil models were pub­ lished from Japan and provided experimental data supporting the role of H. pylori infection in the occur­ rence of gastric cancer. Sugiyama et al. (153) first demonstrated that H. pylori could increase the inci­ dence of MNU-induced gastric cancer in the Mongo­ lian gerbil animal model. A total of 170 male Mongo­ lian gerbils were used in the study. Seven of 19 Mongolian gerbils ( 3 6 . 8 % ) , which were inoculated with the ATCC 4 3 5 0 4 type strain of H. pylori first and then treated with 10 ppm of MNU for 2 0 weeks, developed gastric adenocarcinoma 4 0 weeks after the study commenced. Five of seven cancers (71.4%) were signet ring-cell carcinomas, one was a poorly differentiated adenocarcinoma, and one was a welldifferentiated adenocarcinoma. In cases of treatment with 30 ppm of M N U for 6 weeks first and then inoc­ ulation of H. pylori, 6 of 18 Mongolian gerbils (33.3%) developed gastric adenocarcinomas at 4 0 weeks. Four of six cancers (66.6%) were well-differ­ entiated adenocarcinomas, one was signet ring-cell carcinoma, and one was poorly differentiated adeno­ carcinoma. These observations may suggest that the time of inoculation of H. pylori, the dose of chemical carcinogens, and the order of inoculation of H. pylori and administration of chemical carcinogens are criti­ cal to determine the histological types of gastric can­ cers. This hypothesis, however, has not been con­ firmed in all studies using the Mongolian gerbil model (144). In one study, 2 0 Mongolian gerbils infected with H. pylori ATCC 4 3 5 0 4 alone, 18 Mongolian gerbils treated with 10 ppm of MNU alone for 20 weeks, or 18 Mongolian gerbils treated with 30 ppm of MNU alone for 6 weeks did not develop gastric cancer at all. These findings suggest that while H. py­ lori may induce gastric cancer in Mongolian gerbils, infection plus administration of very low-dose chemi­ cal carcinogens may be needed. In contrast, Watanabe et al. (169) reported that long-term infection with H. pylori alone could induce gastric adenocarcinoma in the Mongolian gerbil model 62 weeks after inoculation. They demonstrated that 10 of 2 7 H. py/on-infected Mongolian gerbils ( 3 7 % ) developed gastric cancers, all of which were

well-differentiated, intestinal type carcinomas. Inter­ estingly, the investigators used an H. pylori strain (TN2GF4) that was originally isolated from a patient with gastric ulcer and then passaged in the stomach of Mongolian gerbils several times before resolution. This strain had vacuolating cytotoxin and cagA genes and appeared extremely spiral in form. A key point was that gastric cancer was not observed in the in­ fected animals 39 weeks or 52 weeks after inoculation with H. pylori. Honda et al. (67) also reported in the same year that two of five Mongolian gerbils ( 4 0 % ) infected with H. pylori ATCC 4 3 5 0 4 , also containing the vacuolating cytotoxin and cagA genes, developed gastric cancer 72 weeks after inoculation that was well-differentiated adenocarcinoma. However, Hirayama et al. (62) reported that only one gastric cancer developed in Mongolian gerbils infected with H. py­ lori ATCC 4 3 5 0 4 at 96 weeks of follow-up (1.8%) and the pathology was a poorly differentiated adeno­ carcinoma. In 1999, Shimizu et al. (145) reported that H. pylori infection plus administration of M N N G , a dif­ ferent chemical carcinogen, enhanced the develop­ ment of gastric cancer at 5 0 weeks, compared to ad­ ministration of M N N G alone or H. pylori infection alone. As M N N G itself has anti-H. pylori effect, highdose administration of M N N G eradicated H. pylori in the animals. Six of 25 Mongolian gerbils ( 2 4 % ) , which were administered 60 mg of M N N G first for 10 weeks followed by H. pylori inoculation, developed gastric cancer. Of the six cancers, three were welldifferentiated adenocarcinomas, one was poorly dif­ ferentiated adenocarcinoma, and two were signet ring-cell carcinomas. In contrast, there was no cancer in animals administered M N N G alone. Of 25 Mon­ golian gerbils that were infected with H. pylori first and then administered 2 0 mg of M N N G for 30 weeks, 15 animals ( 6 0 % ) developed gastric cancer at 50 weeks. Nine of these cancer were well-differentiated adenocarcinomas, two were poorly differentiated ad­ enocarcinomas, and four were signet ring-cell carcino­ mas. In their report they noted that the titers of antiH. pylori antibodies in the cancer-bearing Mongolian gerbils were higher than in cancer-free Mongolian gerbils treated in the same manner. These findings may suggest that host immune response has some linkage to tumor development in these models (10, 28, 37). Tokieda et al. (159) demonstrated that H. pylori infection plus administration of M N N G increased the incidence of gastric cancer, compared to administra­ tion of MNNG alone. Four of six Mongolian gerbils (66.7%) with H. pylori infection followed by M N N G for 2 0 weeks developed gastric cancer 52 weeks after inoculation. The incidence was higher than in Mongo-

CHAPTER 40 • GASTRIC CANCER

491

Table 2. Development of gastric cancer in H. pylori-infected Mongolian gerbils Report (yr)

H. pylori strain

Chemical carcinogen

Time cancer observed (wk)

No. of gerbils with cancer/total no. (%)

Histology" 6 signet 2 poorly 5 well 10 well 2 well 2 poorly 4 signet 1 poorly 9 well 4 well-moderate

Sugiyama (1998)

ATCC 43504

MNU

40

13/37 (35)

Watanabe (1998) Honda (1998) Hirayama (1999) Shimizu (1999)

TN2GF4 ATCC 43504 ATCC 43504 ATCC 43504

None None None MNNG

62 72 96 50

10/27 (37) 2/5 (40) 1/56 (1.8) 15/25 (60)

Tokieda (1999)

ATCC 43504

MNNG

52

4/6 (67)

" Signet, signet ring-cell carcinoma; poorly, poorly differentiated adenocarcinoma; well, well-differentiated adenocarcinoma.

lian gerbils administered the same dose of MNNG alone ( 1 7 . 6 % ) . These six studies were conducted under different experimental conditions, being (i) cotreatment with chemical carcinogens, (ii) use of different strains, and (iii) different observation periods. As the result of these differences, the incidence of gastric cancer and the subtype of gastric cancer might have been influ­ enced (Table 2 ) . The most troublesome issue is the diagnostic criteria for gastric cancer in Mongolian gerbils. In general, gastric cancer in humans is diag­ nosed by three histological characteristics, (i) cellular and nuclear atypia, (ii) aberrant glandular structure, and (iii) invasion (137). H. pylori-infected Mongolian gerbils sometimes exhibit invasive glands into muscu­ lar layer and aberrant glands, with these histological changes being reversed upon H. pylori eradication. These histological characteristics in this model can be confused with well-differentiated adenocarcinomas developed in experiments in which animals are in­ fected with H. pylori alone. On the other hand, treat­ ment with low-dose chemical carcinogens sometimes induces cellular and nuclear atypia. Therefore, a diag­ nosis of well-differentiated adenocarcinoma in Mon­ golian gerbils induced by H. pylori infection needs to be prudent. In terms of these criticisms, the incidence of welldifferentiated adenocarcinomas in experimental models, either infected with H. pylori alone or with coadministration of low-dose chemical carcinogens, may be overestimated. To resolve these issues, com­ mon criteria for diagnosis of gastric cancer in Mongo­ lian gerbils are required or histology needs to be sup­ ported by genetic evidence to confirm the diagnosis of gastric cancer. In relation to cancer prevention, Shimizu et al. (145) reported that H. pylori eradication could de­ crease the incidence of gastric carcinomas in Mongo­ lian gerbils induced by H. pylori inoculation plus ad­

ministration of low-dose chemical carcinogens. Of 23 Mongolian gerbils administered MNU in the drinking water at 30 ppm for a total of 5 weeks and followed by infection with H. pylori, 15 animals developed gastric cancer at 50 weeks ( 6 5 . 2 % ) . The other 2 4 Mongolian gerbils administered MNU and later infected with H. pylori were given treatment to cure the infection at 21 weeks. At week 5 0 , the prevalence of gastric cancer in H. py/on'-eradicated Mongolian gerbils was signifi­ cantly lower (20.8%) than in H. pylori-infected Mon­ golian gerbils. These observations indicate that eradi­ cation of H. pylori in the early phase of the process reduced the occurrence of gastric cancer in this model. This result suggests that H. pylori eradication treat­ ment may be an effective approach in terms of gastric cancer prevention in humans.

GENETIC ALTERATIONS IN GASTRIC CARCINOGENESIS Gastric carcinogenesis in humans is a multistep process as well as multifactorial. An accumulation of genetic alterations may result in development of gas­ tric cancer, having an analogy with that of colon can­ cer (166). Genetic alterations occur in oncogenes, tumor suppressor genes, cell adhesion molecules, telo­ mere and telomerase activity (95), and genetic insta­ bility ( 1 6 , 1 7 1 ) . Different histological types of gastric cancer exhibit different patterns of genetic alterations. Tahara (156) clearly summarized the genetic altera­ tions in well-differentiated intestinal type and poorly differentiated diffuse type gastric adenocarcinomas (Table 3). The p53 mutation in tumor suppressor genes, cyclin E and p21 overexpression in cyclin and CDK inhibitors, transforming growth factor a overexpression (157) in growth factors, and CD44 aber­ rant transcript (176) in cell adhesion molecules are relatively observed in both types of gastric cancers. In

492

ASAKA ET AL.

Table 3. Genetic alterations in both types of human gastric cancer"

Alterations Tumor suppressor genes p53 LOH, mutation APC LOH, mutation DCC LOH LOH of lp LOH of lq LOH of 7q Cyclin and CDK inhibitors Cyclin E overexpression p21 overexpression Oncogenes K-ras mutation c-met amplification K-sam amplification c-erbB-2 amplification Growth factors EGF overexpression TGFct overexpression Adhesion molecules E-cadherin, loss or mutation CD44 aberrant transcript

Well differentiated

Poorly differentiated

(%)

(%)

60 40-60 50 30 44 53

75 0 0 38 0 33

57 77

63 76

10 19 0 20

9 39 33 0

40 60

20 55

0 100

50 100

" Modified from reference 1 5 6 with permission.

contrast, antigen-presenting cell mutation (112) and erbB-2 amplification (61) are likely to occur in welldifferentiated gastric cancers, and K-sam amplifica­ tion and mutation or loss of E-cadherin occurs in poorly differentiated gastric cancers ( 3 1 , 60). Since specific carcinogens have specific mutagenic proper­ ties, the types of genetic mutations present in a specific form of gastric cancer may provide important clues to the mutagenic agent. The p53 is a nuclear protein and functions as guardian of the genome (96). The wild-type p53 binds to the responsive element on DNA and induces WAFl/CIPl/p21, which binds to cyclin-dependent ki­ nase and induces G l arrests (58). Another major func­ tion is to induce apoptosis via induction of Bax, Fas/ Apo-1, and the other apoptosis-related proteins (13, 127). Therefore, the mutant p53 might link to the dysregulation of a cell cycle and apoptosis. In fact, p53 mutation is the most widely described molecular alteration in human cancers (65, 74, 78). The muta­ tion occurs in 4 0 to 7 0 % of diffuse or intestinal types of human gastric cancers. Shiao et al. (141) described that p53 mutated in 5 8 % with dysplasia and 6 7 % with carcinoma of the stomach. Ochiai et al. (117) reported that 4 of 10 incomplete type intestinal meta­ plasias demonstrated p53 mutation on exon 5 or exon 7 by PCR-single strand conformation polymorphism analysis and direct sequencing. Therefore, p53 muta­

tion might occur in the early steps of gastric carcino­ genesis. As described above, the Mongolian gerbils may be the best animal model to investigate the gastric carcinogenesis induced by H. pylori infection, since this model resembles human pathology associated with H. pylori infection, that is, chronic active gastri­ tis, gastric atrophy, intestinal metaplasia, and gastric carcinoma. However, because this animal has not been popular in cancer experiments to date, unfortu­ nately, the genetic data linked to oncogenesis are lack­ ing. In addition, specific antibodies for use in Mongo­ lian gerbils are also lacking. These are some of the disadvantages of the Mongolian gerbil model for the study of gastric cancer. To break through this bottle­ neck, we need the genetic information associated with oncogenesis in Mongolian gerbils, such as the p53 gene, which might be an appropriate gene to investi­ gate the oncogenic mechanisms of both types of gas­ tric cancer induced by H. pylori infection.

PERSPECTIVE Gastric cancer is an example of a cancer associ­ ated with a chronic inflammatory process that results in a metaplastic epithelium and cancer (51). Other examples are Barrett's esophagus, squamous metapla­ sia in the bronchus in smokers, chronic inflammation in ulcerative colitis, or bladders infected with schisto­ somes. In general, the risk is related to the extent and severity of the atrophic changes present. All of the problems listed by Evans and Mueller regarding mak­ ing causal associations between viruses and cancer (35) are applicable to H. pylori and gastric cancer, including (i) there is a long incubation or induction between infection and cancer, (ii) the candidate agent is common and the cancer is relatively rare, (iii) the need for cofactors, (iv) the cause of cancer may vary depending on geographic areas or age, (v) different strains may have different oncogenic potential, (vi) the human host plays a critical role in susceptibility (age at infection, genetic characteristics, status of im­ mune system), (vii) cancer is multifactorial and the agent may play roles at different points, and (viii) there are often other causes of the cancer. Differences in the incidence of gastric cancer in populations with a similar high prevalence of H. py­ lori infection can be related to the differences in the age of acquisition of chronic atrophic gastritis (Fig. 3), which, in turn, is related to an interaction between environmental factors, especially diet, and H. pylori infection. The incidence of gastric cancer varies in dif­ ferent regions and can fall rapidly, even in the same population in relation to levels of sanitation, stan­ dards of living, food storage and the use of salt,

CHAPTER 40 • GASTRIC CANCER

Carcinoma

3.

4.

5.

Carcinoma Atrophic Pan-Gastritis

6.

7.

8.

Figure 3. Status of the gastric mucosa in two populations with a high incidence of H. pylori infection. One population (A) experi­ ences a rapid transition of superficial gastritis to atrophic pangastritis. Gastric cancer begins to be seen in patients in their 40s, and the incidence increases thereafter. This pattern is typical in Japan or Korea. The population represented in panel B has a low rate of development of atrophic pangastritis, and gastric cancer is a rare disease. Such a pattern is typical in tropical countries such as Bang­ ladesh.

9.

10.

11.

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CHAPTER 40 • GASTRIC CANCER

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Helicobacter pylori: Physiology and Genetics Edited by H. L. T . Mobley, G. L. Mendz, and S. L. Hazell © 2 0 0 1 ASM Press, Washington, D.C.

Chapter 41

Markers of Infection DAVID Y . GRAHAM AND WAQAR A . QURESHI

Markers for the presence of a Helicobacter pylori in­ fection consist of features or events that suggest that the infection is, or has been, present and include alter­ ations in gastroduodenal structure and function, as well as tests to detect the bacterium itself or the pres­ ence of bacterial enzymes. Initially, the primary marker suggesting an H. pylori infection was the pres­ ence of one of the diseases associated with the infec­ tion, including histologic gastritis, peptic ulcer dis­ ease, and gastric adenocarcinoma. It is now recognized that primary mucosa-associated gastric lymphoma (MALT lymphoma) and approximately 1 0 % of cases of nonulcer dyspepsia are also directly related to H. pylori infection. Once it was confirmed that H. pylori was not a colonizer of inflamed gastric mucosa, but was rather the cause of gastric inflamma­ tion (gastritis), it was only a matter of time before the previous associations with gastritis could be trans­ ferred to H. pylori and prior associations clarified.

L A B O R A T O R Y ABNORMALITIES Serum Gastrin or Pepsinogen H. pylori infection is associated with progressive and complex abnormalities of gastroduodenal physi­ ology. There are a number of biochemical markers suggestive of the presence of an active or past H. py­ lori infection. For example, one biochemical marker of an active H. pylori infection is an increase in fasting and in meal-stimulated serum gastrin levels (40, 6 3 , 64, 9 0 , 100). Prior to the discovery of H. pylori, ele­ vated pepsinogen I levels were thought to be a genetic marker for risk of developing duodenal ulcer disease (94). Clarification of the role of H. py/on-associated gastritis in gastrin release showed that an elevated serum pepsinogen was not a genetic marker relating to duodenal ulcer but rather was related to the pres­

ence of the infection (4). Intrafamilial clustering of H. pylori infection led to the erroneous conclusion that it was a genetic marker. Marked or extreme hypergastrinemia is associated with destruction of the stomach, leading to profound hypochlorhydria, and can be seen in long-standing infection with H. pylori, but it is not specific because it is also present in autoimmune gas­ tritis (99). Several investigators have suggested that measur­ ing the change in gastrin or pepsinogen levels follow­ ing treatment might be a useful method to evaluate whether the infection had been cured ( 1 2 , 1 5 , 16, 3 8 , 40, 4 1 , 6 6 , 79). For example, Furuta et al. (35) sug­ gested using a 2 5 % increase in pepsinogen I/II ratio as the cutoff for determining whether the infection had been cured. They reported a sensitivity of 9 5 . 7 % and a specificity of 8 9 . 7 % . This hypothesis was tested recently using paired sera to evaluate the changes in gastrin levels, as well as in serum pepsinogen I and II levels and pepsinogen I/II ratios and IgG titers after successful treatment (2). The decline in meal-stimu­ lated gastrin was significant 2 weeks after therapy. Cure of infection also resulted in a significant fall in levels of both fasting and postprandial pepsinogen I and II levels. The fall in pepsinogen I/II ratio was the most marked change. Nevertheless, none of the pa­ rameters proved useful to detect cure of the infection in an individual patient because, despite statistically significant changes in pepsinogen and gastrin levels for the group as a whole, no cutoff value or percent change was found that reliably identified whether the infection had been cured (2). Measurement of a change in levels of pepsinogen or gastrin requires the use of paired sera and waiting for more than 6 months (likely more than 1 year) to obtain reasonably accu­ rate results, and none of these tests can be recom­ mended for clinical use, either diagnostically or to test whether the infection has been cured. There are a

David Y. Graham and Waqar A. Qureshi • Department of Medicine, VA Medical Center, and Baylor College of Medicine, Houston, TX 77030.

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number of noninvasive tests that can provide an accu­ rate assessment of H. pylori status pre- and posttherapy (see below). Inflammation H. pylori causes chronic inflammatory destruc­ tion of the stomach and, as one would expect, there are markers of this inflammatory process (88). Some are subtle and can be found by comparison with con­ trol groups of uninfected individuals or occur after H. pylori eradication. For example, several studies have suggested that there is a reversible fall in leukocyte count after cure of the infection (42, 51). In a recent study looking at the effect of H. pylori infection and CagA status on leukocyte counts and liver enzymes, pretreatment white blood cell counts and serum as­ partate transaminase (AST) were higher in infected patients (42). The AST levels were higher in infections with CagA-positive strains compared to CagA-negative strains. When treatment resulted in a cure, there was a significant fall in the total white blood cell count from 7,413 per m m to 6,738 per mm (P = 0.04). This drop was accounted for largely by a reduction in the polymorphonuclear leukocyte count. The AST levels were unaffected. It is important to note that the white blood cell count and the AST levels were within the normal range before and after therapy, suggesting that the "normal" range may reflect use of many in­ fected individuals. The fact that the AST did not change suggests that it was actually not related to the infection or that it is a marker for susceptibility to infection with CagA-positive H. pylori. 3

an increase in corpus inflammation irrespective of the method used to reduce acid secretion. Typically, in H. pylori infection there are reversi­ ble abnormalities in acid secretion, with acid secretion being prolonged after meals due to a defect in the reflex inhibition of acid secretion as the intragastric pH falls below 3 (19). Eradication of the infection restores the normal inhibitory pathways and may have other effects on acid secretion depending on the pattern of gastritis. For example, inflammation of the gastric corpus inhibits acid secretion, and following cure of the infection, the acid secretion produced by a stimulus such as pentagastrin increases markedly (44). In contrast, when the corpus is minimally in­ flamed, there is typically either no change or a slight decrease in pentagastrin-stimulated acid over time, possibly related to the resolution of the increase in gastrin secretion (19). It is thought that the inflamma­ tion-reduced acid secretion from the corpus is related to production of the cytokine interleukin 1 (IL-1). Histologic Markers of the Infection

3

Effect on Acid Secretion and Histology H. pylori infection damages the gastric mucosa and has an effect on gastric acid secretion. The overall effect is related to the predominant form of gastritis. The stomach can be divided conveniently into an acidsecreting portion (the body or corpus) and the distal stomach where acid-secreting cells are either absent or sparse (the antrum). H. pylori organisms are pres­ ent over the entire surface of the stomach, but the interaction of the bacterium and the surface cells dif­ fers among the regions. The severity of damage in the antrum is always equal to and typically more severe than that in the corpus (36). High levels of acid secre­ tion are associated with mild corpus inflammation and low H. pylori density in the corpus. This is typical of patients with duodenal ulcer disease and has been termed the antral predominant or corpus-sparing pat­ tern of gastritis. Inhibition of acid secretion allows the H. pylori to interact with the mucosa, producing

Histological gastritis is the primary manifesta­ tion of the infection. The typical histology is infiltra­ tion with a combination of acute and chronic inflam­ matory cells along with development of intramucosal lymphoid aggregates and follicles (88). H. pylori cells are present in sufficient quantity to be seen with highpower or oil-immersion magnification of histologic sections or of Gram stains of smears of gastric mucus. The typical histologic stain, hematoxylin and eosin (H&E), is excellent for identifying the presence of gas­ tritis, and the clinical sequelae of the infection such as gastric carcinoma or MALT lymphoma, but is rather unreliable for identifying the actual bacterium. Spe­ cial stains to identify H. pylori ate preferred to reduce error. Special stains are especially helpful posttherapy when there is still considerable remaining chronic in­ flammation. The Warthin-Starry and modified Giemsa stains are widely used to make identification of the bacteria easier, but neither is ideal. Triple stains which combine H8cE, Alcian blue, and another stain to identify the bacterium such as the Genta or ElZimaity triple stains allow easy identification of H. pylori and excellent visualization of gastric morphol­ ogy ( 1 3 , 2 4 , 2 5 , 2 7 , 3 7 ) . The original Genta stain used uranyl nitrate, which is not available widely, and the staining technique was not suitable for use with an autostainer. Recent modifications include replacing uranyl nitrate with a lead nitrate-gum mastic solution and using a microwave oven for the sensitization, sil­ ver impregnation, and reduction steps. This reduced the staining time to 28 min. The technical time can be reduced to 9 to 10 min if deparaffinzation and all

CHAPTER 41 • MARKERS OF INFECTION

steps following reduction are done with the help of an autostainer (27). Our laboratory routinely uses the Genta or ElZimaity triple stains, which both require only one slide. For those laboratories that use a special stain only when there is a possibility of H. pylori (e.g., when the mucosa shows inflammation) and posttherapy, we recommend the use of the Diff-Quik stain as it is read­ ily available, rapid, and inexpensive (25). When only a few bacteria are present and there is question about whether a case is positive or negative, we use the ElZimaity dual stain, which combines periodic acidSchiff (PAS) and a silver stain (26) (Fig. 1). This is the only practical way to visualize H. pylori in the duodenal mucosa. Failure to include a special stain may cause a false-negative result in up to 2 5 % , espe­ cially in posttreatment patients. Immunohistochemistry is used in some laboratories, but it is inferior to the triple stains or the dual stain for H. pylori diagnosis. A group of expert pathologists recommended that for diagnosis of H. pylori infection and character­ ization of the pattern of gastritis, two biopsies should routinely be taken from the antrum and two from the body of the stomach for histological evaluation (18). These biopsies should be taken from the most normalappearing mucosa. For detection of H. pylori, we rou­ tinely take three biopsies, one each from the distal antrum, the angulus incisura, and the greater curve of the mid corpus as this has an essentially 1 0 0 % accuracy for detection of H. pylori infection (22, 23). Whenever possible, large-cup biopsy forceps should be used. Since the organisms are most abundant in the mucous layer on the surface of the tissue, it is important not to wash off this layer. We recommend that biopsy specimens not be handled or oriented by

Figure 1. Example of the El-Zimaity dual stain of gastric mucosa (26). The stain combines periodic acid-Schiff and a silver stain and is the preferred stain when the density of H. pylori is very low or when searching for H. pylori in the duodenum.

501

Table 1. Recommendations for collecting gastric mucosal biopsies for detection of H. pylori Use jumbo or large-cup forceps. Biopsy normal-appearing mucosa. Take multiple (at least four) biopsies: two antral and two corpus. Do not handle or orient specimens; rather, "shake off" into for­ malin. Encourage pathologists to use a special stain and to report results using the new Sydney system.

the endoscopist but rather be "shaken off" the forceps by shaking the opened forceps in formalin (Table 1). The biopsy samples are fixed immediately. It is impor­ tant to train the technicians to mount the specimens "on edge" so that the surface can be visualized. Sev­ eral cuts are examined. False-negative results may occur due to incorrect sample collection, such as bi­ opsy from an area of atrophy or metaplasia, or recent treatment with proton pump inhibitors or antibiotics. Taking specimens from the gastric corpus, which is less frequently involved by intestinal metaplasia, is especially important in geographic regions where atrophic gastritis is common.

MARKERS BASED ON H. PYLORI

ENZYMES

Rapid Urease Tests H. pylori contains abundant urease, making the presence of urease activity a useful marker for the presence of the organism. When a biopsy specimen containing H. pylori is introduced into a medium con­ taining urea, urease splits the urea into ammonia and carbon dioxide. The ammonia released results in an increase in pH, which can be detected by a color change of a pH indicator (Fig. 2 ) . The accuracy of rapid urease testing is high, such that a correlation between histology and rapid urease testing provides a simple measure to gauge the accuracy of the pathol­ ogy department. In a study with 143 patients, we found the sensitivity and specificity of different rapid urease tests to be in the range of 95 and 1 0 0 % , respec­ tively (22). There is little difference among the avail­ able rapid urease tests, so that cost and availability are the prime determinants of which is used. The speed of the reaction is enhanced when large biopsies or multiple biopsies are placed in a single test well and when a warmer is used to speed the reaction (61, 62, 113, 114). The sites with the highest yield are the gastric angle and the greater curve of the corpus (109). One approach is to place a sample from each of these sites into the same well of the rapid urease test. Rapid urease tests require a high bacterial density

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Figure 2. Examples of three commercial rapid urease tests including the original CLO test and two second-generation tests. One of each pair is the control, the other is after the color change.

such that recent use of antibiotics, bismuth-containing compounds, or proton pump inhibitors may cause false-negative results. Because of the high cost of his­ tologic examination in the United States, it has been suggested that it may be cost effective to retain the biopsy specimens for histology in the endoscopy labo­ ratory until after the results of the rapid urease test are known. If the rapid urease test is negative after 2 4 h, the biopsy specimens are sent to the laboratory. If the rapid urease test is positive, they are discarded. Of course, biopsy samples from abnormal-appearing mucosa must always be sent to the laboratory for pro­ cessing and examination.

term effects are unpredictable, making the C test preferred, where available. The C - U B T has proven to be an extremely relia­ ble test and yields satisfactory results despite almost every conceivable modification that has been tried. The urea breath test is a qualitative, not a quantita­ tive, assay for the presence of gastric urease activity. The concepts underlying the test are straightforward, as are the factors that could potentially lead to falsepositive or false-negative results (Table 2). Urease ac­ tivity is not present in mammalian tissue but is widely distributed among bacteria (e.g., the microbiota of the mouth contain many urease-containing bacteria). A number of factors could theoretically affect the C UBT results (Table 2 ) , including exposure of urea to 1 3

13

1 3

Urea Breath Testing The urea breath test is the noninvasive method of choice to determine H. pylori status either pre- or posttherapy. This test is based on the organism's ure­ ase activity, which liberates C 0 from labeled urea, resulting in the production of labeled C 0 that can be easily detected in the breath. Two forms of labeled urea are commercially available: one contains the sta­ ble, nonradioactive isotope C and the other contains the radioactive isotope C . Although the amount of radiation exposure with the [ C]urea breath test ( C-UBT) is small, none is best and the test is cont-raindicated in children, as well as pregnant women and, possibly, women of childbearing age. The amount of radiation exposure is approximately equiv­ alent to one day's background radiation, but as the label can be potentially incorporated into the bicar­ bonate pool and the half-life is very long, the long-

Table 2. Considerations regarding the accuracy of the C-urea breath test 13

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Substrate Quantity of substrate Formulation (tablet, powder, etc.) Breath collection Device used (storage, stability, etc.) Analyses Device (mass spectroscopy, infrared, etc.) Expression of results Local factors in the stomach Duration of substrate within the stomach Area of contact between bacterial urease and substrate Type of test meal Presence of oral or intestinal bacteria with urease activity Density of H. pylori Presence of urease inhibitors

CHAPTER 41

oral microbiota, either in the mouth or the stomach, which could hydrolyze the substrate and cause a falsepositive urea breath test result. T o overcome this problem, some investigators have encapsulated the urea or administered it as a tablet ( 6 , 4 5 ) . These routes of administration have, in turn, raised other issues of concern related to the dissolution and distribution of tablets and capsules within the stomach as well as the possibility of emptying prior to dissolution, leading to false-negative results (43). Other investigators have instructed patients to cleanse their mouths before in­ gesting the urea to reduce the number of bacteria, but this procedure would not eliminate exposure to those organisms already ingested ( 7 7 , 8 5 ) . The pH optimum for the urease of non-H. pylori gastric contaminants is generally 7 or above, and urea hydrolysis will not occur in the stomach unless the quantity of bacteria and the pH are both high. The potential impact of mouth bacteria can be overcome by delaying the first post-urea breath sample for 15 or 30 min in order to dilute any swallowed bacteria and to allow the acid in the stomach to inactivate mouth bacterial ureases. Administering the urea after a test meal provides simi­ lar benefits since the meal causes dilution of the gastric contents and secretion of acid, which lowers the pH. Despite these precautions, both the quantities of bac­ teria and the pH may be high in patients with gastric atrophy and result in false-positive urea breath tests associated with negative serologic results (80). The specificity and sensitivity of the standard U.S. protocol for the C - U B T have repeatedly proved to be excellent, providing reliable information about H. pylori status before or after therapy (55, 5 6 , 5 8 ) . Recent studies have shown that it is possible to retain the accuracy of the C - U B T while simplifying the test. Simplifications included elimination of a special breath collection bag and collection of breath samples using a straw to blow into a test tube, elimination of fasting for more than 1 h before testing, and elimina­ tion of the solid test meal. These changes have had the practical advantage of allowing a shorter test pe­ riod as well as enhancing the convenience to the sub­ ject and the person administering the test. Replace­ ment of the solid test meal with a citric acid solution eliminated meal preparation and results in a solution with a pH below 4 to inhibit ureases other than H. pylori and to stimulate urease activity, probably by increasing the permeability of the membrane through the postulated Urel channel (29, 7 8 , 9 6 ) . The amount of substrate used in the C - U B T has not proven to be a critical factor for achieving accu­ rate results. Theoretically, there is a lower limit below which the proportion of false-negative tests would in­ crease, but varying the amount of expensive [ C]urea from 250 to 75 mg has been done without a reduction 13

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• MARKERS OF INFECTION

in either specificity or sensitivity. This mass of sub­ strate administered ensures that substrate exhaustion does not occur such that breath sampling can be de­ layed until any pulse of labeled C O 2 from orally hy­ drolyzed urea has passed (58, 6 9 ) . Extra or "cold," or nonradioactive, urea was not added to the commer­ cial C - U B T , which is administered as a capsule con­ taining carrier-free radioactivity. With that test even a small quality of urease can cause substrate exhaus­ tion. 14

Breath collection devices A number of different collection devices have been used to collect breath samples for analysis. The type of breath collection device used is dictated by the requirements of the analyzer used and the need for storage or shipment. Overall, collection devices have not proven to be a problem. Typically, when mass spectrometry is used for analysis, the breath samples are shipped to a central facility in glass or plastic tubes similar to those used for blood collection. The original breath sample is either collected in a bag and a small sample is transferred to the shipping tube or the breath is collected directly in the tube by blowing through a straw inserted into the bottom of the tube, which is then capped (7). When larger breath samples are required, as in the use of infrared instruments, a C0 -impermeable bag of 150 to 4 0 0 ml is used. 2

Detection of

1 3

C0

2

As previously described, the C - U B T detects H. pylori infection indirectly based on the hydrolysis of orally administered [ C]urea. The liberated C 0 ap­ pears in expired breath where it can be detected as an increase in the isotopic ratio of C 0 / C 0 . Thus, in the C - U B T a breath sample is taken before the substrate is administered and another sample is taken 15 or 30 min after, depending on the test kit. Classi­ cally the enrichment of C 0 in the breath has been assessed using a gas isotope ratio mass spectrometer. Such instruments require very little sample and are extremely sensitive and accurate even at very low lev­ els of respiratory C 0 . Newer methods based on in­ frared spectrometry also can be used to determine the ratio of C 0 / C 0 (9, 4 9 , 84, 86). These offer the potential of lower instrumental costs and for analysis at the point of patient care. The choice of analyzer depends on the number of samples to be analyzed as well as the characteristics of the analyzer. Many of the infrared instruments are capable of detecting the enrichment of C 0 in the breath when the amount is well above the cutoff value but are less able to pro­ vide an accurate estimate of samples near this value. 13

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In Western countries, evaluation of dyspeptic patients for the presence of H. pylori is likely to become one of the most frequent indications for requesting a urea breath test. In this population the frequency of H. pylori is typically low (e.g., the proportion of white United States-born individuals is less than 1 2 % ) . Be­ cause the majority of test results will be negative, the test must be accurate in the region around the cutoff value. This region is precisely where mass spectrome­ try excels and some of the newer instruments have the greatest difficulty. In clinical trials this has not proven to be a significant problem; nevertheless, it behooves the physician who contemplates purchase of a pointof-care instrument to ensure that it provides accept­ able accuracy in the range around the cutoff point. One approach to work around this problem is to send samples with results near the cutoff value for reanalysis by mass spectrometry. Expression of results In the United States, the cutoff value for the C UBT in adults was determined using receiver opera­ tion characteristics (ROC) curves to determine the lowest increase in C 0 abundance associated with H. pylori infection in a group of 60 H. pylori-infected and 60 uninfected volunteers (58). The cutoff for a positive test was defined as an increase of > 2 . 4 % , and this was validated against histologic examination and culture of gastric mucosal biopsies (58). Never­ theless, calculation of the results in terms of an in­ crease in C 0 abundance may not be the best method. In other C - U B T applications it has been customary to calculate the proportion of the dose re­ covered in breath. This calculation takes into account the weight of substrate, its molecular weight, number of labeled carbons, the rate of C 0 production, as well as the breath enrichment of C 0 at a given interval after substrate ingestion. Although the simple change in isotopic abundance from the pretest base­ line value to postdose value initially sufficed for diag­ nostic purposes in adults, it poses problems particu­ larly in the use of the test in children. The test outcome hinges on two separate processes, one arising from the organism and the other from the host. H. pylori urease liberates pure C 0 from the administered [ C]urea. As the labeled C 0 combines with respira­ tory C 0 at its natural abundance, the degree to which the C 0 is diluted by host C 0 will determine the degree of enrichment present in the expired breath. C 0 production differs in relation to age (adults greater than children), sex (males more than females), weight, or height. Thus, the identical amount of [ C]urea hydrolyzed in the stomach of a small person (e.g., child) would provide a proportion­ 1 3

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ally higher enrichment than that from a large person. Any cutoff value based on breath enrichment alone may give rise to misleading results when used in popu­ lations whose anthropometric measurements differ significantly from those of normal U.S. adults. This problem can be overcome by using established meth­ ods to estimate an individual's C 0 during a breath test. One method is based on body surface area (in m ) and is calculated from measurements of height and weight. The method assigns a value of 3 0 0 mmol C 0 / m i n / m (48). The second uses equations devel­ oped by Schofield to predict basal metabolic rates from the age, sex, height, and weight and has the ad­ vantage of six age stratifications (95). With the Scho­ field equations the rate of actual urea hydrolysis can be obtained by combining the estimate of host C 0 production rates with urea dose and breath enrich­ ment values. When this is done, the cutoff value is expressed as the urea hydrolysis rate (u,g/min), and a value of 10 u-g/min provides indication of the presence or absence of H. pylori infection that is independent of age, sex, height, or weight (57). Use of this test result expression is recommended and is absolutely essential for children under the age of 5. One cannot use the absolute level of urease activity to predict the number of H. pylori in the stomach in more than the most general way. 2

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Use of the urea breath test The urea breath test, histology, and rapid urease testing all require a relatively high density of H. pylori. Thus, any practice that reduces the intragastric con­ centration of H. pylori could lead to false-negative results, and avoidance of low concentrations of H. pylori is the basis for the recommendation to withhold confirmation testing until 4 or more weeks after end­ ing therapy. Use of bismuth or antibiotics in the pre­ testing period may also result in a false-negative test. Proton pump inhibitor therapy reduces the density of H. pylori through a direct anti-H. pylori effect and approximately 2 0 % of H. py/on-infected patients will have a false-negative result when taking a proton pump inhibitor. We recommend that proton pump in­ hibitor therapy be stopped at least 1 week before test­ ing. In our experience and the experience of others, standard dose H -receptor antagonists do not affect the accuracy of the C - U B T and they can be used up to the time of testing (10). False-positive tests are rare, but when they occur, they are likely due to contamina­ tion of the stomach with urease-positive organisms. This can be reduced by using the citric acid test meal because, as noted above, this reduces the intragastric pH below the pH optimum of these ureases. 2

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CHAPTER 41

MARKERS BASED O N ANTI-H. PYLORI ANTIBODIES Serologic Markers There have been a number of methods developed for the noninvasive detection of anti-H. pylori IgG or IgA, including whole blood, serum, saliva, and urine (17, 30, 3 3 , 9 8 , 107), as well as immunoblotting (5, 11, 32, 5 2 , 54, 82, 9 2 , 1 0 1 ) . Enzyme-linked immuno­ sorbent assays (ELISA) for urine IgG have also proved successful but have the same drawback as serum methods (3, 5 3 , 76). Detection of anti-H. pylori anti­ body is the easiest noninvasive approach to test for the presence of an H. pylori infection. With some tests only a few drops of fingerstick blood are required and the results are available in less than 5 min. Antigens The initial serologic tests were patterned after those developed for Campylobacter jejuni and con­ sisted of a mixture of very crude antigens. Cross-reac­ tivity with other bacterial antigens (e.g., flagellar anti­ gens) was expected and indeed occurred. Secondgeneration tests have used purified antigens such as the high-molecular-weight antigens, which include antigens that are confirmationally determined. Some of the second-generation tests have proven accurate worldwide (28, 7 4 , 1 0 2 , 1 0 6 , 1 0 8 ) . ELISA are widely available because of their low cost, rapidity, and re­ producibility. Complement fixation, hemagglutina­ tion, bacterial agglutination, immunofluorescence, and immunotransference (Western blot) tests are also available but are less accurate and are not widely used. Tests using crude antigens (e.g., "in-house" tests) are generally less reliable and are not recommended for clinical use. Tests for specific virulence factors such as CagA have been developed but have not proven to have any special clinical utility (47) and have generally suffered from poor specificity and sensitivity (111, 112). Interpretation of results Detection of serum IgG against H. pylori typi­ cally indicates a current or prior infection. Recent studies have shown that the prevalence of H. pylori infection is decreasing in all age groups in both Japan and the United States (60, 7 0 - 7 2 ) and likely in all Western countries. Thus, the population of individu­ als with anti-H. pylori antibodies and negative UBTs or no H. pylori seen on histology has increased and will continue to cause an underestimation of the true ability of serologic tests to accurately identify the pres­

• MARKERS OF INFECTION

505

ence of anti-H. pylori antibodies in body fluids. In our experience, those with positive ELISA using secondgeneration ELISAs, such as HM-CAP, typically have histological evidence of an old H. pylori infection (e.g., lymphoid aggregates). When serologic tests that are accurate in Western countries are used in other countries such as in Japan, the cutoff value must often be adjusted, usually upward. We speculate that this is not because the antigens of different H. pylori infec­ tions differ remarkably, rather, the cutoff must be raised to exclude those whose infections have been lost and whose antibody titers are falling. The accuracy of any test is only as good as the tests used to confirm the correctness of the serologic test. For example, in Japan the results of serologic tests were markedly influenced by the histological techniques used to determine the presence or absence of active H. pylori infection (75). It is now apparent that it is insufficient to only be able to accurately diagnose the presence of an active H. pyloriinfection (e.g., histologically or withUBT); to char­ acterize a serologic test one must also take into account evidence of prior H. pylori infections. One can there­ fore never expect serologic tests to provide as accurate results for the presence of an active H. pylori infection as do histology, culture, or the UBT. In addition, in general as the prevalence of the infection falls in a com­ munity, the accuracy of serologic tests suffers with an increase in the proportion of false-positive tests. In that population, a negative test essentially excludes an H. pylori infection (65). Changes in titer following cure of the infection Antibody titers fall following successful eradica­ tion of infection. A number of authors have investi­ gated whether this can be used to identify whether an individual patient has been cured. Again, as with pepsinogen and gastrin levels, it is easy to demonstrate a fall in titer for the group under investigation but not for an individual patient (2). Paired sera and months of follow-up are needed, making this a clini­ cally impractical approach. The decrease in titer after cure is slow and unpredictable, but over time most individuals will serorevert. In our experience this may require decades. For example, Al-Assi et al. found that although there was a drop in anti-H. pylori IgG anti­ body titer during treatment, levels in only one patient (6%) dropped below 5 0 % (2). Kosunen et al., on the other hand, using the 5 0 % drop in titer as an indicator of treatment success 6 months posttreatment, re­ ported a sensitivity of 9 7 % and a specificity of 9 5 % (59). Test for IgA and IgM Anti-H. pylori Antibodies Although tests for IgA and IgM to H. pylori are available in many countries, none are approved by the

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U.S. Food and Drug Administration. Overall, neither IgM nor IgA tests alone are superior to IgG serology, and the sensitivity and specificity of these tests have generally been too low for them to be recommended either alone or in combination with an IgG test.

MARKERS BASED O N T H E PRESENCE OF H. PYLORI ANTIGENS Stool Antigen Testing Recently there has been increased interest in iden­ tifying H. pylori protein antigens in stool as a marker of infection. Premier Platinum HpSA (Meridian Diag­ nostics Inc., Cincinnati, Ohio) has developed an in vitro qualitative enzyme immunoassay commercial kit that is stated to be able to detect H. pylori protein antigens of concentration > 1 8 4 ng/ml of feces. The test uses polyclonal antibodies to H. pylori as capture antibodies, adsorbed to microwells. Samples are di­ luted as per manufacturer's instructions and added to each antibody-coated microwell. After incubation for 1 h with a peroxidase-conjugated polyclonal anti­ body, unbound material is washed off, substrate is added, and the wells are incubated for 10 min at room temperature. Color develops in the presence of bound enzyme that is measured spectrophotometrically after adding a stop solution. Absorbance is measured at 450/630 nm. Overall, studies using pretreatment stool H. py­ lori antigen tests have shown that the sensitivity and specificity of stool antigen testing were comparable to histology or UBT (1, 8, 14, 3 1 , 5 0 , 8 1 , 83, 87, 1 0 3 - 1 0 5 ) . For example, in one prospective multicenter trial with 501 patients with H. pylori infection, proven by histology and a rapid urease test or culture, stool antigen for H. pylori was positive in 2 5 6 of 272 patients (sensitivity, 9 4 . 1 % ; 9 5 % confidence interval, 91 to 9 7 % ) . O f 2 1 9 patients without infection, 201 were negative by HpSA (specificity, 9 1 . 8 % ; 9 5 % con­ fidence interval, 87 to 9 5 % ) . In that study the posttreatment sensitivity and specificity for this marker were lower (90 and 9 5 % , respectively) (104). There are exceptions reporting less satisfactory results ( 3 4 , 7 3 , 9 1 , 9 3 ) , and the optimum cutoff values for optical density have varied slightly between stud­ ies (34, 83, 87). It has now become evident that there may be considerable lot-to-lot variation in stool anti­ gen tests. The most likely explanation is the poly­ clonal sera used for the capture antibody are obtained from rabbits and are thus difficult if not impossible to standardize. Stool antigen testing has proved to be less reliable when used soon after the end of therapy, and it is now generally recommended that one must

wait longer to confirm eradication (39, 6 8 ) . Gener­ ally, posttherapy studies have generally also shown good sensitivity and specificity when testing is delayed at least 4 weeks. The concept of a stool antigen test is a good one, and several companies have stool anti­ gen tests in trial that use monoclonal antibodies as capture antibodies. In general, one can use a UBT or the stool antigen for initial diagnosis, or as confirmation of a positive serologic result with the caveat that one should proba­ bly wait 6 or 8 weeks after therapy when using the stool antigen test. The UBT is preferred where avail­ able.

MARKERS BASED O N T H E PRESENCE OF T H E BACTERIUM Culture H. pylori is fastidious both in its growth and transport requirements. Successful culture relies on the transport medium, time in transit to the pathology laboratory, temperature during transportation, and the medium used; all influence bacterial viability and recovery (46, 110). For transportation and storage, various media have been evaluated. We use cysteineAlbimi broth with 2 0 % glycerol and have found that skim milk with 1 7 % glycerol was equally satisfactory (46). At room temperature there is a decrease in H. pylori titer after 6 h. At 4°C the organism will survive for 1 week and at - 7 0 ° C , or in liquid nitrogen, it will survive indefinitely. Once plated on two media (selective and nonselective), the samples are incubated at 37°C with high humidity in a microaerophilic at­ mosphere for at least 10 or preferably 14 days. The first colonies appear after 3 to 4 days. Identification is by colony morphology, Gram stain, and the en­ zymes the bacteria produce, including urease, oxidase, catalase, and glutamyltranspeptidase. Culturing the organism also allows testing for antibiotic susceptibil­ ity. This is particularly useful in treatment failures or in areas of high antibiotic resistance. The specificity from culture is 1 0 0 % . The sensitivity varies from 50 to 9 9 % depending on the laboratory and interest of the microbiologist. Most laboratories with an interest have extremely high yields. The order in which biopsies are taken (culture or histology) does not make a difference as preimmersion of biopsy forceps in formalin had no detrimental effect on the ability to culture H. pylori (115). Culture from stool has proven to be difficult, with a low yield (89). The frequency of positive culture might be en­ hanced by induced diarrhea (89).

CHAPTER 41 • MARKERS OF INFECTION

PCR Tests for Markers of H. pylori

Infection

9. Braden, B., F. Schafer, W. F. Caspary, and B. Lembcke. 1996. Nondispersive isotope-selective infrared spectroscopy: a new analytical method for C-urea breath tests. Scand. } . Gas­ troenterol. 31:442-445. 10. Bravo, L. E., J. L. Realpe, C. Campo, R. Mera, and P. Correa. 1999. Effects of acid suppression and bismuth medications on the performance of diagnostic tests for Helicobacter pylori infection. Am. J. Gastroenterol. 94:2380-2383. 11. Burnie, J. P., W. Lee, J. C. Dent, and C. A. McNulty. 1988. Immunoblot fingerprinting of Campylobacter pylori. J. Med. Microbiol. 27:153-159. 12. Calam, J. 1997. Helicobacter pylori and hormones. Yale ] . Biol. Med. 69:39-49. 13. Caselli, M., N. Figura, L. Trevisani, P. Pazzi, P. Guglielmetti, M. R. Bovolenta, and G. Stabellini. 1989. Patterns of physical modes of contact between Campylobacter pylori and gastric epithelium: implications about the bacterial pathogenicity. Am.]. Gastroenterol. 84:511-513. 14. Chang, M. C , M. S. Wu, H. H. Wang, H. P. Wang, and J. T. Lin. 1999. Helicobacter pylori stool antigen (HpSA) test—a simple, accurate and non-invasive test for detection of Helico­ bacter pylori infection. Hepatogastroenterology 46: 299-302. 15. Chen, T. S., S. H. Tsay, F. Y. Chang, and S. D. Lee. 1994. Effect of eradication of Helicobacter pylori on serum pepsino­ gen I, gastrin, and insulin in duodenal ulcer patients: a 12month follow-up study. Am. ] . Gastroenterol. 89: 1511-1514. 16. Chittajallu, R. S., C. A. Dorrian, J . E. Ardill, and K. E. McColl. 1992. Effect of Helicobacter pylori on serum pepsin­ ogen I and plasma gastrin in duodenal ulcer patients. Scand. ] . Gastroenterol. 27:20-24. 17. Cohen, H., S. Rose, D. N. Lewin, B. Retama, W. Naritoku, C.Johnson, L. Bautista, H. Crowe, and A. Pronovost. 1999. Accuracy of four commercially available serologic tests, in­ cluding two office-based tests and a commercially available C urea breath test, for diagnosis of Helicobacter pylori. Helicobacter 4:49-53. 18. Dixon, M. F., R. M. Genta, J. H. Yardley, and P. Correa. 1996. Classification and grading of gastritis. The updated Sydney system. International Workshop on the Histopathology of Gastritis, Houston, 1994. Am. ] . Surg. Pathol. 20: 1161-1181. 19. Dore, M. P., and D. Y. Graham. 2000. Pathogenesis of duo­ denal ulcer disease: the rest of the story. Baillieres Best Pract. Res. Clin. Gastroenterol. 14:97-107. 20. El-Zaatari, F. A., A. M. Nguyen, R. M. Genta, P. D. Klein, and D. Y. Graham. 1995. Determination of Helicobacter py­ lori status by reverse transcription-polymerase chain reaction. Comparison with urea breath test. Dig. Dis. Sci. 40:109-113. 21. El-Zaatari, F. A., S. M. Oweis, and D. Y. Graham. 1997. Uses and cautions for use of polymerase chain reaction for detection of Helicobacter pylori. Dig. Dis. Sci. 42: 2116-2119. 22. El-Zimaity, H. M., M. T. Al-Assi, R. M. Genta, and D. Y. Graham. 1995. Confirmation of successful therapy of Helico­ bacter pylori infection: number and site of biopsies or a rapid urease test. Am. J. Gastroenterol. 90:1962-1964. 23. El-Zimaity, H. M., and D. Y. Graham. 1999. Evaluation of gastric mucosal biopsy site and number for identification of Helicobacter pylori or intestinal metaplasia: role of the Syd­ ney system. Hum. Pathol. 30:72-77. 24. El-Zimaity, H. M., H. Ota, S. Scott, D. E. Killen, and D. Y. Graham. 1998. A new triple stain for Helicobacter pylori suitable for the autostainer: carbol fuchsin/Alcian blue/hematoxylin-eosin. Arch. Pathol. Lab. Med. 122:732-736. 13

Bacterial DNA can also be used as a marker for the infection. Under the ideal circumstances, the sensi­ tivity is close to that of culture. The potential advan­ tages of PCR include high specificity, quick results, and the ability to type bacteria without the require­ ment for special transport conditions. The routine use of PCR to diagnose H. pylori infection has proven problematic. PCR is very sensitive to inhibition by factors present in stool (20, 2 1 ) . False-positive results are common, possibly because of contamination in the laboratory. Because PCR assays detect homolo­ gous sequences, if other bacteria contain those se­ quences, it will test positive, causing specificity to be low. Although this test has high sensitivity and is sometimes compared to the UBT, practical considera­ tions and cost have limited its use (20). However, PCR use in H. pylori infection remains very promising and may permit rapid detection of antimicrobial resistance by detecting mutations associated with various antibi­ otics (67, 9 7 ) . At the present time PCR remains a re­ search tool for the diagnosis of H. pylori infection.

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beck, and M. Granstrom. 1997. The diagnostic value of en­ zyme immunoassay and immunoblot in monitoring eradica­ tion of Helicobacter pylori. Scand. J. Infect. Dis. 29: 147-151. Sunnerstam, B., T. Kjerstadius, L. Jansson, J. Giesecke, M. Bergstrom, and J . Ejderhamn. 1999. Detection of Helico­ bacter pylori antibodies in a pediatric population: compari­ son of three commercially available serological tests and one in-house enzyme immunoassay. /. Clin. Microbiol. 37: 3328-3331. Vaira, D., P. Malfertheiner, F. Megraud, A. T. Axon, M. Deltenre, G. Gasbarrini, C. O'Morain, J, M. Pajares Garcia, M. Quina, and G. N. Tytgat. 2000. Noninvasive antigenbased assay for assessing Helicobacter pylori eradication: a European multicenter study. The European Helicobacter py­ lori HpSA Study Group. Am. J. Gastroenterol. 95:925-929. Vaira, D., P. Malfertheiner, F. Megraud, A. T. Axon, M. Deltenre, A. M. Hirschl, G. Gasbarrini, C. O'Morain, J. M. Garcia, M. Quina, and G. N. Tytgat. 1999. Diagnosis of Helicobacter pylori infection with a new non-invasive anti­ gen-based assay. HpSA European Study Group. Lancet 354: 30-33. Vakil, N., A. Affi, J. Robinson, M. Sundaram, and S. Phadnis. 2000. Prospective blinded trial of a fecal antigen test for the detection of Helicobacter pylori infection. Am. J. Gastroent­ erol. 95:1699-1701. van Der, E. A., R. W. Der Hulst, P. Roorda, G. N. Tytgat, and J. Dankert. 1999. Evaluation of three commercial serological tests with different methodologies to assess Helicobacter py­ lori infection. /. Clin. Microbiol. 37:4150-4152. Westblom, T. U., and B. D. Bhatt. 1999. Diagnosis of Helico­ bacter pylori infection. Curr. Top. Microbiol. Immunol. 241: 215-235. Wilcox, M. H., T. H. Dent, J. O. Hunter, J. J. Gray, D. F. Brown, D. G. Wight, and E. P. Wraight. 1996. Accuracy of serology for the diagnosis of Helicobacter pylori infection—a comparison of eight kits. /. Clin. Pathol. 49:373-376. Woo, J. S., H. M. El-Zimaity, R. M. Genta, M. M. Yousfi, and D. Y. Graham. 1996. The best gastric site for obtaining a positive rapid urease test. Helicobacter 1:256-259. Xia, H. X., C. T. Keane, J. Chen, J. Zhang, E. J. Walsh, A. P. Moran, J. S. Hua, F. Megraud, and C. A. O'Morain. 1994. Transportation of Helicobacter pylori cultures by optimal systems. /. Clin. Microbiol. 32:3075-3077. Yamaoka, Y., and D. Y. Graham. 1999. CagA status and gastric cancer: unreliable serological tests produce unreliable data. Gastroenterology 117:745. Yamaoka, Y., T. Kodama, D. Y. Graham, and K. Kashima. 1998. Comparison of four serological tests to determine the CagA or VacA status of Helicobacter pylori strains. /. Clin. Microbiol. 36:3433-3434. Yousfi, M. M., H. M. El-Zimaity, R. A. Cole, R. M. Genta, and D. Y. Graham. 1996. Detection of Helicobacter pylori by rapid urease tests: is biopsy size a critical variable? Gastrointest. Endosc. 43:222-224. Yousfi, M. M., H. M. El-Zimaity, R. A. Cole, R. M. Genta, and D. Y. Graham. 1996. Does using a warmer influence the results of rapid urease testing for Helicobacter pylori? Gastrointest. Endosc. 43:260-261. Yousfi, M. M., R. Reddy, M. S. Osato, and D. Y. Graham. 1996. Culture of Helicobacter pylori: effect of preimmersion of biopsy forceps in formalin. Helicobacter 1:62-64.

Helicobacter pylori: Physiology and Genetics Edited by H. L. T. Mobley, G. L. Mendz, and S. L. Hazell O 2 0 0 1 ASM Press, Washington, D.C.

Chapter 42

Antibiotic Susceptibility and Resistance FRANCIS MEGRAUD, STUART HAZELL, AND YOURI GLUPCZYNSKI

The realization that peptic ulcer disease was caused by an infectious agent has led to the still ongoing search for the most appropriate therapy. Many thera­ pies have been tried, with the conclusion that multiple drug combinations are at present essential to the achievement of acceptable outcomes; cure of infec­ tion in > 8 0 % of patients is based on intention to treat. One class of drugs, which has been a constant from the early work of Marshall and Warren, is the 5-nitroimidazoles, principally tinidazole and met­ ronidazole (103), agents that had been used in the treatment of anaerobic bacterial and selected proto­ zoan infections (133). Two other drugs were also used with success in the following years: a macrolide compound, clarithromycin, and a B-lactam com­ pound, amoxicillin.

5-NITROIMIDAZOLE ACTIVITY A N D MECHANISM O F RESISTANCE

The 5-nitroimidazoles are prodrugs, in that they need to be activated within the target cell. The 5-nitroimidazole antibiotics have a very low reduction po­ tential and are activated by a one-electron reduction process. The reduction of the nitro group (Fig. 1) leads to the generation of reactive products, primar­ ily free radicals, which damage subcellular structures and cause damage and lethal mutations in DNA (41). Some of the reduction products are mutagenic and their formation can lead to the generation of secondary free radicals, which are also damaging. With the reduction of the drug, a concentration gradient is formed that facilitates the diffusion of more of the particular 5-nitroimidazole into the cell (41). While the process of drug activation may be rep­ resented concisely, the simplicity of such an explana­ tion belies the complexity of the intracellular pro­ cesses. First, it must be understood that the basis for the selective toxicity of the 5-nitroimidazoles relates to the redox potential of the intracellular environment (the midpoint redox potential for the metronidazole couple being 4 1 5 mV) (41). Anaerobes have the re­ ductive capacity to activate these drugs, whereas aero­ bic bacteria and mammalian cells do not. That H. pylori, a microaerophile, was susceptible to 5-nitro­ imidazoles was the first indication that the metabo­ lism of this bacterium was atypical (103). As a micro­ aerophile it was suggested that the mechanism of susceptibility and resistance to 5-nitroimidazoles may be unique, involving processes such as futile cycling, generating superoxide anions leading to cell death.

The MACH-1 (94) and MACH-2 (110) studies provided data that the combination of a proton pump inhibitor together with clarithromycin and metroni­ dazole or clarithromycin and amoxicillin represented the most effective drug combinations for the treat­ ment of Helicobacter pylori infection. Yet these com­ binations are subject to failure due to resistance (4). While the mechanisms of macrolide resistance in H. pylori are well understood, the same cannot be said for resistance to 5-nitroimidazoles. Indeed, the issue of resistance to the 5-nitroimidazoles is confused by apparent problems in relation to the accuracy and re­ producibility of susceptibility testing, the definition of resistance, and the perceived clinical relevance of resistance. The issues relating to drug selection and antibi­ otic resistance as well as the prospect of using geno­ mics for drug discovery will be reviewed in this chapter.

Francis Megraud • Laboratoire de Bacteriologie, Hdpital Pellegrin, 33076 Bordeaux Cedex, France. Stuart Hazell • College of Science, Technology and Environment, University of Western Sydney, Campbelltown, Australia. Youri Glupczynski • Laboratoire de Microbiologic, Clinique Universitaire de Mont-Godinne, Yvoir, Belgium.

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N

(CH ) OH 2

2

Figure 1. Chemical structure of the prodrug metronidazole. Metro­ nidazole is activated by the reduction of the nitro group (arrow) leading to the generation of a reactive radical.

However, several studies demonstrated that this hy­ pothesis was not correct ( 8 0 , 1 3 8 ) . One of these stud­ ies provided direct evidence for intracellular reduction of metronidazole in susceptible isolates and reduced levels of reduction in resistant isolates (80). H. pylori possesses enzymes having distinctly "anaerobic" characteristics. One of these enzymes, pyruvate:flavodoxin oxidoreductase (porGDAB) (72), catalyzes the formation of acetyl-CoA from py­ ruvate. Pyruvate:flavodoxin oxidoreductase had pre­ viously been considered an enzyme restricted to hyp­ erthermophilic bacteria and members of the domain Archaea (72, 73). The interest in pyruvate:flavodoxin oxidoreductase arises because in anaerobes the related enzyme system, pyruvate:ferridoxin oxidoreductase, is central to the activation of 5-nitroimidazoles (41). Hughes et al. were able to obtain indirect evidence that the pyruvate:flavodoxin oxidoreductase of H. pylori was able to activate metronidazole (72, 73). Although pyruvate:flavodoxin oxidoreductase is es­ sential, it was difficult to determine the exact contri­ bution to drug activation and resistance (72, 73). In addition to the above findings, there was evi­ dence for the involvement of not only pyruvate:flavodoxin oxidoreductase but also 2-oxoglutarate:acceptor oxidoreductase {oorDABC) in 5-nitroimidazole resistance (70, 72, 7 3 , 81). Kaihovaara et al. (81) noted that under anaerobic conditions, but not aero­ bic conditions, electrons could be transferred from flavodoxin (fidA) to metronidazole. This was consis­ tent with the observations of Smith and Edwards, who showed that metronidazole-resistant isolates of H. py­ lori could be rendered susceptible by a period of an­ aerobic incubation (137). That other systems may be linked to 5-nitroimid­

azole activation and resistance became apparent as further data were accumulated. Smith and Edwards (137) identified NADH oxidase activity as being asso­ ciated with metronidazole resistance, in that there was a threefold greater NADH oxidase activity in cyto­ solic fractions from metronidazole-susceptible iso­ lates compared with resistant isolates. NADH oxi­ dases are enzymes that reduce molecular oxygen to hydrogen peroxide or water without an intermediate electron acceptor. The H. pylori genome has revealed no genes homologous to typical NADH oxidase; how­ ever, NAD(P)H oxidase activity may be associated with enzymes that are not primary NAD(P)H oxi­ dases. Examples of enzymes that exhibit nonspecific NAD(P)H oxidase activity include a number of flavoproteins, for example, alkyl hydroperoxide reductase (17), thioredoxin reductase (7, 9 3 , 125, 169), gluta­ thione reductase (18), mercuric reductase, and dihydrolipoamide dehydrogenase (19). An important advance in our understanding of 5-nitroimidazole resistance comes from the work by Hoffman et al. On the basis of substantial molecular evidence, they reported that mutations in the gene en­ coding for an oxygen-insensitive nitroreductase (rdxA) lead to increased resistance due to the inactivation of nitroreductase (53). Another study from the same group established that 5-nitroimidazole resis­ tance in H. pylori strain ATCC 4 3 5 0 4 was due to an insertion sequence (a mini-IS605) and deletions in the rdxA gene (30). In addition, this group noted no mu­ tation in this strain compared with a susceptible strain in other putative "resistance" genes (catalase [katA], superoxide dismutase [sodB], flavodoxin [fldA], ferridoxin [fdx], pyruvate:flavodoxin oxidoreductase [porGDAB], and RecA [recA]). Tankovich et al. (147) reported four mechanisms of rdxA inactivation in clinical isolates from France and North Africa: frameshift mutations, missense mutations, deletion of bases, and the presence of an insertion sequence (miniIS605). Important as the findings of Hoffman's group were, it was soon apparent that RdxA was not the complete story. Apart from the proposed involvement of pyruvate:flavodoxin oxidoreductase and other en­ zyme systems in 5-nitroimidazole activation, Jenks et al. (79) found metronidazole-resistant isolates of H. pylori without apparent mutations in rdxA, support­ ing the belief that additional mechanisms of 5-nitro­ imidazole resistance must be present in some isolates of H. pylori. This conclusion has been supported by Kwon et al. (90), who indicated that in addition to rdxA, mutations in a number of genes may contribute to 5-nitroimidazole resistance. Their data support the concept of multiple enzymes within the bacterium being able to activate these drugs, i.e., the redox po-

CHAPTER 42 • ANTIBIOTIC SUSCEPTIBILITY AND RESISTANCE

tential of the cell influences the capacity of an organ­ ism to activate 5-nitroimidazole and the maintenance of an appropriate redox potential may be governed by multiple enzyme systems. Kwon et al. (90) observed that by measuring mRNA with a reverse transcriptase PCR, there appeared to be a decrease in the level of transcrip­ tion of nitroreductase (rdxA), ferredoxin oxidore­ ductase (2-oxoglutarate:acceptor oxidoreductase; oorDABC), and ferredoxin-like protein (fdxB) in strain 26695 (150) in the presence of metronidazole. Two other studies by the same group established that, in addition to RdxA, ferredoxin-like protein (fdxB) and an NAD(P)H flavin oxidoreductase (frxA) can contribute to the activation of metronidazole in H. pylori and that the resistance phenotype is associated with mutations in these genes (86, 87). An important observation from these studies was that inactivation of fdxB, frxB, and rdxA resulted in different levels of metronidazole resistance in H. pylori, consistent with clinical observations (110). Kwon et al. proposed that the spectrum of MIC data seen with clinical isolates of H. pylori was indeed due to "a partial and/or com­ plete inactivation of the genes fdxB, frxA, and rdxA." They also speculated that other systems might also contribute to metronidazole activation and resistance (86, 87). Resistance to 5-nitroimidazole is widespread. In the developed countries, it varies between 10 and 5 0 % whereas in developing countries it may concern virtually all strains (107). It seems to be the conse­ quence of the consumption of 5-nitroimidazole for gynecological and dental infections in the former and parasitic infections in the latter. However, as will be discussed later in this chapter, the results may be largely influenced by the method of susceptibility test­ ing used. When 5-nitroimidazole-based therapy is given to a patient harboring a resistant strain, the chance of success decreases by 2 0 % (110).

513

complex (52). Resistance by efflux did not seem to play a role in H. pylori when the three putative restriction-nodulation-division efflux system operons pres­ ent in the genome were submitted to mutagenesis. No effect on the in vitro susceptibility of H. pylori to 19 antibiotics including macrolides was noted (9). The well-known mechanism of macrolide resis­ tance that involves methylation of an adenine residue and is named M L S b (for macrolides, lincosamides, and streprogramin B) has not been found in H. pylori (31, 74). Another mechanism achieving the same goal has been described more recently, first in Escherichia coli (135) and later in other species such as Mycobac­ terium avium, Mycobacterium intracellulare, and My­ coplasma pneumoniae. Versalovic et al. were the first to demonstrate that this mechanism was involved in H. pylori resistance (158). They showed that point mutations (adenine —• guanine) in two positions, namely, 2 1 4 2 (A2142G) and 2 1 4 3 (A2143G) (for­ merly designated as the 2058 and 2 0 5 9 cognates in E. coli, then 2143 and 2 1 4 4 in H. pylori nomenclature before being correctly renamed based on the ribo­ somal sequence of H. pylori), were associated with macrolide resistance (149). Later, Occhialini et al. showed that these mutations were also associated with a lack of binding of macrolides to isolated ribo­ somes: the amount of bound antibiotic increases pro­ portionally with the amount of purified ribosomes from the susceptible strain but not from the resistant strain (126) (Fig. 2 ) . Therefore, a causal association is most likely involved and the mutations are targetstructural mutations. Originally, the two transition mutations A2142G and A2143G were found in clinical specimens. An­ other mutation has been described, the transversion A2142C, which seldomly occurs. Debets-Ossenkop et

22 -, O

20 4

MACROLIDE ACTIVITY AND MECHANISMS OF RESISTANCE To be active, a macrolide must penetrate the bac­ terial cell and bind the ribosomes. However, there is no evidence for a role of ribosomal proteins in this binding. The target is a domain of the 23S rRNA, the peptidyl transferase loop in domain V. Binding domain V leads to an interruption in protein elonga­ tion; consequently the bacterium can no longer syn­ thesize protein. Goldman et al. have shown that clar­ ithromycin, its parent compound erythromycin, and its 14-hydroxy metabolite have the tightest binding interaction observed to date for a macrolide-ribosome

Ribosome Concentration (DO-260 nm) Figure 2. Binding of labeled erythromycin on increasing concentra­ tions of H. pylori ribosomes.

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al. were able to generate other mutations (A2143C, A2142T, A2143T) in vitro, by site-directed mutagen­ esis of a clarithromycin-susceptible strain. However, these mutant strains were unstable and had a reduced growth rate; similar results were reported by Wang et al. (160). In addition, the mutant A2143T had an MIC of only 0.5 mg/liter. It has been hypothesized that the change in nucleotide sequence in these cases induces a change in free energy and conformation within the ribosome greater than for A2142G and A2143G, which has an impact on bacterial fitness. The mutations that are not found are most likely le­ thal. It is not clear why the mutant A2142C, which is apparently of the same type as the two previous ones, is found so seldomly (28). The simultaneous mu­ tations A2115G and G2141A described by Hulten et al. have never been found again (74). The impact of specific mutations on the MIC has also been studied. In most studies, A2142G mutations were significantly more likely to be present in isolates for which the MICs were > 6 4 mg/liter than in those for which the MICs were < 6 4 mg/liter (65 versus 3 0 % , P = 0.01) (156, 159). MICs of clarithromycin are usually lower than MICs of erythromycin and, even in rare cases, the MICs for strains with the A2143G mutation may be under the cutoff for clar­ ithromycin resistance while the strains are clearly re­ sistant to erythromycin (43, 160). Notwithstanding, we do not think that it is advisable to use clarithro­ mycin for treatment in such cases. J F I T . pylori contains two 23S rRNA operons (6, 150) with resistance mutations, when present, usually in both; heterozygosity has only been detected four times ( 7 4 , 1 4 3 , 1 5 8 ) . It is now understood that muta­ tions occur spontaneously and are selected after expo­ sure to the drug. Indeed, the presence of a low number of bacterial cells with a given point mutation has been shown within a population of otherwise susceptible bacteria, even though the patient was not supposed to have previously consumed macrolides (98, 102, 105, 156). Both predominant mutations have also been ob­ served in the same population of a given strain. The frequency of mutations inducing sponta­ neous resistance to erythromycin was found to be in the range of 3.2 X 1 0 " to 6 X 1 0 in vitro (57). However, this figure may be different in vivo. Indeed, the bacterial mutation rate increases under stress con­ ditions (antibiotic or pathogenic stress) (104). Fur­ thermore, a homolog of the mutS gene, which codes for mutation repair in E. coli, may have a different role in H. pylori, leading to the hypothesis that H. pylori is a spontaneously hypermutable bacterium (14). No association was found with known patho­ genic properties of the strains (cagA, vac A genotype) (156). 7

- 8

The stability of mutants A2142G and A2143G has been questioned, since resistance usually has a biological cost (92). In two studies, in which the strains were subcultured 10 to 5 0 times in vitro or obtained from a given patient after an interval of sev­ eral months, and the identity of the pre- and postisolates was confirmed by random amplified polymor­ phic DNA, resistance was still present, indicating the stability of the mutations (74, 108). In contrast, the authors of another study claimed that a reversion to­ ward the susceptible phenotype was possible (167). As resistance of H. pylori to macrolides is chromosomally mediated and essentially transmitted verti­ cally to the bacterial descendants, its spread has been relatively limited. Its prevalence ranges from 0 to 2 0 % , depending mainly on the policy of use of these drugs in the given area (107). In the case of failure of a clarithromycin-based therapy, a clarithromycin strain is isolated in as many as 6 0 % of the cases. When a clarithromycin-based triple therapy is given to a pa­ tient harboring a resistant strain, the chance of success decreases by more than 5 0 % .

RESISTANCE T O B-LACTAMS Amoxicillin is the only B-lactam used to treat H. pylori infection and it is included in most current ther­ apeutic regimens. This is due to its very low MIC against this bacterium, usually < 0 . 0 3 |xg/ml. Until re­ cently, amoxicillin-resistant H. pylori strains were not thought to exist although resistance to this compound could be selected in vitro after serial passages in in­ creasing subinhibitory concentrations of amoxicillin (57, 139). Clinical H. pylori strains for which the MICs were 0.25 to 0.5 u.g/ml, i.e., up to 100 times those of the most susceptible strains, were occasion­ ally found in individual resistance surveys (161), but the impact of this finding on therapy was not consid­ ered. Recently, Dore et al. (38) reported the occurrence of amoxicillin resistance in 17 H. pylori strains iso­ lated from patients in Sardinia (Italy) and in the United States. These resistant isolates, identified from subjects enrolled in a clinical trial on amoxicillin plus omeprazole therapy, showed pretreatment amoxicil­ lin resistance; the MICs were > 2 5 6 u.g/ml and were apparently associated with a marked reduction in treatment efficacy (39). The amoxicillin resistance phenotype was unstable and lost after storage at - 80°C, but plating these strains on amoxicillin gra­ dient plates could restore resistance. The rescued iso­ lates had MICs and minimum bacterial concentra­ tions (MBCs) ranging from 0.5 to 32 |xg/ml and 32 to > 1 , 0 2 4 ixg/ml, respectively, and the MBC/MIC

CHAPTER 42 • ANTIBIOTIC SUSCEPTIBILITY AND RESISTANCE

ratio for all tested strains was > 3 2 , indicating a toler­ ance to amoxicillin. A report from the Netherlands (156) described an H. pylori strain with stable resistance to amoxicil­ lin (MIC, 8 |xg/ml) that was isolated several times from a patient having received multiple courses of amoxicillin therapy. Amoxicillin resistance could be transferred from the resistant strain to a susceptible one by natural DNA transformation at a frequency of 1 0 bacteria; this led to the production of stable resistant transformants for which the MICs were 4 0 0 times greater than the value for the susceptible strain and similar to that for the naturally resistant strain. In contrast to the report by Dore et al. (36), amoxicillin resistance remained stable after repeated cycles of freezing and culture. Another report by Han et al. (60) described both stable and unstable amoxicillin resistance (256 u.g/ml) in seven H. pylori clinical iso­ lates, with three unstable isolates losing their resis­ tance after freezer storage and reculture. Variable degrees of cross-resistance to other peni­ cillins have been observed among amoxicillin-resistant H. pylori isolates, depending on the level of resis­ tance to amoxicillin (38). Overall resistant strains for which the amoxicillin MICs were > 3 2 (xg/ml were significantly resistant to other B-lactams compared to those for which the MIC was 2 |xg/ml (low-level resis­ tance). Moreover, several antibiotic resistance pheno­ types to B-lactams were observed among amoxicillinresistant strains, which suggests the existence of mul­ tiple resistance mechanisms in H. pylori (60). Resistance to B-lactam antibiotics by gram-nega­ tive bacteria is most commonly due to the production of B-lactamase, either chromosomally encoded, or, more often, plasmid mediated (95). In H. pylori, amoxicillin resistance could not be ascribed to the ac­ quisition or expression of a B-lactamase, since B-lac­ tamase activity was not detected by the chromogenic cephalosporin nitrocefin assay in any of the resistant strains (36, 60, 156). This finding is not surprising, since no B-lactamase homolog genes were found in either of the two H. pylori strains sequenced (6, 50). Other important mechanisms of resistance to B-lac­ tam antibiotics include alterations in the penicillinbinding proteins (PBPs), decreased permeability of the antibiotic into the bacterial cell wall, or a combination of the two strategies (78). The PBPs are a set of en­ zymes found in the cytoplasmic membrane of bacteria that are involved in the terminal stages of peptidogly­ can biosynthesis (46). They are integral components in the determination and maintenance of cellular mor­ phology and are, as the nomenclature suggests, the target proteins for penicillin and other p-lactam anti­ biotics ( 1 4 0 , 1 4 2 ) . PBPs are present in almost all bac­ teria but vary among species in number, size (ranging - 5

515

from 15 to 150 kDa), and affinity to B-lactams (46). Covalent binding of the B-lactams to specific PBPs in susceptible organisms results in the inability of the bacterium to build a complete cell wall and leads to rapid cell lysis and death. Alterations in the PBPs, which affect the ability of the B-lactams to bind, can confer increased resistance to these antibiotics (59, 100). Alterations in PBPs that result in resistance to Blactams have mainly been described in gram-positive bacteria such as Streptococcus pneumoniae (56, 76) but have also been reported among different gramnegative bacterial species including Haemophilus in­ fluenzae (111), Neisseria gonorrhoeae, Neisseria meningitidis (141), and Proteus mirabilis (122). Ini­ tial reports in H. pylori suggested the existence of three major PBPs (PBP 1, 2, 3) with molecular masses of 6 6 , 6 3 , and 60 kDa (PBP 1, 2, and 3, respectively) (33, 3 5 , 77). In one study, Dore et al. (35) found that amoxicillin resistance was dependent on a previously undetected PBP (PBP-D) with a molecular mass of 32 kDa and a lower affinity for amoxicillin. This lowmolecular-mass protein was also found by Krish­ namurthy et al. (83), who characterized it as PBP 4. Using H. pylori membrane preparations labeled with digoxigenin-ampicillin, Harris et al. (61) were able to identify a total of eight PBPs with molecular masses ranging from 72 to 28 kDa. Interestingly, amoxicillin was found to bind almost exclusively to the 72-kDa PBP (PBP 1A) with a higher affinity than the digoxi­ genin-ampicillin, suggesting that this PBP might also be involved in amoxicillin resistance. DeLoney and Schiller (34) isolated in vitro an amoxicillin-resistant strain of H. pylori after several subcultures in increas­ ing concentrations of amoxicillin. Comparative anal­ ysis of the PBP profiles generated from isolated bacte­ rial membranes of the susceptible parental strain and of the resistant strain revealed a significant decrease in labeling of PBP 1 by biotinylated amoxicillin in the amoxicillin-resistant strain. In line with these two reports, Kusters et al. (85) showed that point muta­ tions in the PBP 1A gene were associated with stable amoxicillin resistance in their clinical H. pylori strain isolated from a patient in the Netherlands, and that this resistance could be transferred in a susceptible strain by natural DNA transformation. The overall prevalence of resistance to amoxicil­ lin still remains extremely low in most countries. In 1999 in a large European multicenter trial, no amoxi­ cillin resistance was found among the 4 8 5 H. pylori isolates tested (110). Likewise, other surveys per­ formed in several European countries (29, 6 6 , 97, 165) failed to identify any amoxicillin-resistant strains. By contrast, high resistance rates were re­ ported in certain studies: 2 6 % in Sardinia, Italy (130); 2 9 % in Brazil (112); and even 4 1 % in China (166).

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The reasons for these large variations remain puz­ zling. It is not known whether they reflect true epide­ miologic differences in amoxicillin resistance or tech­ nical factors (testing method applied, size of inoculum, type of medium, atmosphere, and duration of incubation) or to differences in criteria used to de­ fine resistance (as there is currently no agreed break­ point value of resistance for amoxicillin). Whatever its true occurrence, the emergence of high-level resis­ tance to amoxicillin (MIC, > 2 5 6 |xg/ml) may repre­ sent a major threat regarding an effective H. pylori eradication therapy. For these reasons, susceptibility of H. pylori to amoxicillin must be monitored, espe­ cially whenever treatment failure with amoxicillincontaining regimens occurs.

RESISTANCE T O O T H E R ANTIBIOTICS Tetracyclines Tetracyclines are a component of the bismuthbased triple therapy regimen recommended in the early 1990s for treating H. pylori infection and have also been used extensively to treat other infections, such as nongonococcal urethritis, in recent years. Cur­ rently, tetracyclines are essentially used for the treat­ ment of H. pylori infection as part of a quadruple therapy, which also includes metronidazole, a bis­ muth salt, and an antisecretory agent (either a proton pump inhibitor or an H -receptor antagonist) (32). This regimen has been found to be very effective and is currently recommended as a second- or third-line "rescue" treatment after initial failure ( 7 1 , 1 0 3 , 1 0 9 ) . Tetracycline activity is due to its binding to the 3 OS ribosomal subunit at a position that blocks the fixation of the aminoacyl-transfer RNA to the accep­ tor site on the messenger RNA-ribosome complex. High-level resistance to tetracycline (MIC, > 2 5 6 u,g/ ml) was reported for the first time in 1996 in Australia in a patient in whom H. pylori eradication had failed with triple therapy (117). Overall resistance of H. py­ lori to tetracyclines has been found to be low and is estimated to be less than 2 % ( 1 3 , 1 5 , 2 9 , 1 6 5 ) . Higher resistance rates ( 5 % ) have, however, been reported in other studies from Japan and Korea (88). Interest­ ingly, all tetracycline-resistant isolates exhibited cross-resistance to metronidazole, and the cross-resis­ tance (including tetracycline resistance) could be transferred to tetracycline-susceptible H. pylori strains. The exact mechanism of tetracycline resistance has yet to be determined. Three genes expressed both in vitro and in vivo and presenting homology with potential efflux systems have recently been identified 2

in H. pylori (9). In uptake studies, there was no ob­ servable energy-dependent tetracycline or chloram­ phenicol efflux activity in H. pylori. Likewise, muta­ genesis experiments of the three putative efflux operons had no effect on the in vitro susceptibility of H. pylori to tetracycline and to a panel of several other antibiotics. Globally these data suggest that tetracy­ cline resistance may not be caused by active efflux in H. pylori and also that it is unlikely to be part of multidrug resistance mechanisms as in other gramnegative bacteria (124). Fluoroquinolones Fluoroquinolones inhibit the A subunit of the DNA gyrase. This enzyme is a tetramer consisting of two A subunits and two B subunits encoded by gyrA and gyrB, respectively. The main function of this en­ zyme is to relax the supercoiling of DNA to allow its replication (131). In H. pylori (as in several other bacterial species) resistance to fluoroquinolones is as­ sociated with mutations in gyrA (120). Four classes of mutations have been described in ciprofloxacinresistant H. pylori strains with substitutions at amino acid positions 87, 8 8 , and 91 and a double substitu­ tion at amino acids 91 and 97 of the resistance-deter­ mining region of GyrA. These point mutations were associated with an increase in the MICs from ^ 0 . 2 5 to s 4 u.g/ml. When homology comparisons were made, the strongest similarity was found between the gyrA gene of Campylobacter jejuni and H. pylori (76% identity at the amino acid sequence level). Inter­ estingly, Moore et al. (120) were able to transform ciprofloxacin-susceptible strains using the amplified fragment from resistant strains as a DNA donor. Fluoroquinolones are not commonly used in H. pylori treatments. However, when they have been used either alone or in combination with other antibi­ otics, low eradication rates were achieved (91, 96) and secondary resistance was consistently found (144). These compounds are not considered for use in current regimens even in the setting of second-line therapies following treatment failure. As a conse­ quence, there have only been a few surveys concerning H. pylori resistance to fluoroquinolones. This resis­ tance has been reported to be very low and to vary from 0 to 4 . 7 % in three recent studies from Greece (116), the Netherlands (30), and Bulgaria (13). How­ ever, fluoroquinolone resistance averaged 11 % in one survey from Portugal (15). The rise in H. pylori resis­ tance to these drugs is likely to reflect the increasing exposure of adults to fluoroquinolones, which are now widely used for the treatment of community-ac­ quired infections in many countries. New fluoroquin­ olones are nevertheless being tested (44, 146).

CHAPTER 42 • ANTIBIOTIC SUSCEPTIBILITY AND RESISTANCE

Rifamycins Rifabutin (a spiro-piperidyl derivative of rifamycin-S) and other derivatives of rifampin are inhibitory in vitro against H. pylori at much lower concentra­ tions than rifampin (MIC at which 9 0 % of isolates are inhibited [ M I C ] , 0.008 u.g/ml) (2, 65) and have been proposed as possible candidates for second- or third-line eradication therapy. A triple therapy includ­ ing rifabutin in combination with amoxicillin and a proton pump inhibitor has been shown to be effective in the eradication of H. pylori after failure of other therapies and in spite of resistance to other antibiotics (11, 127). The mechanism of action of rifabutin against H. pylori has recently been discovered. Similarly, as oc­ curring in strains of E. coli and Bacillus subtilis, rifa­ butin inhibits the B-subunit of the DNA-dependent RNA polymerase encoded by the rpoB gene (65). Lab­ oratory mutants of H. pylori, which exhibit amino acid substitutions in codon 5 2 4 - 5 4 5 or in codon 586 of the rpoB gene, are resistant to rifabutin (65). Until now, resistance to rifamycins has been reported only exceptionally in clinical isolates of H. pylori. One re­ cent report described a clinical isolate that developed resistance to rifabutin during therapy (66). This iso­ late carried an rpoB gene that retained a wild-type cluster region sequence but had acquired a novel codon V 1 4 9 F mutation. In transformation experi­ ments, this mutation was shown to confer high-level rifabutin resistance (increase in rifabutin MIC from 0.002 to 8 ixg/ml). Interestingly, an equivalent muta­ tion inducing high-level resistance to rifamycins was also present in several resistant isolates of Mycobacte­ rium tuberculosis (66). It is probable that the high clinical effectiveness of rifabutin in eradicating H. py­ lori can be related to the limited use of rifamycins in general and of rifabutin in particular. Indeed, the U.S. Food and Drug Administration-approved indications of rifabutin are restricted to the cure or prevention of M. avium complex disease patients with advanced human immunodeficiency (HIV) infection (84). 90

Nitrofurans Furazolidone and nitrofurantoin are nitroheterocyclic and nitroaromatic compounds that share many similarities with metronidazole both in their struc­ tures and modes of action (106). Like that of 5-nitro­ imidazoles, the biological activity of nitrofurans in­ volves enzymatic reduction of the parent compound to generate electrophilic radicals that cause damage to the DNA strands, thereby causing cell death (106, 162). These compounds have good in vitro activity against H. pylori ( M I C , 0.5 |xg/mL for furazolidone 90

517

and 1.0 (i,g/mL for nitrofurantoin) (50), and antimi­ crobial combinations that include nitrofurantoin have been shown to retain an excellent bactericidal activity against metronidazole-resistant strains of H. pylori (27). In addition, H. pylori does not appear to acquire readily resistance to this group of antimicrobial agents (57). Because of the high rates of metronidazole resis­ tance in H. pylori in many areas and the preserved activity of nitrofurans against metronidazole-resistant strains, these compounds have been recommended as possible alternative agents in H. pylori eradication regimens. Furazolidone combination triple therapy in particular has proved effective (55). Resistance to furazolidone and to nitrofurantoin was considered to be nonexistent, but two recent re­ ports have reported rates of resistance of 2 % in South Korea (89) and 4 % in Brazil (112). Interestingly, in the study by Kwon et al. (89), all seven furazolidoneand nitrofurantoin-resistant H. pylori isolates (MIC, 4 (Jig/ml) were also resistant to metronidazole (MIC, 2:16 ixg/ml). The resistance mechanisms are currently unknown but may differ from that of metronidazole since inactivation of rdxA, frxA, and fdxB genes, which are known to be involved in metronidazole re­ sistance, do not result in furazolidone or nitrofuran­ toin resistance (89).

ANTIMICROBIAL SUSCEPTIBILITY TESTING When Should It B e Performed? Culture and susceptibility testing for H. pylori are not commonly performed in routine practice. This may partly be due to the specific requirements of H. pylori in terms of growth and transport that render it difficult to culture and/or to the lack of availability of internationally agreed susceptibility methods for H. pylori. Economics has also had an effect, with all recent international consensus statements (99, 123) advocating empirical management of H. pylori infec­ tion in primary care, based on the use of indirect diag­ nostic tests (urea breath test, stool antigen test, or serology). The requirement for culture has been re­ stricted to selected situations in which resistance is more likely to be encountered, e.g., for patients in whom previous empirical therapies have failed. This strategy is fraught with the risk that the re­ sistance data gathered in many places might not re­ flect the background resistance rates in the commu­ nity over time. Because of the low number of treatment options available in patients harboring re­ sistant strains and owing to the increasing trend of resistance, one might question whether this probabi­ listic approach still holds true. Rather, pretherapy an-

518

MEGRAUD ET AL.

tibiotic susceptibility testing might have to be consid­ ered for guiding individual patient treatment, at least in areas with a high prevalence of H. pylori antibiotic resistance (e.g., more than 4 0 to 5 0 % resistance to metronidazole and more than 10 to 1 5 % resistance to clarithromycin). In any case, as a minimal require­ ment, surveillance of antibiotic resistance needs to be monitored over time to determine local/regional trends and to guide empirical treatment. Which Susceptibility Tests Should Be Used? The methods used for H. pylori susceptibility testing can be divided into standard phenotypic meth­ ods on the basis of diffusion or dilution, and genotypic methods. Phenotypic methods The dilution susceptibility testing methods can be performed in either agar or broth and yield a quan­ titative result. They are applicable to all antimicrobial agents that are tested as twofold serial dilutions of various concentrations, depending on the agents tested, and the MICs obtained are expressed in u,g/ ml (or in mg/liter). Agar dilution method. Agar dilution is a reliable technique, which is usually carried out as the reference method for evaluating the accuracy of other testing methods. Although this method proves well suited in large studies on stored strains, it is laborious, timeconsuming, and thus nonadapted for use in everyday practice. Recently, the National Committee for Clini­ cal Laboratory Standards (NCCLS) approved the agar dilution method as the test of choice for H. pylori (121). Standardization of clarithromycin susceptibil­

ity testing was proposed regarding the appropriate medium, supplementation, size of inoculum, incuba­ tion atmosphere, and appropriate time to read the plates. A quality control strain (H. pylori ATCC 43504) and breakpoint limits for several antibiotics were also proposed. In Europe, guidelines aimed at standardizing the antimicrobial susceptibility testing of H. pylori have also been drawn up by the European H. pylori Study Group (49), although these recom­ mendations have not been yet published in full. Com­ parison of the NCCLS and European standardized agar dilution methods is shown in Table 1. Susceptibility and resistance breakpoints (i.e., MICs at which an organism is deemed susceptible or resistant to the antibiotic using standard dosing regi­ mens containing that drug) have been defined for clar­ ithromycin (susceptible, < 0 . 2 5 u.g/ml; intermediate, 0.25 to 1 ixg/ml; resistant, > 1 ixg/ml) (121). Clarithro­ mycin resistance as determined by these criteria has indeed been found to accurately predict treatment failure (109). Metronidazole resistance measurement does not seem entirely reliable in foreseeing treatment failure. In contrast to clarithromycin, which displays a bimodal distribution of MICs with a clear separation be­ tween susceptible (MICs usually are ^ 0 . 0 3 u,g/ml) and resistant strains (MICs are > 1 u,g/ml), the distri­ bution of MICs of metronidazole is more gradual and continuous (110). Hence, the prevalence of resistance may vary greatly depending on the M I C at which the breakpoint is set. A resistance breakpoint of 8 u,g/ml was initially suggested for metronidazole (54). This cutoff value was derived from the breakpoint values recommended by the NCCLS for anaerobes and seemed to correlate with treatment outcomes in the initial studies in which a bismuth salt-metronidazole dual therapy was used (54). On the other hand, the

Table 1. Suggested modifications of standard methods for susceptibility testing of H. pylori Parameter Method Medium Inoculum Incubation Reading Quality control strain(s) Quality control limits

USA-NCCLS Agar dilution Mueller-Hinton agar plus aged (£2 weeks old) sheep blood (5% vol/vol) 1 X 1 0 - 1 X 10 CFU/ml (2.0 McFarland density) prepared from a 3-day blood agar plate 7

8

35°C; microaerobic atmosphere (gas systemgenerated microaerobic atmosphere) 3 days H. pylori ATCC 43504 Amoxicillin, ciprofloxacin, clarithromycin, metronidazole, tetracycline

European H. pylori Study Group Agar dilution Mueller-Hinton agar plus horse blood (10% vol/vol) 0.5 to 1 X 10 CFU/ml (4.0 McFarland density) prepared from a 2-day blood agar plate 37°C; microaerobic atmosphere 9

3 days H. pylori CCUG 38770, H. pylori CCUG 38771, H. pylori CCUG 38772 Amoxicillin, clarithromycin, metronidazole

CHAPTER 42 • ANTIBIOTIC SUSCEPTIBILITY AND RESISTANCE

impact of metronidazole resistance on the efficacy of current triple regimens seems more variable ( 3 7 , 1 1 0 , 153). Currently, there is no consensus as to the break­ points that should be recommended for metronida­ zole or for amoxicillin in the treatment of H. pylori infection. Broth microdilution method. This method has been used infrequently because of the difficulty of culturing H. pylori in broth (26). By supplementing brucella broth or Mueller-Hinton broth with fetal calf serum, this technique provides very acceptable results (58, 128). The advantage of this method over agar dilution is that it is more adaptable to automation and therefore decreases the workload. Correlations between the MICs determined by the broth microdilu­ tion method and the Epsilometer (E)-test have gener­ ally been found to be excellent for amoxicillin and clarithromycin (58, 1 2 8 ) . On the other hand, agree­ ment of results between the two methods was less satisfactory with metronidazole ( 7 1 % agreement within ± 1 log2 dilution). Moreover, major errors (i.e., H. pylori susceptibility by the E-test and resis­ tance by the microbroth dilution method) occurred in 15 (12.3%) of the 122 H. pylori strains. Although no direct comparisons were performed with the reference agar dilution method in this study, it is likely that these discordances reflect an overestimation of metro­ nidazole resistance by the E-test (see below). Breakpoint susceptibility testing. This method is a simplified version of either the agar or broth dilu­ tion method previously mentioned. Colonies of H. py­ lori are inoculated on a plate or in broth containing the critical concentration of antibiotic necessary to define resistance. Two different concentrations can be used when needed. This method is interesting because, being a direct measure of antimicrobial activity, it avoids errors associated with extrapolating disk diffu­ sion zone sizes and MIC results. It is also much easier to perform than the standard agar dilution method. One limiting factor is that ready-to-use plates are not commercially available and that preparation of anti­ biotic dilutions should be made extemporaneously at the site of testing. The breakpoint agar dilution method has been evaluated only for testing metroni­ dazole susceptibility compared to the disk diffusion method (12) and to the E-test (154). Overall, agree­ ment between methods was observed in 9 4 % of iso­ lates in both studies. In most cases, discordances were observed when subpopulations of H. pylori grew as isolated colonies within the inhibitory zone. The re­ producibility of the breakpoint agar method was not assessed in either of the two studies. Additional vali­

519

dation studies are required before this method can be recommended for susceptibility testing of H. pylori. Disk diffusion testing. The disk diffusion method is the easiest and most economical method for susceptibility testing of single isolates in routine practice. In this test, up to six disks can be placed on an agar plate; after incubation the zone of inhibition is measured and the isolate is recorded as resistant or susceptible on the basis of a cutoff point validated in the laboratory. The disk diffusion method has not usually been recommended for bacterial species re­ quiring long incubation periods ( > 2 4 h), because of the unstable patterns of antibiotic release from the disks. On the whole, studies comparing the disk diffu­ sion method to agar dilution and/or E-test have pro­ duced conflicting results for metronidazole ( 3 , 2 2 , 5 8 , 118, 154). This could be expected, as H. pylori is a slow-growing microorganism that needs specific growth conditions and thus methodological problems may often be encountered. For example, the choice of the medium, the age of the colonies, the inoculum size, the duration of incubation, and the threshold at which the interpretative breakpoint is set may all influence the outcome of the susceptibility tests (62, 68). Overnight anaerobic preincubation of the cul­ tures subsequently followed by microaerobic incuba­ tion (for 48 h) has also been shown to increase metro­ nidazole activity, suggesting that the redox potential may also influence the result (1, 2 0 ) . As mentioned in Table 2, all the parameters previously cited have often been found to vary extensively between studies. Nevertheless, an excellent agreement has usually been found between disk diffusion and the other testing methods for most classes of antibiotics other than the nitroimidazoles ( 2 1 , 58). One recent multicenter study carried out in France validated the disk diffusion method for eryth­ romycin and clarithromycin susceptibility testing of H. pylori compared to the E-test (42). Using an MIC resistance breakpoint of 1 |xg/ml for both macrolides, there was a 1 0 0 % agreement between both methods at breakpoint inhibition zones of 22 and 17 mm for clarithromycin and erythromycin, respectively. This excellent concordance between methods obviously re­ lates to the bimodal MIC distribution of the bacterial populations with a large gap between susceptible and resistant strains, as is the case for macrolides. At pres­ ent no standardized methods have been proposed for the disk diffusion test and it is felt that further work is needed before recommendations can be made for H. pylori, in particular for the susceptibility testing of metronidazole despite a promising study (168).

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>. rt -a 1 0 0 subcultures in vitro (62). The extent to which repeti­ tive in vitro culture reduces the infectivity of H. pylori in animals, again, appears to be bacterial strain and host dependent. The basis for decreased infectivity in in vitroadapted H. pylori isolates is poorly understood. Sev­ eral authors described changes in the phenotypes of H. pylori isolates having undergone either repeated subcultures in vitro or growth under different culture conditions. Repeated subculture of H. pylori resulted in various changes, including loss of lipopolysaccha­ ride (LPS) O side chains (58) and of catalase activity (52). Nevertheless, animal-adapted H. pylori isolates that had been passaged repeatedly in vitro did not exhibit differences in the LPS-associated Lewis anti­ gen phenotype (80) or in the production of major cel­ lular proteins (62). In addition, the use of random amplified polymorphic DNA analysis, a very sensitive fingerprinting technique for detecting rearrangements or point mutations in bacteria, did not reveal any major modifications in DNA arrangement of isolates following multiple passages in vitro (62, 80). Various studies have also been unable to show significant differences at the DNA level in H. pylori isolates after long-term colonization ( 2 0 , 4 9 ) . The ap-

585

CHAPTER 46 • IN VTVO ADAPTATION TO THE HOST

Table 1. Colonization of animal hosts by H. pylori isolates Animal host

Colonization''

0

H. pylori

r

Host

s t r a i n

G27 HAS-141

rf

HAS-145'' N6 SPM326/IL-8

+

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SSI

Mice: Swiss C57BL/6 Swiss C57BL/6 Swiss C57BL/6 C57BL/6 Swiss BALB/c BALB/c C57BL/6 C57BL/6 BALB/c BALB/c C57BL/6 C57BL/6 BALB/c

C57BL/6

Gl.l 26695

Swiss Gerbils Mice (Swiss) Piglets

Type

SPF SPF SPF SPF SPF SPF Euthymic SPF SPF SPF SPF SPF SPF SPF SPF SPF SPF SPF SPF Euthymic Germ-free Germ-free SCID SPF SPF SPF SPF Euthymic SCID SPF SPF Gnotobiotic

wu

Time p.i. (wk)

. (mean ± SD)

No. infected/total no.

2.88 ± 0.18