Pseudomonas Infection and Alginates: Biochemistry, genetics and pathology [1 ed.] 978-94-010-7319-6, 978-94-009-1836-8

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Pseudomonas Infection and Aiginates

PSEUDOMONAS INFECTION AND ALGINATES Biochemistry, genetics and pathology Edited

by

P. GACESA and N. J. RUSSELL Department of Biochemistry, University of Wales College of Cardiff, Wales

CHAPMAN AN 0 HALL LONDON· NEW YORK· TOKYO· MELBOURNE· MADRAS

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First edition 1990

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1990 Chapman and Hall

Softcover reprint of the hardcover 1st edition 1990 Typeset in IOpt Baskerville by SLarborough Typesetting ServiLes

ISBN-13: 978-94-010-7319-6 e-ISBN-13: 978-94-009-1836-8 DOl: 10,1007/978-94-009-1836-8 All rights reserved. No part of this publiLation may be reproduced or transmitted, in any form or by any means, electronic. mechanical, photocopying, recording or otherwise, or stored in any retrie"al system of' any nature, without the written permission of the copyright holder and the publisher, application for which shall be made to the publisher. British Library Cataloguing in Publication Data

Pseudomonas infection and alginatcs. I. Man. Pathogens: P.leutfO/l/OIl([.I aerugillo.l{/ I. GaLes a, P. (Peter). II. Russell, N.J. (!'.;icholasJ.) ＶＱＧＮｏｉＴｾ＠

ISBN-13: 978-94-010-7319-6 Library of Congress Cataloging-in-Publication Data

Pseudomonas infection and alginates: biochemistry, gcnetics, and pathology/edited by P. Gacesa and N. J. Russell. p. cm. I ncludes bibliographical references.

ISBN-13: 978-94-010-7319-6 I. P.leur/O/l/OIl([.1 aerllgillo.l{/ infections - Pathogenesis. 2. AlginatesPhysiological effect. I. Gacesa, P. (Peter). II. Russell, !'.;icholasJ. [DNLM: I. Alginates. 2. Pseudomonas Infections. WC 3301'974:)] QR201.P74P74 1990 616'.0145 -dc20 DNLMIDLC 89-23913 for Library of Congress CIP

Contents

Contributors Preface Introduction: pathogen P. H. CLARK

\,11 IX

Pseur/o/l/III/as anuginvs{/,

2 Clinical aspects of mucoid R. DINWIDDIE

an opportunist

PIl'uriorJ/onas (ll'l"ugillllsn

infections

:) The structure and properties of alginate P. GACESA and N. J. RUSSELL 4 Characteristics of mucoid

PSl'udomonas aeruginosa in vitro

I:)

29

and

/rl (lll'O

50

J. R. W. GOVAN 5 The microcolon} mode of growth in ,liDO - an ecological perspective J. W. COSTERTON, M. R. W. BROWN, I. LAM, K. LAM and D. M. G. COCHRANE 6 Adherence and the role of alginate N. R. BAKER

76

9!l

7 Immunology of alginate and other surface antigens in m lIcoid and non -mucoid PSI' Ildonw IWS ncruf!:i 1I0sa S. S. PEDERSEN, G. H. SHAND and N. H01BY

109

H The contribution of PsPUdOfll.IJIUlS (1Prugillosa alginate to evasion of host defence s. E. SMITH and J. A. SIMPSON

1:)5

9 Interactions of alginate with exoenz)'mes J. WINGENDER

I()O

vi

Contents

10 Biosynthesis of alginate A. NARBAD and P. GACESA, N. J. RUSSELL

181

11 Genetics of alginate biosynthesis in Pseudomonas aeruginosa D. E. OHMAN and J. B. GOLDBERG

206

12 Future prospects N. J. RUSSELL and P. GACESA

221

Index

229

Contributors

N. R. BAKER Department of Microbiology, The Ohio State University, Columbus, Ohio, USA M. R. W. BROWN Department of Pharmaceutical Sciences (Microbiology Research Group), Aston University, Birmingham, UK. P. H. CLARKE Department of Biochemistry, Cniversity College London, London, UK. D. M. G. COCHRANE Department of Pharmaceutical Sciences (Microbiology Research Group), Aston University, Birmingham, UK. J W. COSTERTON Biological Sciences, University of Calgary, Calgary, Alberta, Canada. R. DINWIDDIE The Hospital for Sick Children, London, UK. P. GACESA Department of Biochemistry, University of Wales, Cardiff, UK. J B. GOLDBERG Department of Microbiology and Immunology, University of California. Berkeley, California, USA. J R. W. GOY AN Department of Bacteriology, University of Edinburgh Medical School, Edinburgh, UK. N. H0IBY Department of Clinical Microbiology. Statens Seruminstitut, Copenhagen, Denmark. J LAM Biological Sciences, University of Calgary, Calgary, Alberta, Canada. K. LAM Biological Sciences, University of Calgary, Calgary, Alberta, Canada. A. NARBAD Department of Biochemistry, University of Wales, Cardiff, UK. D. E. OHMAN Department of Microbiology and Immunology, University of California, Berkeley, California, USA. S. S. PEDERSEN Department of Clinical Microbiology, Statens Seruminstitut. Copenhagen, Denmark. N. J RUSSELL Department of Biochemistry, University of Wales, Cardiff, UK. G. H. SHA:".ID Department of Clinical Microbiology, Statens Seruinstitut, Copenhagen, Denmark. J A. SIMPSON School of Biology and Biochemistry, Brunei University, Uxbridge, Middlesex, UK.

viii

Contributors

S. E. SMITH School of Biology and Biochemistry, Brunei University, Uxbridge, Middlesex, UK. J. WINGENDER Abteiling Mikrobiologie, Rheinisch-Westfalisches Institut fur Wasserchemie und Wassertechnologie, Mulheim, Ruhr, FRG.

Preface

The concept of this book arose out of an international workshop, which we organized and held at the University of Wales Conference Centre at Gregynog. The workshop was the first occasion on which researchers from all the different disciplines concerned with the extracellular virulence factors of mucoid strains of Pseudomonas aeruginosa in relation to cystic fibrosis (CF) had met to discuss this multifaceted problem. It was deemed a particularly timely moment to gather together experts for the exchange of facts, ideas and hypotheses. No formal abstracts were presented and no proceedings were published. But during the succeeding months the organizers were persuaded by a number of participants that a wider audience should benefit from what had proved to be such a fruitful cross-fertilization of expertise. Thus we moved from being workshop organizers to book editors, sure in the knowledge that at least we had a willing and enthusiastic set of contributors! It should be stressed, however, that this book is not a transcript of that workshop. Not all those participants are authors, and some new names have been added. Instead we have focused on alginate as an extracellular virulence factor of P. aeruginosa in CF pulmonary infections. Recent advances in the biochemistry and molecular genetics of alginate biosynthesis, as well as in our understanding of the basic defect in CF and isolation of the gene, mean that the book is even more timely than when first planned. We have sought to provide the first comprehensive coverage of mucoid P. aeruginosa dealing with clinical aspects of the organism and lung infections in CF; the non-mucoid to mucoid transformation in vitro and in vivo are discussed in relation to host responses to alginate; the structure, properties and biosynthesis, including genetics, of alginate are dealt with in detail and related to its chemical, physical and biological properties. No book is produced without a few tribulations, but these have been minor and we should like to thank our contributors publicly for their efforts. They have produced authoritative and up-to-date coverage of their particular areas. We have exercised a modicum of editorial change, aiming for consistency of treatment of the various aspects without dulling individual style. Previously, clinicians, bacteriologists, biochemists and

x

Preface

geneticists had not considered the topic in an integrated fashion. We hope, therefore, that the book will appeal to a wide audience, both clinicians and non-clinicians, from biologists to chemists. But above all we hope that it will help to further understanding of mucoid P. aeruginosa infections and provide much needed benefit to those who need it most the children and adults suffering from CF.

1

Introduction: Pseudomonas aeruginosa, an opportunist pathogen P. H. CLARKE

1.1 THE GENUS PSEUDOMONAS The genus Plewiolllonas is the major one of the family Pseudomonadaceae, which comprises a heterogeneous group of rod-shaped, Gramnegative, polarly flagellated bacteria. Pseudomonas strains are ubiquitous in soil and water and are able to grow in very simple media at the expense of a great variety of organic compounds; some are pathogenic for plants and animals. There have been many studies on the biochemical characteristics of pseudomonads and these properties have been widely used for classification and taxonomy. An important contrihution in this respect was the survey of the physiological and nutritional properties of the aerohic pseudomonads carried out hy Stanier et al. (1966); other taxonomists have followed this approach. However, there are still many problems in classification at both the genus and species levels. :\Iore recently, studies have been made on nucleic acid homologies. The G + C ratio for the genus as a whole ranges from 58 to 70o/c. Species recognized as Psewlollwflas were assigned to five sub-groups on the basis of rRNAIDNA homologies (Ballard 1'1 !Lf.. 1970; Palleroni et al., 1973; Palleroni, 1986). Many strains, which had been classified as Pseudomonas species on other criteria. could not readily be fitted into one of these sub-groups, which indicated that they might not be very closely related. De Vos and De Ley (1983) examined the intra- and intergeneric similarities of the rRNA of PsewlO1rwnas and XUlltholl101WS species. They concluded that the genus PSfUdotnonas had become a dumping ground for a variety of unrelated bacteria that were aerobic, Gram-negative and had polar flagella, and that it should be split into two or three separate genera. Woese et al. (1984) applied oligonucleotide cataloguing of 16S rRNA to PII'Ilt/olflonas species and confirmed the existence of the five distinct sub-groups of Palleroni 1'1 al. (1973). However, they concluded that none of the sub-groups were exclusive to recognized PSl'udolrlonas species and

2

Introduction

that 'not only is Pseudomonas an invalid phylogenetic unit in comprising five separate, unrelated groupings, but each ofthe separate groupings are [sic] non-valid as well in being too restricted a definition to encompass all the related bacteria that genealogically belong therein'. A more detailed account of current views on Pseudomonas taxonomy will be found in reviews by Palleroni (1975, 1984, 1986). Pseudomonas pyo(vanea, later renamed Pseudomonas aeruginosa, was chosen as the type species and various authorities agree that in any reorganization, in which other species are added or subtracted from the genus Pseudomonas, the type species should continue to be P. aeruginosa. In contrast to all the other species examined, P. aeruginos{l was found by Stanier et at. (1966) and Jessen (1965) to have a distinctive and internally uniform biotype. Because of its importance as a potential human pathogen, it is not surprising that P. {lfruginosa has received more attention from bacteriologists than have other less well-defined Pseudomonas speCIes. P. (lfl"uginosa can survive and multiply in dilute aqueous solutions and strains can be isolated not only from soil and water samples but also from contaminated solutions of eye drops, weak disinfectants, hand creams, hospital humidifiers and ventilators, brushes, mops and sinks (Lowbury, 1975). It is frequently found in clinical specimens from burns, surface wounds, urinary tract, ear and eye infections and also from cerebrospinal fluid in meningitis. Mucoid strains are most commonly isolated from the lungs of patients with the inherited disease cystic fibrosis (Doggett I'l al., 1966; Reynolds I't al., 1976). However, at nearly all sites of infection by P. al'l'llginosa there have been reports of the isolation of mucoid strains (Doggett, 1969; McCarthy et al., 1986; McAvoy I't al., 1989). The underlying condition in several of these types of infection is a chronic disease and it seems reasonable to assume that the mucoid forms are adaptations to the chronic disease state and are not induced by an abnormality specific to cystic fibrosis patients. In general, P. al'l'llginosa presents no problems to healthy individuals but is a potential pathogen for patients whose resistance is already impaired. It may become a particularly dangerous hazard in hospitals since it survives in so many reservoirs. In clinical bacteriology, references to Psl'tldolllOnas infection usually imply that P. aerugillosa has been identified and not one of the other Pseudolllonas species. 1.2 MORPHOLOGY AND GROWTH

P. {Ll'rllginosa isolates normally produce large, smooth colonies which have a tendency to spread over the surface of the agar. However, most strains are pleiotrophic and smaller colonies may appear in laboratory culture,

Morphology and growth

3

Fig. 1.1 No n-muco id and muco id morphology of P. aeru/5inusa co lonies on solid medium. (From Govan and Harris, 1986.)

particularly on minimal agar. Th e small cololIY \'arialI ts can usuall y be maintained as st.able sub-strains and are often selected for genetic studies, After prolonged growth on media wit h a high Glrbon content (alld/or low nit.roge n) very slimy colonies are produced by l1I ost strains. Th ese slimy co lonies are quit e diffe re nt from th e mllcoid co lonies characteristic of cyst.ic fibrosis a nd other infection s. Th e t.el'ln 'nlu c! Jid ' is restrict.ed to st.rains which prod uce copious amount s of alginate on COlli pl ex agarbased m edia aft e r overnight growth (Phillips, 19h9; Fyfe and Gman , 19HO). Fig, 1.1 shows th e char;lcteristi c a ppea ra nce of large Illu coid colonies al1long a background of norl1lal colonies of P. Ill'l"IlgillO.l(1. Isolates from cyst ic fibr osis and other infections usually include both Illucoid and no n-Illu co id fo I' III s, Ea rli e r c1assificat.ion s included pigl1lentation as a generic cha racte r but this has beco llle less siglIificant \\·it.h th e inclusion of Illan y non-pigm e nted species in the genus. SOl1le of t.he fluor escent. pigl1lents prodll ced by

4

Introduction

P. aeruginosa, and related Pseudornonas species, are strong iron chelators and are produced in large amounts in media of low iron content. The iron-siderophore complex is readily taken up by the cells and thus facilitates growth at low iron concentrations. The most well-known pigment produced by P. aeruginosa is the blue-green pyocyanin, which gave the species the earlier name of P. pyocyanea. This is one of a number of phenazine pigments which may be produced in various amounts in different media. The phenazines are secondary metabolites derived from the biosynthetic pathway for the aromatic amino acids. The amount of pyocyanin, and the intensity of colour, is related to the amount of phosphate in the medium and the major carbon source for growth. Some strains of P. aeruginosa produce little or no phenazine pigments under any growth conditions. P. aeruginosa is distinguished from other fluorescent pseudomonads in being able to grow at 43°C, but not at 4°C, with an optimal growth temperature of 37°C. Electron micrographs reveal polar pili which act as receptors for various bacteriophages (Bradley, 1972) and may assist in the attachment of invasive bacteria to cell surfaces. Transferable plasmids have been shown to determine conjugative pili, which are also receptors for donor-specific phages (Bradley, 1980, 1983). P. aerugino.'(l is known to have intrinsic resistance to many antibiotics and it has been suggested that this may be due to impermeability of the outer cell layers. The cell envelope has the general structure of Gramnegative bacteria with an inner cytoplasmic membrane, a rigid peptidoglycan layer and a complex outer membrane comprised of the three major constituents: phospholipids, lipopolysaccharide and proteins. Studies on the composition and function of these structures were reviewed by Meadow (1975) and Nikaido and Hancock (198f)). The core structure of the lipopolysaccharide and its biosynthesis have been investigated by the use of defective mutants, with altered sensitivities to phages and bacteriocins, for the analysis of the oligosaccharides (Rowe and Meadow, 1983). The outer membrane contains a large number of proteins with a few dominant species. Nicas and Hancock (1983) concluded that protein F was the major porin protein and showed that a mutant lacking protein F had the normal permeability to a chromogenic cephalosporin reduced by 80%. It was thought earlier that the porin channels of protein F were larger in diameter than those of the enteric bacteria and this made it difftcult to explain the relative impermeability of P. aeruginosa. However, it is now suggested that most of the porin channels may be dosed or that a large fraction are small in diameter. Nikaido and Hancock (1986) suggest that the lipopolysaccharide molecules may be very dosely packed, as the result of divalent cation binding of the many phosphate -residues, and that this, together with the

Bacteriophages and pyocins

5

small size of the porin channels, makes the outer membrane impermeable to both hydrophobic and hydrophilic antibiotics.

1.3 PLASM IDS

Plasm ids occur frequently in many Pseudomonas species. Those studied in most detail are the catabolic plasm ids and the conjugative and drugresistance plasmids. They have been classified into 13 different incompatibility groups (Jacoby, 1986). The drug-resistance plasmid RPI was first isolated from a P. {lI'Tuginosa infection of burns (Lowbury et al., 1969). The resistance genes carried on plasmids add appreciably to the already considerable intrinsic resistance of P. aeTuginosa. Plasmids in this group present considerable problems ill clinical infections since they have a broad host range and can be transferred into P. aeruginosa from bacteria reservoirs such as Klebsiella. It has been found that the various incompatibility groups contain plasmids determining a variety of phenotypes. For example, the conjugative plasmid FP2, used for genetic mapping, belongs to the IncP-l group, which contains several drug-resistance (R) plasmids. Homologies have also been reported between R plasm ids and plasm ids carrying genes for degradative pathways, suggesting a common evolutionary origin. Some of the transferable drug-resistance plasmids can also promote chromosomal transfer (see below).

1.4 BACTERIOPHAGES AND PYOCINS

Many strains of P. aeruginosa are lysogenic and release temperate bacteriophages under particular growth conditions. Virulent (nontemperate) phages can be isolated from sewage and other natural sources. Many types are known, including DNA phages with complex structures, filamentous single-stranded DNA phages and spherical RNA phages (see Holloway and Krishnapillai, 197;'». Several temperate (or semitemperate) phages have been used for transduction. ｾｲｯｰｨ｡ｧ･ｳ＠ may exist within the bacteria as autonomous entities or be integrated into specific sites on the chromosome. The bacteriocins of P. aeruginosa are referred to as aeruginocins or pyocins, the latter being the most commonly used designation. They have been classified into two main types: the S-type, of amorphous appearance and sensitive to proteolytic enzymes; and the R-type, having the structure of degenerate phages. The genetic determinants of several pyocins have been mapped on the chromosome (Holloway and Krishnapillai, 1975; Sano and Kageyama, 1984). Pyocins are very easy to obtain and, together with lytic phages, have been used for typing clinical strains (Lowbury,

6

Introduction

1975). Mutants that are resistant to specific pyocins or phages were found to be altered in the composition oftheir lipopolysaccharides (Meadow and Wells, 1978).

1.5 METABOLISM P. aeruginosa is an aerobic organism with characteristically oxidative

metabolism. Nitrate can replace oxygen as an electron acceptor, although growth is slower. Substrate-level phosphorylation OCCllrs in one step of arginine catabolism and this is the only exception to the capture by oxidative phosphorylation of the chemical energy released in catabolic pathways. Glucose is used as a carbon source for growth and is metabolized predominantly via the Entner-Doudoroff pathway, which was first established in P. sacdwrojJhila (Entner and Doudoroff, 1952). In this pathway glucose is metabolized via glucose 6-phosphate and 6-phosphogluconate to 2-keto 3-deoxy 6-phosphogluconate, which is split by the Entner-Doudorofl pathway aldolase enzyme to give pyruvate and glyceraldehyde 3-phosphate (see Chapter 10). Glucose can also be oxidized directly to gluconate and 2-ketogluconate prior to phosphorylation. A few other sugars are catabolized by similar reactions. Although few carbohydrates are utilized by P. aemginosa there are many other carbon compounds, including aliphatic hydrocarbons, carboxylic acids, hydroxy acids and aromatic compounds, which can be used for growth. The tricarboxylic acid cycle is the main route for terminal respiration and also provides intermediates for biosynthesis. The glyoxylate cycle, with its key enzymes - isocitrate lyase and malate synthasereplenishes the pool of tricarboxylic acid cycle intermediates during growth on two-carbon compounds, and other pathways fulfil a similar role (Clarke and Omston, 1975). Many amino acids can be used as growth substrates, and regulatory systems have evolved which prevent the recycling of newly synthesized amino acids but allow catabolism to take place when amino acids are freely available in the environment (Clarke, 1979). Intermediates of the catabolic pathways for these various compounds provide the building blocks for biosynthetic reactions. Pathways for the biosynthesis of amino acids are broadly similar to those of the enteric bacteria but there are some interesting variations (Palleroni, 19S6; Phillips, 1986). Monosaccharides are constituents of a variety of macromolecules including carbohydrates, glycoproteins and lipopolysaccharidcs. Studies with mutants have elucidated the core oligosaccharide of membrane lipopolysaccharides (Rowe and Meadow, 1983). More recently, genetic and biochemical studies have been carried out on alginate biosynthesis (see Chapters 10 and II).

Genetics

7

1.6 GENETICS

Genetic analysis of PSI'IU{OIllIlIl(lS is still a long wa) frOIll the fille detail of that of ｪ＼ｾｳＨＧｨ･ｲｩ｡＠ coli but there is an increasing understanding of genetic structure and functioll within this group. Although there arc oln'iuus similarities with E. co/i, there are also differences \\hich reflect di£lerellces in ecologv and metabolism. Genetic studies han' focused mainly Oil P. (Jl'I'liginusll, but P. jmtid({, P. ('('/JlLcia and the plant pathogens have also been studied. 1 .6.1 Conjugation

Genetic analysis of P. (J(')'ugirw.I(l began with the demollstration b\' Holloway (1955) that auxotmphic markers could be trallsferred b\ conjugation. The conjugative plasmid FP2, which also carries IIllTUl1\ resistance, mobilizes the chromosome of strain PAO frOIll a single site and was used to map the early markers. Several other conjugati\e plasmids were isolated but these had the sallle origill of trailS fer, IIOlle gave libstrains and markers entering later than ;)() min could not be mapped accurately. A few F plasm ids were found to transfer later genes but have not been used systematically for genetic mappillg. A major advance was the discover), that va riant s of the d rug ｾ＠ resistance plasmid R6H could mobilize the PAO chromosome (Haas and Iiollo\\,;[v, 1976). Plasmid R6HAj mobilizes the chrolllosome from lllany poinh, transferring short regions, of about 1() min, in either directioll. Although this plasmid could not be used to map all regions of the chro1llosonw, it did make it possible to measure lillkage of earh and late markers and to establish circularity (Royle ft 0/., 19H I). An additiollal use of R plasm ids for mapping ca1lle from the finding that ｬ･ｰｲ｡ｴｵｾｳｮｩｶ＠ nllltallts of plasmid R6H could illsert into the chromosollle ullder selectioll al Ihe ｮｯｾｰ･ｲｬｩｳｶ＠ temperature to give ｉｬｦｲｾｴｹｰ･＠ strains .. \nothel' Illethod of obtaining Hfr donors inl'()lved the illsertion of a plasllIid illto the chromosome by having copies of a trallsposoll present on both the chromosome and the plasmid (IloIlO\\a\, 19H(i). "\11 extellsioll of these methods was the generation of Htr straills In the insertion of a ｴ･ｬｰｲ｡ｵｾｳｮｩｶ＠ Illutallt of RliH carnillg a transposon (see below). Transposon llIutagenesis has also been used to generate Illutallts of chromosomal and plasmid genes. 1.6.2 Transduction

The most widely used transducing phage fin straill PAO is F 1 Hj, or its \'ariant F 1 l6L which gives higher transduction frequencies for lin ked

8

Introduction

markers. These phages promote generalized transduction in strain P AO and have also been used for other P. aeruginosa strains (e.g. Brammar et al., 1967). Transduction has provided detailed information on gene linkage over regions comprising 1-2% of the chromosome and has been useful in investigations of the chromosomal arrangement of genes for catabolic and biosynthetic pathways. 1.6.3 The chromosome map

With a combination of the several conjugation methods, supplemented with transductionallinkage analysis, over 200 genes have been located on the chromosome of P. aeruginosa P AO. Details and further references will be found in reviews by Holloway and Matsumoto (1984) and Holloway (1986). O'Hoy and Krishnapillai (1987) have now revised the time-scale of the PAO map (see Fig. 11.2). Hfr donors were constructed using a temperature-sensitive mutant of R68, which was loaded with transposon Tn2521, and by selecting for streptomycin resistance at 43°C. The donors obtained were stable, had a high efficiency of transfer and the transfer origins were dispersed around the map with either polarity. Thus it was possible to obtain precise time-mapping data and O'Hoy and Krishnapillai (1987) have recalibrated the complete map to 75 min. This is a major achievement and will give greater precision over all regions of the chromosome. Plasmid R68.45 and related chromosome-mobilizing plasmids are able to pick up chromosomal genes to form R-prime plasmids. This has advantages in investigating chromosomal homology between different strains of P. aeruginosa. With a detailed map for strain PAO available, it is now possible to take other strains of P. aeruginosa and to select R-prime plasmids which can be used to investigate particular regions of the chromosome. 1.7 REGULATION OF GENE EXPRESSION

Biosynthetic genes of P. aeruginosa are seldom as tightly clustered as in E. coli and this reflects the different patterns of regulation. For example, the genes for tryptophan biosynthesis, which form a single linkage group in E. coli, are arranged in P. aeruginosa as three groups of two genes and one single gene. The early enzymes coded by the gene pairs trpEG and trpDC are repressed by tryptophan, but tryptophan synthetase, coded by trpAB, is induced by its substrate, indole glycerol phosphate. The regulation of tryptophan catabolism is also of interest and the early enzymes of the pathway are induced by an intermediate of the pathway, kynurenine (Clarke and Ornston, 1975; Crawford, 1986; Phillips, 1986).

The wider perspective

9

The genes for catabolic pathways are usually arranged in clusters which are regulated independently. For complex pathways the enzymes for the early steps of a peripheral pathway may be induced by their substrates, or early intermediates, as is the case for tryptophan catabolism. This allows many different compounds to be fed into a common pathway whose enzymes are regulated by the later intermediates (Clarke and Ornston, 1975). Positive control of gene expression has been described for Pseudomonas species, for example the amidase of P. aeruginosa PAC (Farin and Clarke, 1978; Cousens et al., 1987). 1.8 GENE CLONING

Cloning of P. aeruginosa genes in E. coli has the advantage of employing well-defined systems but the disadvantage that many P. aeruginosa genes are poorly expressed in E. coli. Several cloning vectors based on P. aeruginosa plasmids have been developed (see Mermod et ai., 1986). The most useful appear to be those derived from the broad host-range plasm ids since they can be maintained in both P. aeruginosa and E. coli. This is a field which is developing very rapidly and will make it possible to sequence many more genes and to study the regulation of expression of P. aeruginosa genes in vitro. 1.9 VIRULENCE FACTORS

P. aeruginosa strains produce a range of toxins, enzymes and pigments that contribute to pathogenesis. These include exotoxin A and exoenzyme S, both of which inhibit protein synthesis in susceptible cells. Several extracellular proteinases may be produced, including elastase and an alkaline proteinase. Other products related to virulence include phospholipase A and haemolysins. The significance of the various P. aeruginosa virulence factors was reviewed by Nicas and Iglewski (1986). The role of alginate in pathogenicity is the main theme of this volume and will be discussed in the following chapters. Recent advances in the biochemistry and genetics of P. aeruginosa are contributing to an understanding of the structure and function of alginate and are helping to elucidate it's mode of action in disease. 1.10 THE WIDER PERSPECTIVE

The importance of understanding the role of P. aeruginosa as a pathogen should not allow us to lose sight of the fact that these bacteria thrive in soil and water, utilizing organic compounds at very low concentrations. By metabolizing many different aromatic and aliphatic compounds they

10

Introduction

contribute to general metabolic recycling and playa role in cleaning up the natural environment. The enzymes involved in these reactions also have considerable potential for use in biotechnology, for biotransformation reactions which are difficult to carry out by conventional chemical methods.

REFERENCES Ballard, R. W., Palleroni, N. J., Doudoroff, M., Stanier, R. Y. and Mandel, M. (1970) Taxonomy of the aerobic pseudomonads: Pseudomonas cepacia, P. marginata, P. allicola and P. caryophylli. j. Gen. Microbial. 60, 199-214. Bradley, D. E. (1972) Shortening of Pseudomonas aeruginosa pili after RNA-phage adsorption. j. Gen. Microbial. 72, 303-19. Bradley, D. E. (1980) Morphological and serological relationships of conjugative pili. Plasmid 4, 155-69. Bradley, D. E. (1983) Specification of the conjugative pili and mating systems of Pseudomonas plasmids.j. Gen. Microbial. 129,2545-56. Brammar, W. J., Clarke, P. H. and Skinner, A. J. (1967) Biochemical and genetic studies with regulator mutants of the Pseudomonas aeruginosa amidase system. j. Gen. Microbiol. 47, 87-102. Clarke, P. H. (1979) Regulation of enzyme synthesis in the bacteria: a comparative and evolutionary study. In: Goldberger, R. F. (ed.) Biological regulation and development. Vol. 1: Gene expression, Plenum, :'IIew York, pp. 109-70. Clarke, P. H. and Omston, L. N. (1975) Metabolic pathways and their regulation: Part I. In: Clarke, P. H. and Richmond, M. H. (eds) Geneticsalld Biochemistryo{ P.wudomonas. Wiley, Chichester, pp. 191-261. Cousens, D.]., Clarke, P. H. and Drew, R. E. (1987) The amidase regulatory gene (amiR) of Pseudomonas aeruginosa.j. Gen.1Hicrobiol. 133,2041-52. Crawford, I. P. (1986) Regulation of tryptophan synthesis in Pseudomonas. In: Sokatch, JR. (ed.) The Bacteria. Vol. X: The biology of Pseudomonas, Academic Press, New York, pp. 251-63. De Vos, P. and De Ley,]. (1983) Intra- and intergeneric similarities of Pseudolllonas and Xantlul1Iwnas ribosomal ribonucleic acid cistrons. llll.j. S.vsl. Baclniol. 33, 487-509. Doggett, R. G. (1969) Incidence of mucoid Pseudomonas aerugillosa from clinical sources. Appl. Microbiol. 18,936-7. Doggett, R. G., Harrison, G. M., Stillwell, R. N. and Wallis, E. S. (1966) An atypical Pseudoillonas aeruginosa associated with cystic fibrosis of the pancreas. j. Pediall'. 68, 215-21. Entner, N. and Doudoroff, M. (1952) Glucose and gluconic acid oxidation of Pseudomonas s(lcc/wrojJhila.j. Bioi. Cheill. 196,853-62. Farin, F. and Clarke, P. H. (1978) Positive regulation of amidase synthesis in Pseudoillonas aeruginosa. j. Bacleriol. 135, 379-92. Fyfe,]. A. M. and Govan,]. R. W. (1980) Alginate synthesis in mucoid PseuriolllOll(lS aeruginosa: a chromosomal locus involved in control. j. Gen. Microbiol. 119, 443-50. Govan,]. R. M. and Harris, G. S. (1986) Pseudoillonas aeruginoS(l and cystic fibrosis: unusual bacterial adaptation and pathogenesis. i'dicrobiof. Sci. 3, 302-8.

References

11

Haas, D. andllolloway, B. 'Ii\'. (I ｾＩＷＶ＠ Chromosome mobilization by the R plasmid R68.45: a tool in P.Il'udolllOllll.l g-enetics. ,'101. Cm. Gm!'t. 158, 229-:l7. Holloway, B. \\1. (195}) Cenetic recombination in PI!'lldOlnOIiIlS aemgillosa. J. Cm. M icrobiol. 13, ＵＷＲｾＸＱＮ＠ Holloway, B. W. (1986) Chl'Ol1Iosome mobilization and genomic organization in X: The biology 01 Psewlm//o/Ul.I. In: Sokatch .J. R. (ed.) Th!' 8orteria. ｾﾷｉＮ＠ PSeudOIllO//({.\, Academic Press, New York. pp. ＲＱＷｾＴＨＩＮ＠ Holloway, B. W. and Krishnapillai. V. (197!)) Bacteriophag-es and bacteriocins. In: Clarke, P. I I. and Richmond, :VI. II. (eels) Gelle/irs and Biochelllistry of PSI'I1r/III/IO//(/.I, Wiley, Chichester, pp. Ｙｾ＠ 132. [[olloway, B. W. and :\Iatsllmoto, I I. (1984) P.II'UdO/l/OIW.\ alTllgillos{I. Cn/f/. Mllps 3, ｉｾｈＷ＠ . .Jacoby, G. A. (1986) Resistance plasm ids of PSI'/UIOIlIOIIII.I. [n: Sokatch,.J. R. (cd.) The Boc/nio. Vol. X: The I,iolog)' of PI'('udOlr/OIlIl,\. Academic Press, New York. pp. ＲＶＵｾＹＳＮ＠ Jessen, O. (19fi5) P.li'lulo 11/0 II 0,\ alTl/gil/o,la ol/(l ollin Grfl'll Fll/oIP,I(NII P.II'WiOINOllwl.l. A /axol/omic .I/I/dy, Munksg-ard, Copenhag-en. Lowbury. E. J. L. ( 1975) Biological importance of P\(/1do/t/ol/(/1 (lI'IIIfi/"OI!l: medical aspects. In: Clarke, P. II. and Richmond, :V1. I I. (eds) Gmetics (Iud Bio(lIelll/.111\' ' oIP.,eur/oIl/IIIIOS, Wiley, Chichester, pp. ＺｬＷｾＶＵＮ＠ Lowbury. E . .J. L., Kidson, A .. Lilly. II. A., Avdiffe. C. A . .J. and/ones, R. J (1%9) Sensitil'ity of Pleur/OI/WIlIlS OfrugiIlO.II/ to antibiotics: emergence of strains highly resistant to carbenicillin. LIIIIIPI ii, ＴＸｾＢＲＮ＠ McAvov, M. J, J\:e\\'ton, V., Paull. A., Morgan. J, Gaccsa, P. and Russell, !\'. J (1989) Isolation of mllcoid strains of' !'snlllollWI/IlSlIemgillo,11I from non-cystic fibrosis patients and ch,u'acterization of the structure of their sC(Teted alginate ..J. Med. ;l"linIJhiol. 28,183-9. ｾＡ｣ｃ｡ｲｴｨｹＬ＠ Y. P., Rosenberg, G., Rosenstein, B. J and lIubbard. \'. S. (1986) :\1 lIcoid Plelidoll/OIlIl.1 111'1 IIglll 0.1 II frolll a patient ,,'ithout cystic fibrosis: implications and rev'iew of the literature. PFililllr. Infeci. Dis. 5, ＲＵＶｾＸＮ＠ :\leadoll'. P. :V!. (1975) \"all and membrane structures in the g-enus p.lewlo/I/o/III,I. In: Clarke, P. I I. and Richmond, 1\1. II. (eds) (;l'IItlil'l 1I11r/ Bio{helll/sirv of P.lfUr/OIllOIlIlS. Wiley, Chichester, pp. !i7 - ｾＩＸＮ＠ :\leadow. 1'. :\1. and Wells. P. L. (I ｾＩＷＸ＠ Receptor sites for R-type p}"ocins and bacteriophage E7') in the core part of the lipopolysaccharide of I'smrlolllollll,\ lII'rugi II 0.1 II PAC I ..J. (;1'1/. ,Hirl'llhiol. 108, :\:):)-43. Mermod. !\'" Lehrbach. P. R., Don. R. I I. and rimmis. K. 1\. (I !)8fi) Gene cloning and manipulation in I'.lelidolllOIlIl,I. In: Sokatch,.J. R. (ecl.) The BII(INili. Vol. X: Tltl' hilllll,!,'" o/,PII'//{io/l/OIIII.I, Academic Press, I\e\\ York, pp. ＳＲＵｾＮ＠ :\icas, T. L. and Ilancock. R. E. W. (I ｾＩＸＳ＠ PII'IIr/O/l/OIllIS IIl'l"IIgillolll outer membrane permeabilitv: isolation of a porin F-deficient mutant..J. B(lr/Niol.

153. ＲＸＱｾＵＮ＠

Nicas, T. Land Ig-Ie\\'ski, B. I I. (1986) Toxins and virulence factors of PseudolllolillS III'mgillosli. In: Sokatch.J R. (ed.) The Bllrlfrill. Fol. X: 1'111' billlog)' O/,VII'lidoIllOIlIlS, Academic Press. l\ew York, pp. ＱＹＵｾＲ＠ :). l\;ikaido, H. and Hancock, R. E. \\'. (1986) Outer membrane permeability of PlfllllolIlIJllllS (1I'lI/giIiUSIi. In: Sokatch.J. R. (ed.) The Bllrltrill. Fol. X: The billlu,!,'" 11/1'.1'1'1111011101111.1', Academic Press. J\ew York, pp. ＱＴＵｾＹＳＮ＠ O'Hov, K. and Kl'ishnapillai, V. (I ()87) Recalibration of the PI/,"do/l/Ollll.lllfrugillola strain 1';\,0 (hromosotlle map in time units using- ｨｩｧＭｦｲ･ｱｵｮ｣ｶｯｾ＠ recombination dOllors. Gelldin 115, (i ｉｾ＠ 18.

12

Introduction

Palleroni, ]\; . .J. (1975) General properties and taxonomy of the genus P5eudomona.l. [n: Clarke, P. H. and Richmond, M. H. (eds) Genetic5 and Bioehemi5try of" Pseudomonas, Wiley, Chichester, pp. 1-36. Palleroni, :--.I . .J. (1984) Genus l. P.Il:udorrwna,l. In: Krieg, K R. and Holt,.J. C. (eds) Bergey's i'vlanual of 5ystmwtie Baeteri%gy, VoJ. I, Williams and Wilkins, Baltimore, pp. 141-99. Palleroni, N. J. (1986) Taxonomy of the pseudomonads. In: Sokatch, J. R. (ed.) The Bacteria. Vol. X: The biology of" Pseudomona5, Academic Press, ]\;ew York, pp.3-25. Pallcroni, ;\J.J., Kunisawa, R., Contopoulou, R. and Doudoroff,:\1. (1973) Nucleic acid homologies in the genus Pseudomonas. Int.]. 5yst. Baetrriol. 23, 333-9. Phillips, A. T. (1986) Biosynthetic and catabolic features of amino acid metabolism in Fwudumonas. In: Sokatch,J. R. (ed.) The Bacteria. Vol. X: The biulogy Pseudomonas, Academic Press, ]\;ew York, pp. 385-437. Phillips, I. (1969) Identification of PseurlurrwlIl1.\ anuginosa in the clinical laboratory.]. ,'vIed. Alicrubiol. 2, 9-16. Reynolds, H. Y., di Sant'Agnese, P. A. and Zierdt, C. H. (197G) Mucoid P,ll'IIdumonas aeruKinusa: a sign of cystic fibrosis in young adults with chronic pulmonary disease.]. Am. Mell. A5soe. 236, 2190-2. Rowe, P. S. N. and Meadow, P. M. (1983) Structure of the core oligosaccharide from the lipopolysaccharide of Pseudomonas aenLKino.l{/ PAC I R and its defective mutants. 1:"ur.]. Biocizem. 132,329-37. Royle, P. 1.., Matsumoto, H. and Holloway, 8. W. (1981) Genetic circularity of the PseudumoNas aerugin())(1 PAO chromosome. I Raeteriol. 145, 145-55. Sano. Y. and Kageyama, M. (1984) Genetic determinant of pyocin AP41 as an insert in the Pseudomonas ae/'uginosa chromosome. I Bacteriol. 158,562-70. Stanier, R. Y., Palleroni, N. J. and Doudoroff, M. (1966) The aerobic pseudomonads: a taxonomic study.]. Gen. Micrubiol. 43, 159-271. Woese, C. R., 8lanz, P. and Hahn, C. M. (19H4) What isn't a pseudomonad: the importance of nomenclature in bacterial classification. SySI. AjJpl. Alicrobiol. 5, 179-95.

or

2

Clinical aspects of mucoid

Pseudomonas aeruginosa infections R. DINWIDDIE

2.1 CYSTIC FIBROSIS - CAUSE AND SYMPTOMS Cystic fibrosis (CF) is an autosomal recessive disease which is most commonly found in Caucasian peoples. In the UK it affects about 1 infant in 2000 (Kuzemko, 1986). The recently isolated gene for CF (Rommens, et al., 1989) is located on the long arm of chromosome number 7 (Wainwright et aZ., 1985), and genetic probes which lie close to it are already in use for the purposes of prenatal diagnosis (Farrall et aZ., 1986). The basic biochemical abnormality probably lies at the cellular level but has not yet been identified (Williamson et ai., 1983). It is thought to involve a defect in the transport of chloride ions across membranes (Quinton, 1983). The major clinical and diagnostic features arise almost entirely from the abnormalities affecting the exocrine glands. The systems most affected are the respiratory tract and the digestive tract. The sweat shows high levels of sodium and chloride ions and the patient often tastes salty when kissed. These abnormalities of sweat electrolytes form the basis of the most specific diagnostic test so far available (Gibson and Cooke, 1959). The disease is life-long and ultimately results in progressively deteriorating lung function as a consequence of chronic infection with a number of bacterial pathogens, particularly Staphylotli(cus aureus, Haemophilus injiuenzae and Pseudomonas aeruginosa. Mucoid strains of P. aeruginosa secrete large quantities of an extracellular polysaccharide called alginate; in conjunction with viscid lung secretions this significantly impairs pulmonary function and makes it difficult for the patient to eradicate the organism from the lungs. This infection often has a relentless and insidious course and is a major factor in the pulmonary disease ofCF. The vast majority of patients also have nutritional problems secondary to abnormal pancreatic function. As more and more reach adult life other problems such as cirrhosis of the liver and male infertility become evident. Despite these many difficulties the long-term prognosis has improved dramatically in the last thirty years.

14

Clinical aspects of mucoid infections

2.2 GENETICS AND PRENATAL DIAGNOSIS OF CYSTIC FIBROSIS The incidence of CF varies in different racial groups and in different countries. Among Caucasoid Europeans the incidence has been reported as between I: 2100 and I: 8000 (Table 2.1). The incidence among Caucasians in the USA is similar to that in Europe. It is much less comlllon, although not rare, among Negroes in the USA. It is probably not uncommon among the peoples of Arabia and the Indian subcontinent, but it is very rare in the Chinese and Japanese. The current incidence in the UK suggests a carrier rate for the gene of 1 in 20. Because carriers are entirely healthy and since there is as yet no reliable detection test, this is only an estimate. It has been postulated that the carrier state may have been associated with an increased resistance to pulmonary tuberculosis in the past and that this could have been due to an increased secretion of hyaluronidase by the CF carrier's fibroblasts (Meindl, 1987). However, the persistence of the gene at such high incidence is not dearly understood since there appears to be no other oln'ious current heterozygote advantage. It can be calculated that at this incidence in the countries of the European Community and the USA there must be approximately 25 million carriers of the CF gene. Prenatal diagnosis, utilizing DNA polymorph isms linked to the CF gene, is now possible at 8-10 weeks gestation by chorionic villus sampling. Amniocentesis is also used at 18 weeks in order to detect abnormalities of gut microvillar enzymes and is increasingly successful (Brock, 1988) . .\ieonatal screening for CF is available; this makes use of the fact that trypsin leaks back from the foetal pancreas into the bloodstream even in utPrO, which leads to elevated enzyme levels at birth and during the first 3 months of life. This is best estimated by measuring immunoreactive trypsin in the dried blood spots routinely collected for neonatal screening for other diseases, including phenylketonuria and hypothyroidism (Kuzernko and Heeley, 1983). In the affected infant immunoreactive

Table 2.1

Incidence of cystic fibrosis in different countries and races

Rare

Country

Incidence

Caucasians Caucasians Caucasians Caucasians Jewish Negro Mongoloid

UK Italy Sweden USA Israel USA Hawaii

1/2100 1/2600 1/8000 1/2400 1/5000 1/17000 1/90000

Reference

Kuzemko (1986) Righetti et al. (1976) Selander (1962) Kram et al. (1962) Levin (1963) Kulzycki and Shauf (1974) Wright and Norton (1966)

Pathophysiology ofpulmonary infection in cystic fibrosis

15

trypsin levels are elevated during the first 3 months of life, following which they fall to normal or sub-normal levels. As yet there is no conclusive evidence that diagnosis by neonatal screening significantly improves long-term survival. There is also no evidence that neonatal screening reduces the age or frequency of colonization of the lungs with P. aeruginosa (Williams et ai., 1988a). 2.3 PATHOPHYSIOLOGY OF PULMONARY INFECTION IN CYSTIC FIBROSIS Lung disease remains the major complication of CF. The physiology of the lung disease in this condition is extremely complex but mucoid strains of P. aeruginosa remain the principal pathogenic organism (Hl'liby, 1982). Recent work has led to an increased understanding of the complex mechanisms involved in the changes in the lung occur from the stage of early bacterial infection to the later onset of major progressive deterioration in lung function. This work has also led to the development of new and more specific treatment regimes and the evolution of more effective antibiotics for the management of the pulmonary complications. These advances have resulted in an increase in life expectancy for patients with CF in both childhood (Wilmott et ai., 1983) and adulthood (Penketh et al., 1987; Batten, 1988). Despite these advances a great deal remains to be learned about the lungs in CF if further reduction in morbidity and mortality from the condition is to be achieved. 2.3.1 Bacterial infection The lungs in the CF child are thought to be normal at birth (Reid and De Haller, 1964). Bacterial colonization, however, frequently occurs at an early stage and this sets in train a sequence of complications that are secondary to inflammatory obstruction and non-distribution of ventilation, which lead to chronic bronchitis and bronchiolitis, common presenting features of the disease in this age group. A response to this problem is an increase in mucus secretions within the lung; these have a high viscosity and lead to further airway obstruction, resulting in micro-abscess formation with local areas of over-inflation and air trapping (Tomashefski et al., 1983). These changes contribute to the progressive decline in lung function which is seen as age increases and which is ultimately fatal in many cases. The role of other pathogens such as Staph. aureus in the induction oflung damage is important but there has been a relative decrease in the incidence of this organism in recent years, while the rate of isolation of P. aeruginosa has increased (Mearns, 1980). This organism initially colonizes the airway in a non-mucoid form, but

16

Clinical aspects of mucoid infections

soon changes to the mucoid type (see Chapter 4), which is associated with a worsening of respiratory function (Pitcher-Wilmott et at., 1982). The prevalence of P. aeruginosa infection at the Hospital for Sick Children, Great Ormond Street, is 36% (Dinwiddie, 1986). The prevalence at different ages varies from 20% at the age of 6 years to 60% by the age of 9 years (Wilmott, 1985). The clinical manifestations of P. aeruginosa infection are extremely variable and initially there may only be an intermittent cough, which is present during intercurrent respiratory tract infections. P. aeruginosa itself may only be cultured occasionally at these times and there can be long periods when it is not seen in the sputum. This stage probably does not represent true colonization, which is defined as three positive cultures obtained on sequential clinic visits, usually at two-monthly intervals. When the organism has become established there is a more persistent cough and an increase in sputum production. This sequence varies in severity and many children initially have considerable periods without significant symptoms despite the presence of P. aeruginosa in the lungs. As the pulmonary condition advances, the cough becomes more persistent and sputum production becomes a daily event. The volume and stickiness of the sputum varies from time to time and tends to be worse during exacerbations which are frequently caused by intercurrent viral respiratory tract infections. It is at this stage that more aggressive therapy is often instituted - certainly when there is evidence of clinical deterioration as assessed by failure to gain weight, or weight loss, anorexia, increased cough and sputum, and poorer performance in lung function tests. A number of clinics, however, adopt a more aggressive approach to the control of P. aeruginosa, with three-monthly courses of intravenous therapy using agents such as ceftazidime (Szaff et at., 1983; Philips and David, 1987). The clinical aspect of this more intensive regime does mean that the patient is often in hospital for periods of up to 8 weeks per year and this can have significant effects on both school and work, in addition to exposing the patient to hospital organisms and to others with CF, who may have multiply resistant P. aeruginosa present. Efforts have been made to overcome this problem by increasing the number of courses given in the home after a short stay in hospital for placement of the intravenous cannula. Use of in-dwelling subcutaneous catheters such as the Portacath has also facilitated such treatment. As the condition advances, older patients begin to demonstrate other complications, such as pneumothorax, haemoptysis or cor pulmonale. Pneumothorax occurs increasingly in the older patient, in as many as 19% (Batten and Matthew, 1983). The management of this condition has become increasingly difficult in recent years because pleurodesis, although effective for pneumothorax, is an absolute contra-indication to

Pathophysiology of pulmonary infection in cystic fibrosis

17

heartllung transplantation. Haemoptysis may occur in as many as 60% of older patients with chronic P. aeruginosa infection but it is usually self-limiting. In a small number, about 8o/c, it is severe and can be life threatening (Rauen, 1988). A few of these patients require more intensive treatment, including embolization of the affected bronchial artery or lobectomy. Cor pulmonale is a late sign, indicating severe and advanced pulmonary hypertension and its onset is usually associated with a significant reduction in life expectancy. The immune response to the presence of P. aeruginosa in the lung is assuming increasing importance in current thinking about the pathophysiology of lung damage (see Chapter 7). :-'fany CF children have circulating immune complexes which may be important in the mediation of lung damage induced by P. aPruginosa (H0iby and SchiyHz, 1982). It is thought that the tissue damage which occurs in the peripheral airways is caused by a type-3 hypersensitivity reaction in the presence of activated com plement (Zach, 1988). These aspects are reviewed extensively in Chapters 4 and 7. 2.3.2 Allergy and fungal infection

The role of allergy in CF is still unclear, but is thought probably to be a secondary phenomenon to P. (lfyugirwsa infection rather than a primary predisposing factor (Wilmott, 1985). Wheezing is, however, very commoll in CF children, occurring in as many as 50-60% of cases. This occurs through narrowing of the airways secondary to infection, localized oedema, airway obstruction, asthma or a primary increase in bronchial hyperreactivity. Warner et al. (1976) found positive skin tests in 59% of children with CF, the commonest reaction being to A.\jJerp;illusJumigat/1.\, which was found in 56o/c of positive cases. The role of A. Jwnigatu.1 in exacerbations of P. (leruginosa infection is unclear, because true bronchopulmonary aspergillosis is a relatively uncommon complication of CF lung disease when compared with the incidence of p. (lnllginosa itself. The incidence of other allergic manifestations, such as asthma, exerciseinduced bronchospasm, hay fever and eczema, varies from 7 to 20'* (Wilmott, 19H5). Rronchiallability is another factor which may be relevant in CF, although the responses to bronchial challenge with methacholine, histamine or exercise tests have given variable results, especially when performed repeatedly over a period of time (Holtz et al., 1981). The clinical implication of these findings is that wheezing is due to a number of different factors and, therefore, that bronchodilators such as ｾＭ｡ｧｯｮｩｳｴ＠ may have a varying response. Thus, it is important to evaluate the patient for bronchial responsiveness to such agents whenever possible using pulmonary function testing before they are introduced as a regular part

18

Clinical aspects of mucoid infections

of treatment. The clinical implication of wheezing is that it can limit exercise tolerance and, therefore, the ability to partake in normal physical activities and sports. As part of the evaluation it is useful to establish by exercise testing those who show exercise-induced wheezing. This can readily be blocked by the inhalation of salbutamol or cromoglycate beforehand, thereby imprO\'ing the patient's exercise tolerance and sense of physical well-being.

2.3.3 Viral infection

The young child with CF experiences a large number of the common respiratory viral infections which form part of the normal pattern of immunological development, particularly in the pre-school years. The role of viral infection in the induction or exacerbation of P. aerugillosainduced lung damage has been the subject of a number of recent studies (Stroobant, 19HfJ). Peterson pt al. (19H I) found evidence of non-bacterial pathogens in 20o/c of cases with acute pulmonary deterioration. The most common virus isolated was respiratory syncytial virus. Stroobant (1986) compared two groups of children with and without P. (ll'mginma colonization and found that, in all cases where a virus was identified significant pulmonary deterioration occurred and that this was worse in those who had had preceding P. (lrruginosa colonization. It is well known that viruses, particularly respiratory syncytial virus, can cause considerable local tissue damage which could eit.her allow access of P. oerugillosa to the lower I'espiratory tract or result ill worsening of local imlllune-mediated tissue damage which is worse in the presence of this organism. The clinical implicatiolls of recurrent upper respiratory infections of viral origin, especially in young children, are that they should be treated vigorously, including the use of antibiotics, in order to prevent lower respirator), tract colonization with bacteria such as Staph. mal'HS or P. (lfl'ugino.'(l. I fthere are signifIcantly enlarged adenoids or tonsils and these affect the appetite, general well-being of the patient and particularly weight gain, then there is no contra-indication to their removal in the normal way even in the presence of chronic P. {/prugillosa colonization. Nasal polyps are a further common complication of CF and Illay be seen in as many as 26'k of cases (Stern et al., 19H2). There has been no specific correlation with P. aerllgillosa infection, and ill fact some have suggested that nasal polyps are associated wit h a better prognosis overall (Drake-Lee and Pitcher-Wilmott, 1982). However, the polyps should be removed whenever they are causing significant nasal obstruction as this too will affect taste, appetite, weight gain and physical well-beillg.

Clinical monitoring

19

2.4 CLINICAL MONITORING

The CF patient with chronic pulmonary infection is usually monitored by regular lung function tests in addition to sputum cultures Cfable 2.2). Lung function is known to be worse in those who are chronically colonized with P. aeruginosa. The availability of portable lung function apparatus, including peak flow meter and vitalograph, makes it possible to measure lung function at every clinic visit Crable 2.2). Chest X-ray (posten>anterior and lateral) is performed every 6-12 months and may usefully be scored according to the system described by Chrispin and Norman (1974). The frontal and lateral chest X-rays are inspected for the typical abnormalities seen in CF. These include forward bowing ofthe sternum, diaphragmatic depression and spinal kyphosis. They are each given a score of 0, 'not present'; I, 'present but not marked'; or 2, 'marked', depending on the degree of change. 'rhe lung fields are then divided into four zones: right upper, right lower, left upper, left lower. A score of 0, 1 or 2, as described above, is given according to severity for each zone in relation to each of the following pathological changes: increased bronchialline shadowing due to thickening of the walls of the larger airways; mottled shadowing caused by small round shadows indicating sputum accumulation at microlobular level; circumscribed ring shadows in peripheral areas due to bronchiectasis at the bronchiolar level; and large confluent pulmonary shadows indicating lobar collapse/consolidation.

Table 2.2

Respiratory monitoring of the cystic fibrosis patient

Treatment ThroaUcough swab or sputum Lung function: Peak expiratory flow rate Vitalograph FVC* FEV+ Flow/volume curve Full lung function - plethysmography Chest X-ray - frontal + lateral view Other tests: Skin tests for allergy Full blood count Liver function tests/prothrombin time Bronchodilator response Aspergillus antibodies • Forced vital capacity,

t Forced expiratory volume in 1 second.

Frequency Every visit Every visit Every visit Every visit Every visit 6-monthly 6-monthly

As indicated clinically

20

Clinical aspects of mucoid infections

This system is useful in following the development of radiographic changes in the lung over a period of years. In clinical practice scores above 20 indicate advanced disease.

2.5 CLINICAL MANAGEMENT The clinical management of the CF patient with chronic colonization requires an overall approach to the general condition and includes a number of other important aspects in addition to the primary treatment oflung infection. The most important factors are shown in Table 2.3. Table 2.3

Management of P. aeruginosa infection in cystic fibrosis Medical Social Psychological Educational Occupational

The medical aspects specifically relating to the lungs are covered under physical treatment and are shown in Table 2.4. Attention should be paid to the patient's general nutritional state and the need for a high energy input during treatment. The patient who has active infection is known to be in a catabolic state and to have a high protein turnover (Morton et at., 1988). It is important, therefore, to provide the patient with a highly nutritious diet and a large intake of calories (Dinwiddie and Madge, 1988). Sometimes, more invasive nutritional support is needed, including nasogastric feeding or enteral feeding with gastrostomy or jejunostomy. Although this produces an effective increase in energy intake and weight gain during treatment, there is little evidence that it is of value in improving respiratory status over long periods (Lancet, 1986). The effect on body image of invasive enteral feeding can be quite significant, particularly in the adolescent CF patient. The social aspects of chronic infection involve the patient and the family in frequent visits to the hospital for out-patient review and admission at regular intervals for intensive intravenous treatment. This has significant effects on the family in terms of the time involved both in travelling and staying at the hospital and also in absence from full-time education or employment. There are also considerable financial implications for the family as the parents or the patient may be unable to earn during periods of illness and mayor may not be eligible for financial help such as the Attendance or Mobility Allowance in the UK, which can alleviate some of these problems. Although children are able to receive

Clinical management Table 2.4

21

Treatment of chronic lung infection in cystic fibrosis

Treatment

Method

Physical

Chest physiotherapy Forced expiratory technique Positive expiratory pressure Physical exercise

Nutrition

120-150% of recommended daily calorie intake

Antibiotics

Intermittent Continuous Intravenous Oral Nebulized

Other drugs

Bronchodilators Sodium cromoglycate Steroids Inhaled mucolytics

prescriptions free of charge in the UK, this does not apply to adults with CF at the present time, nor to children in some other Fe countries and North America. The presence of chronic lung infection brings with it a number of serious psychological effects, in terms of the chronic nature of the illness and the frustration of being unable to lead a normal life, as well as in terms of the increasing awareness that the long-term prognosis is worsening, a situation which is exacerbated when other friends with CF and chronic mucoid P. aemginosa infection die. The incidence of psychological disturbance in the parents is also high. Bywater (I!:Hl 1) found that 447c of the mothers of CF children were depressed and that marital breakdown was also a major factor in 18o/c of families studied at this time.

2.5.1 Physical treatment Physiotherapy and postural drainage remain the mainsta), of treatment for the lungs in CF. Almost all patients will require some form of physical chest clearance at least once a day. It is useful to continue this even when there are no symptoms because it has a preventive action in keeping the chest clear of secretions and reducing any tendency to atelectasis. In addition, there is a training effect such that tbe chest wall and muscles are used to receiving the treatment so that when it is even more important, for example, during exacerbations of infection, it is llIore effective. Older children and adults often prefer to perform their own treatment and this Illay be carried out successfully with the use of the forced expiratory

22

Clinical aspects of mucoid infections

technique (Prior et at., 1979). This is a particular method of assisting removal of the secretions from the bronchial tree by the use of forced expiration (huffs from mid-lung volume followed by a period of relaxation and breathing control). It must be taught by a physiotherapist trained in the technique and many older children and adults are able to perform effective chest clearance by this method (Hodson and Gaskill, 1983). Recently, the use of the positive expiratory pressure mask has been advocated for patients with CF. This is helpful as a supplement to physiotherapy and over a short period appears to be quite successful (Tyrell et ai., 198tJ).

2.5.2 Physical exercise Physical exercise is very helpful in the pulmonary management of CF and all patients should be encouraged to take as much exercise as they are physically capable of performing, because there is no doubt that this assists in the clearance of lung secretions (Zach et at., 1982). It also helps to maintain a sense of general well-being and is a pleasurable activity when performed within defined limits for each patient. The ability to continue in sports is both physically and psychologically helpful to the CF patient. Those with mild to moderate lung function abnormality are usually able to undertake normal physical activity (Cerney, fl at., 1982; Cropp et at., 1982), whereas those with severe lung disease (i.e. forced expiratory volume in one second is < 309c predicted) will have significantly reduced capacity for physical work. The use of transcutaneous oximetry now provides a simple non-invasive method for assessing oxygenation during different levels of exercise in the CF patient. It is best to design a programme of sub-maximal exercise which can be sustained for periods of 10-20 minutes as the patient will find this less uncomfortable and is more likely to persist with the programme. Exercise-induced bronchospasm can limit exercise tolerance in those who have over-reactive airways. In these cases a l3-agonist such as salbutamol or terbutaline, or an agent such as sodium cromoglycate, should be inhaled before exercising in order to reduce or abolish this complication (Geddes, 1984). The effects of exercise on sputum production are also beneficial and hence will be of particular value in those who have increased sputum volume in relation to their chronic infection.

2.5.3 Antibiotic therapy Antibiotic therapy in CF is vital to control the progression of the bacterial lung infection. Initially, whenStajJh. aurfusor H. injluenzaeis common, oral

Clinical management

23

antibiotics such as flucloxacillin, erythromycin, trimethoprim or amoxycillin may be used with good effect. A number of antibiotic regimes are in common use, especially in the early years of life. These include continuous therapy throughout life, continuous therapy in pre-school years with intermittent therapy thereafter, and intermittent therapy at all ages. The aim of continuous therapy is to prevent chronic lung damage, which will delay the onset of bronchiectasis and possibly the predisposition to chronic P. aemgirlOsa colonization. There is, however, no evidence to date that continuous anti-staphylococcal antibiotics in infancy change the acquisition rate of chronic P. aerugirlOsa colonization of the lungs. A controlled trial of continuous versus intermittent oral therapy to delay the onset of chronic staphylococcal infection is presently being undertaken (Williams et al., 1988a). Although this has demonstrated a reduced colonization rate with Stat}h. aul'pus during the first 2 years of life, there has not as yet been any reduction in the rate of colonization with P. aeTl1ginosa. The role of antibiotics in the clinical management of chronic infection is central to the control of P. al'ruginosa and its damaging effects on the lungs. Until recently all the agents active against it had to be given either parenterally or by nebulizer. The introduction of ciproAoxacin, an agent active by the oral route, has proved effective in the treatment of exacerbations ofCF lung disease (Hodson et at., 1987). Although this is an important advance in treatment, resistance to the antibiotic has already appear'ed (Scully et al., 1986). CF patients who have chronic infection usually have accelerated deterioration in lung function. Frequent or regular antibiotic therapy is thus an important part of treatment. Two regimes of intensive therapy are in use: these involve either regular therapy at three-monthly intervals whether or not there is evidence of clinical deterioration (Szaff et at., 1983), or intermittent therapy when there is clinical deterioration such as increased cough and sputum production, anorexia, weight loss and a worsening of pulmonary function tests (Williams pt ai., 1988b). Both treatment regimes have been associated with the general improvement in prognosis which has occurred in CF during the past twenty years (Szaff et al., 1983; Wilmott et ai., 1985). There has not yet been a properly controlled trial to determine which regime is more effective. A course of intravenous therapy is most effective if given over a period of 10-14 days. This usually involves simultaneous therapy with two agents during exacerbations. There is a wide choice of antibiotics available but the most common combinations are an aminoglycoside, such as gentamicin or tobramycin, with an acyl-ureido penicillin, such as azlocillin or a third-generation cephalosporin such as ceftazidime. The need for continuous venous access means that most of the treatment is given in

24

Clinical aspects of mucoid infections

hospital, where it can be combined with more intensive physiotherapy and a dietary review including specific calorie counting (Dinwiddie and Madge, 1988). The availability of implantable venous access devices such as the Portacath will simplify this form of treatment in the future. A number of CF centres utilize monotherapy with agents such as ceftazidime on a three-monthly basis, whether or not there is any clinical deterioration, in order to control chronic infection in the lungs (Phillips and David, 1987). All of these regimes have an immediate effect on the patient's general condition. Although the organism is seldom eradicated from the sputum, there is usually a significant improvement in lung function, appetite, weight gain, decreased sputum production and a feeling of general well-being. Pedersen et al. (1987) have reported a reduction in annual mortality rate among patients colonized chronically to an incidence of 1-2% per year. The frequency and intensity of these approaches to treatment have the significant disadvantage of disruption to daily life, especially during school time and among those who are in employment. This has led to increasing numbers of patients receiving parenteral therapy at home. Such a treatment regime is entirely practical given suitable support from local family practitioner services with hospital back-up for cannula placement. This allows patients to lead a much more normal life and yet maintain an intensive treatment programme. An alternative approach to control of chronic infection is by the use of nebulized antibiotics in addition to the usual physical treatments. Hodson et al. (1981) showed significant improvement in lung function and a reduction in the need for acute treatment in hospital utilizing regular aerosol carbenicillin and gentamicin in combination. There is also some evidence that the minimal inhibitory concentration of anti-pseudomonal antibiotics, such as azlocillin, may be reduced in the presence of a mucolytic agent (Heaf etal., 1983). Thus, in some patients aerosol therapy with a mucolytic agent prior to physiotherapy, in conjunction with aerosol antibiotic after treatment, may also be effective in controlling the organism. Since aerosols are easily delivered by portable nebulizer equipment this form of treatment is eminently suitable for home use. 2.6 CONCLUSION

The acquisition of P. aeruginosa by the lungs of the CF patient is a major milestone in the progress ofthe disease. Although in some patients it may have relatively little effect on the progressive deterioration of lung function with age, in most it is associated with a more rapid decline and increased symptoms. The prognosis for those with chronic infection is significantly worse than for those without (Wilmott, 1985). It is for this

References

25

reason that a more intensive approach to therapy is required, and the overall treatment is outlined in Table 2.4. The aim of therapy is to encourage the patient to lead as healthy and normal a life as possible. The antibiotic treatment has to be frequent and often parenteral. Present regimes are increasingly aimed at facilitating intravenous access and minimizing the inevitable disruption to family and social life which frequent admission to hospital involves. The development of increased home-based care and the advent of a wider range of oral anti-pseudomonal agents will undoubtedly improve the quality and length of life for these patients.

REFERENCES Batten,]. (1988) Cystic fibrosis in adolescents and adults. Excerpta Medica, Asia Pacific Congress Series 74, 187-94. Batten,]. C. and Matthew, D.]. (1983) The respiratory system. In: Hodson, M. E., Norman, A. P. and Batten,]. C. (eds) Cystic/ibmsis, Bailliere Tindall, London, pp.105-31. Brock, D.]. H. (1988) Prenatal diagnosis of cystic fibrosis. Arch. Dis. Child. 63, 701-4. Bywater, M. (1981) Adolescents with cystic fibrosis: psychosocial adjustment. Arch. Dis. Child. 56, 538-43. Cerney, F. ]., Pullano, T. P. and Cropp, G. ]. A. (1982) Cardiorespiratory adaptations to exercise in C. F. Am. Rev. Resp. Dis. 126,217-22. Chrispin, A. R. and Norman, A. P. (1974) The systematic evaluation of the chest radiograph in cystic fibrosis. Paediatr. Radiol. 2, 101-6. Cropp, G.]., Pullano, T. P., Cerney, F.]. and Nathanson, T.]. (1982) Exercise tolerance and cardiorespiratory adjustments at peak work capacity in C.F. Am. Rev. Resp. Dis. 126,211-26. Dinwiddie, R. (1986) Management of the chest in cystic fibrosis.]. Roy. Soc. Med. 79,6-9. Dinwiddie, R. and Madge, S. (1988) Intensive calorie counting in C.F.: an effective way of achieving weight gain? 10th International Cystic Fibmsis Congress, Excerpta Medica, Asia Pacific Congress Series 74,165. . Drake-Lee, A. B. and Pitcher-Wilmott, R. W. (1982) The clinical and laboratory correlation of nasal polyps in cystic fibrosis. Int.]. Pediatr. Otorhinolaryngol. 4, 209-14. FarraH, M., Law, H.-Y., Rodeck, C. H., Warren, R., Stanier, P., Super, M., Lissens, W., Scambler, P., Watson, E., Wainwright, B. and Williamson, R. (1986) First-trimester prenatal diagnosis of cystic fibrosis with linked DNA probes. Lancet i, 1402-5. Geddes, D. M. (1984) Physical exercise and cystic fibrosis. In: Lawson, D. (ed.) Cystic Fibmsis: horizons, Wiley, Chichester, pp. 117-33. Gibson, L. E. and Cooke, R. E. (1959) A test for the concentration of electrolytes in sweat in cystic fibrosis of the pancreas utilizing pilocarpine by iontophoresis. Pediatrics 23, 545-9.

26

Clinical aspects of mucoid infections

ileaL D. P., Webb, G. J. and :\!atthew, D. .J. (198:)) /1I1'itro assessment of combined antibiotic and mucolytic therapy for PleuriOit/OllaS aeruginosa in cystic fibrosis. Alch. Dis. Child. 58, 824-6. Hodson, M. E. and Gaskill, I). V. (1983) Physiotherapy. In: Hodson, M. E., Norman, A. P. and Batten,J. C. (eds) C)'stir Fibrosis, Bailliere Tindall, London, pp.219-41. I lodson, :\1. E., Penketh, A. R. Land Batten,J. c:. (1981) Aerosol carbenicillin and gentamicin treatment for Pseudoit/ollas Ilf1'llginosa infection in cystic fibrosis. Ｑｾｻｬｉｲ･ｴ＠ ii, I I:) 7-9. Hodson,:\1. E., Butland, R.J. A., Roberts, c:. M., Smith,j. and Ballen,J. C. (1987) Oral ciproHoxacin compared with cOl1\'entional intra\'enous treatment for Psnuioit/ou(fs aerll!{illosa infection in patients with cystic fibrosis. Lance/ i, 235-7. H0iby, N. (1982) Microbiology of lung infections in cystic fibrosis patients. Acta P{J('(liatl. S('{lllri. SUf!f!I. 301,33-54. 110iby, N. and Schi0tz, P. O. (1982) Immune complex mediated tissue damage in the lungs of cystic fibrosis patients with chronic Pseudolilonas aNugil/osa infection. ;\c/a Paedia/I. Swmi. SUjijJl. 301,6:)-7:). Holtz, F. j., Olinsky, A. and Phelan, P. D. (1981) Variability of airways hyperreactivity and allergy in cystic fibrosis. Anh. Dis. Child. 56, 495-9. Kram, E. R., Crane,:\1. M" Sirkin,:\1. G. and Brown,:\1. L. (1962) A cystic fibrosis pilot survey in three New England States. Am.]. Public Health 52, 2041-57. Kulzycki, L. L. and Shauf, V. (1974) Cystic fibrosis in blacks in Washington DC. Am.]. Dis. Child. 127,64-7. Kuzemko,J. A. (1986) Screening, early neonatal diagnosis and prenatal diagnosis. ]. Roy. Soc. AII'd. 79 (Supp!. 12),2-5. Kuzemko, j. A. and Heeley, A. F. (1983) Diagnostic methods and screening. In: Hodson, M. E., Norman, A. D. and Hatten,j. C. (eds) Cystic FiblOSis, Bailliere Tindall, London, pp. 21-30. [.ancet ([ QS6) Editorial. Supplementary nutrition in cystic fibrosis. Lancet i, 249. Levin, S. (1963) Fibrocystic disease of the pancreas. In: Goldschmidt, E. (eeL) Genetirs ofiVligrant aud IsoLated Populations, Williams and Wilkins, Baltimore, p.293. Mearns, :\1. B. (1980) Natural history of pulmonary infection in cystic fibrosis. In: Sturgess, j. N. (eeL) Penpectives in Cystic Fibrosis, Canadian Cystic Fibrosis Foundation, Toronto, p. 325. Meindl, R. S. (1987) Hypothesis: a selective advantage for cystic fibrosis heterozygotes. Alii.]. Ph),s. An/hmjJOI. 74, 39-54. :\[orton, R. E., H utchings,J., Halliday, D., Rennie, M . .J. and Wolman, S. L. (1988) Protein metabolism during treatment of chest infection in patients with cystic fibrosis. Alii.]. Clin. NutI'. 47, 214-19. Pedersen, S. S., Jensen, T., II0iby, :-J., Koch, C. and Flensborg, E. W. (1987) Management of PspuriolllOnas ael'l1giuosa lung infection in danish cystic fibrosis patients. Acta Paediatr. Scand. 76, 955-61. Penketh, A. R. L., Wise, A., Mearns, :\;1. B., Hodson, 1\1. E. and Batten,.J. C. (1987) Cystic fibrosis in adolescents and adults. Thorax 42,526-32. Peterson, N. '1'., H0iby, l\'., Mordhorst, C. II., Lind, K., Flensburg, E. W. and Rune, R. (1981) Respiratory infection in cystic fibrosis patients caused by virus, chlamydia and mycoplasma. Possible synergism with PSl'udoTnou{ls aeJ'nginosa. Acta Paediatr. Scmul. 70, 623-S.

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Phillips, B. M. and David, T.]. (1987) Management of the chest in cystic fibrosis.]. Roy. Soc. Med. 80 (Supp!. 18),30-7. Pitcher-Wilmott, R. W., Levinsky,]., Gordon, 1., Turner, M. W. and Matthew, D . .J. (1982) Pseudomonas infection allergy and cystic fibrosis. Arch. Dis. Child. 57, 582-6. Prior,]. A., Webber, B. A., Hodson, M. E. and Batten,]. C. (1979) Evaluation of the forced expiratory technique as an adjunct to postural drainage in treatment of cystic fibrosis. Brit. Med.]. 2,417-18. Quinton, P. M. (1983) Chloride impermeability in cystic fibrosis. Nature 301, 421-2. Reid, L. and De Haller, R. (1964) Lung changes in cystic fibrosis. In: Hubble, D. (ed.) C)stic fibmsis, London Chest and Heart Association, London, p. 21. Righetti, A. B. B., Mighavacca, M., Prampolini, L. and Guinta, A. (1976) Extensive neonatal screening of CF. Proffedings of Vll Intemational C)stic Fibrosis Conference, p. 153. Rommens,.J. M., Iannuzzi, M. C., Kerem, B.-S. (1989) Identification of the cystic fibrosis: chromosome walking and jumping. Science, 245,1059-65. Scully, B. E., Neu, H. C, Parry, M. F. and Mandell, W. (1986) Oral ciproAoxacin therapy of infections due to Pseudomonas aemginosa. Lancet i, 819-22. Selander, P. (1962) The frequency of cystic fibrosis of the pancreas in Sweden. Acta Paediatr. Scand. 51,65-7. Stern, R. C., Boat, T. F., Wood, R. E., Matthews, L. W. and Doershuk, C. F. (1982) Treatment and prognosis of nasal polyps in cystic fibrosis. Am.]. Dis. Child. 136,1067-70. Stroobant,]. (1986) Viral infection in cystic fibrosis.]. Roy. Soc. Med. 79,19-22. Szaff, M., Hltiiby, N. and Flensborg, E. W. (1983) Frequent antibiotic therapy improves survival of cystic fibrosis patients with chronic Pseudomonas infection. Acta Paediatr.Scand. 72,651-7. Tomashefski, G. F., Vawter, G. F. and Reid, L. (1983) Pulmonary pathology. In: Hodson, M. E., Norman, A. P. and Batten,]. C. (eds) Cystic Fibrosis, Bailliere Tindall, London, pp. 31-51. Tyrell,]. C., Weller, E. J. and Martin,]. (1986) Face mask physiotherapy in cystic fibrosis. Arch. Dis. Child. 61, 598-60 I. Wainwright, B. J., Scam bier, P. J., Schmidtke,]., Watson, E. A., Law, H.-Y., Farrall, M., Cooke, H.]., Eiberg, H. and Williamson, R. (1985) Localization of cystic fibrosis locus to human chromosome 7cen-q22. Nature 318, 384-5. Warner,]. 0., Taylor, B. W., Norman, A. P. and Soothill,]. L. (1976) Association of cystic fibrosis with allergy. Arch. Dis. Child. 51,507-11. Williams, J., Alfaham, M., Ryley, H. C, Goodchild, M. C., Weller, P. H. and Dodge, J. A. (1988a) Screening for cystic fibrosis in Wales and the West Midlands. 10th International Cystic Fibrosis Congress, Excelpta Medica, Asia Pacific Congress Series 74, 20-1. Williams,J., Smith, H. L., Woods, C. G. and Weller, P. H. (1988b) Silastic catheters for antibiotics in cystic fibrosis. Arch. Dis. Child. 63, 658-9. Williamson, R., Crampton,J. M. and Clarke, B. E. (1983) Research perspectives: the basic defect in cystic fibrosis. In: Hodson, M. E., :\Torman, A. P. and Batten,J. C. (eds) C)stic Fibrosis, Bailliere Tindall, London, pp. 260-72. Wilmott, R. W. (1985) Allergy and infection in cystic fibrosis. In: Milner, A. D. and Martin, R. J. (eds) Neonatal and Pediatric Respiratory Medicine, Butterworths, London, pp. 190-210.

28

Clinical aspects of mucoid infections

Wilmott, R. W., Tyson, S. L., Dinwiddie. R. and Matthew, D. J. (1983) Survival rates in cystic fibrosis. Arch. Dis. Child. 58, 835-6. Wilmott, R. \V., Tyson, L. S. and Matthew, D. J. (1985) Cystic fibrosis survival rates. The influence of allergy and Pseudomonas afnlginosa. Am.]. Dis. Child. 139,669-73. Wright, S. W. and Norton, N. E. (1966) Genetic studies in cystic fibrosis in Hawaii. Am.]. Humall Gen. 20, ＱＵＷＭＶｾＮ＠ Zach. M. S. (1988) Lung disease in cystic fibrosis: current concepts. t'xcerpta iHedim, Asia Parifir Congress Series 74, 72-9. Zach, M.S., Oberwaldner, B. and Hausler, F. (1982) Cystic fibrosis: physical exercise versus chest physiotherapy. Arlit. Dis. Child. 57, 587-9.

3

The structure and properties of alginate P. GACESA AND N. J. RUSSELL

3.1 INTRODUCTION Prokaryotes present to the external environment a cell surface that consists of a complex mixture of macromolecules, most of which are composed entirely or partly of sugar residues (Hammond 1'1 at., 1984; Shockman and Wicken, 1982). These molecules are of considerable importance for the survival of prokaryotes and perform a number of distinct functions. They provide antigenic determinants on the cell surface (see Chapter 7) and are involved in colonization events during the establishment of infections (see Chapter 6) and in cell-cell interactions associated with the body's imIJIune response to an infection (see Chapter 8).

Conventionally, these glycans or glycan-containing molecules have been categorized into three well-defll1ed groups: I. peptidoglycans 2. lipopolysaccharides 3. extracellular polysaccharides. These three classes of molecules are quite distinct in terms of their structure and location relative to the outer surface of the plasma membrane (the inner membrane of Gram-negative bacteria). The peptidoglycans are comprised of peptide and carbohydrate and form the main rigid structural component of the prokaryote cell wall, giving it a characteristic shape. Both Gram-positive and Gram-negative bacteria have peptidoglycan in their cell wall, although there is much more in Gram-positive bacteria and there are structural differences between different genera and species. Gram-positive bacteria have a teichoic acid or teichuronic acid polymer covalently bound to the peptidoglycan (Ward, 1981). The lipopolysaccharides, as their name implies, contain a lipid and a glycan component. They are made up of three parts: viz. lipid A, a core

30

The structure and properties of alginate

oligosaccharide and a repeating O-antigen oligosaccharide chain. Lipopolysaccharides are an integral part of the outer membrane of Gramnegative bacteria but are not found in Gram-positive bacteria. They are located in the outer leaflet of the Gram-negative outer membrane, the ｦｾｬｴｹ＠ acids of the lipid A moiety of lipopolysaccharide providing the hydrophobic outer half of the membrane bilayer; there is usually very little phospholipid in the outer leafiet, which contains almost exclusively protein and lipopolysaccharide. The core and O-antigen side-chains are exposed, therefore, to the external surface. It is the side-chain structure of the lipopolysaccharides that is serologically significant and responsible for the O-antigen specificity of the enterobacteria and other Gramnegative organisms. Mutants of bacteria, including P. al'ruginosa, have been isolated which over-produce lipopolysaccharide and possess a characteristically smooth morphology when grown on solid media (see Chapter I). The different colonial morphology types of P. aeruginosa are considered in more detail in Chapter 4. The extracellular polysaccharides are the macromolecules of major concern in this review. In some bacteria the polysaccharide forms part of a discrete capsule, whereas in others an apparently less organized slime layer is produced. Typically, bacteria that produce extracellular polysaccharide have a smooth and gelatinous appearance when grown on agar plates. However, it is important to emphasize the difference between those organisms that have a 'smooth' colonial morphology as a result of their lipopolysaccharide structure and those that genuinely produce exopolysaccharide (see Chapters I and 4).

3.2 BACTERIAL POLYSACCHARIDES 3.2.1 Composition of extracellular polysaccharides

Bacterial polysaccharides vary considerably in their structural complexity. The simplest examples are the homopolymers, e.g. cellulose, which are comprised of a single type of monosaccharide unit linked by one type of glycosidic bond. In contrast, the heteropolymers may be branched molecules and contain a variety of monosaccharides joined by different glycosidic linkages. However, all of the polysaccharides have an organized and usually repeating structure. For example, one of the most complex polysaccharides is that secreted by strains of Rhizobium trifolii, which has a repeating unit of eight sugar residues (McNeil et al., 1986). It is important to emphasize the wide diversity of polysaccharide structure. For instance, there are no less than 83 different types of capsular polysaccharide (the K antigens) in the genus Klebsiella (Atkins

Bacterial polysaccharides

31

HOCH 2 4

ｾ

HO

Ｕ＠

OOH

OH 3

1

2

OH Fig. 3.1

The numbering system for hexoses.

et al., 1979). A similar situation occLirs in many other bacterial genera. It is instructive to consider how this diversity can occur and a useful comparison can be drawn between polysaccharide and protein structures. If one considers the structure of a simple dipeptide containing one type of amino acid then there is only one structure that can result, i.e. the two . amino acid residues linked bv a peptide bond. This does not take into account the possibility of iso-peptide bonds, but these normally occur in only a few specialized proteins (e.g. elastin) and so they have been excluded from consideration. However, a much wider range of different disaccharides can be made from a single type of munosaccharide unit. This is because any une of five different glycosidic bonds (1-1, 1-2, 1-3, 1-4 or 1-6, where the numbers refer to carbon atoms of the sugar residues) may be formed. In addition, the anomeric carbon atom at position I may be in either the ex or f) conformation; i.e. the hydroxyl grou p on C 1 is either below or above the plane of the D-series sugar ring structure (Fig. 3.1). Thus a total of 11 different disaccharide structures are possible. When considering polysaccharide structure it would be more correct to omit the 1-1 linkage from the calculation because this glycosidic link, which occurs most commonly in the disaccharide trehalose, would result in chain termination: even allowing for this, there are still eight possible disaccharide structures. Therefore, there is an inherent eightfold greater diversity in polysaccharide structure as compared to proteins, i.e. one variant of dipeptide versus eight variants of disaccharide. A further increase in diversity is introduced because there are at least 30 different monosaccharides that may be found in polysaccharides compared to the 20 amino acids in proteins; thus the total range of possible polysaccharide structures is immense. Furthermure, polysaccharides may be substituted with different 'non-carbohydrate' groups (see below). This variety in the primary structure of polysaccharides is reflected in the diversity of three-dimensional molecular conformations as determined by X-ray crystallography (Isaac, 1985). and it is highly likely that many interactions between micro-organisms and their external environment are governed by the biological specificity of these polymers. The presence of negative charges is a feature of most bacterial polysaccharides. U suall y, these negative charges are the result of the

32

The structure and properties of alginate

Table 3.1

The structures of some bacterial polysaccharides

Species Leuconostoc mesenteroides Acetobacter xylinum Alcaligenes faecalis var. myxogenes Streptococcus mutans Streptococcus salivarius Streptococcus pneumoniae (Type III) Azotobacter vinelandii Pseudomonas aeruginosa Streptococci (Haemolytic Group A) Klebsiella pneumoniae (Type 28)

Polysaccharide Dextran Cellulose Curdlan Mutan Levan Alginate Alginate Hyaluronan

Structure (1-"6 glucose)" (12i4 glucose)" (1-1'3 glucose)" (1-"3 glucose)" (2-1'6 fructose)" (-3GIcA 12i4Glcl-")" 1-4-linked ManA and GulA 1-4-linked ManA and GulA (-3 GlcNAcl2i4GIcA 1--")" (-2Gal 1-"3 Man 1-"2 Man 1-"2Glc1-l')" 2

I"

1 GicA

3

I"

1 Gic

Xanthomonas campestris

Xanthan

(-4GIc1--l'4GIc 12i4Glc 1-l')"

3

I"

1 ManA(OAc)

2

I"

1 GIcA 4

I"

1 Man

46

II

Pyr Abbreviations: Gal, galactose; GIc, glucose; GIcA, glucuronic acid; GuIA, guluronic acid; Man, man nose; ManA, mannuronic acid; GlcNAc, N-acetylglucosamine; GAc, O-acetyl; Pyr, pyruvate.

incorporation of uronic acids into the polymer. U ronic acids are hexoses in which the primary alcohol group at C6 has been oxidized to the corresponding carboxylic acid; thus D-glucuronic acid is derived by oxidation of D-glucose. The hexoses are oxidized in the form of sugar nucleotides, although there is scope for some limited interconversion between uronic acids in the completed polysaccharide polymer (see Chapter 10). Incorporation of aldonic acids, i.e, those in which the aldehyde group at CI has been oxidized to the corresponding acid, would not normally occur because of the inability of these compounds to form glycosidic bonds via Cl. Negative charges may also be introduced into

Bacterial polysaccharides

33

polysaccharides by substitution of some of the constituent monosaccharides with pyruvyl, succinyl, acetyl or formyl groups. This preponderance of negative charge, coupled with the characteristically high molecular weight of many of the polysaccharides, usually results in the formation of highly hydrated gel-like networks in which a small amount of polymer interacts with a comparatively large volume of water (see section 3.4.3). Bacterial polysaccharides vary enormously in their structure and range from simple homopolysaccharides to complex, highly substituted and branched heteropolysaccharides Crable 3.1). Attempts to analyse chemically the structures of these polysaccharides has, in the past, often produced results that were difficult to interpret. A major reason for this is that glycosidic bonds associated with uronic acids are particularly resistant to acid hydrolysis. Consequently, conditions that were harsh enough to hydrolyse these bonds also resulted in partial decarboxylation of the uronic acids. Also, other hexoses and substituents may be degraded by the strong acids used in the hydrolysis procedures. This leads to underestimation or misidentification of sugar products. However, as analytical techniques have developed it has proved possible to analyse satisfactorily many more bacterial polysaccharides. The exact function of many of the bacterial polysaccharides is still open to debate, although there has been no shortage of suggested biological roles for these materials (Dudman, 1977). Also, it is not yet clear why some bacteria form discrete capsules, e.g. Klebsiella pneumoniaI', whereas others have a much looser association of polysaccharide, e.g. P. {lfl"uginosa. In some bacteria it is fairly easy to ascribe a possible role for the polysaccharide, e.g. as virulence factors, adhesins or protective coats, whereas in others the function remains obscure. In mucoid strains of P. aeruginosa a variety of roles for the polysaccharide have been proposed, including protection against attack by the host immune system (see Chapters 4, 7 and 8), mediation of adhesion (see Chapters 5 and 6) and control of exo-enzyme activity (see Chapter 9). It should be noted that bacterial polysaccharides are usually antigenic and it has been argued that the uronic acids are the major antigenic determinants (Dudman and Wilkinson, 1965). 3.2.2 Polysaccharide shapes

The three-dimensional shape of a polysaccharide is determined by the structures of its component monosaccharides. In this review it is only the structure of the hexoses, and more particularly the pyranose ring form of these sugars, that need concern us. There are four main pyranose ring forms, namely two chair forms and two boat forms (Fig. 3.2). The

34

The structure and properties ofalginate

ｾ＠

J;::7 'Co

°C,

Chair forms

M B,

8

'·'B

0

TYPical boat forms

Fig. 3.2 The chair and boat form, of the pyranose (six-membered) ring structurp of monosaccharides. These structures are in equilibrium with each other and with the furanose (five-membered) ring and straight-chain forms of the monosaccharide. For most hexoses one of the pyranose-ring chair forms is the most stable, and therefore the most likely, conformation.

monosaccharides are able to inten:onvcrt between the various ring forms, although one form is favoured from an energetic point of view. As the boat forms are relalively unstable the hexoses exist predominantly in one of the two chair forms. For most D-series sugars the ICI chair conformation is prefened, whereas the I.-series sugars are usually in the 'c, chair form. This is because the most bulky substituent, i.e. that attached to C5, is held in the energetically favoured equatorial position. Addition of bulky substituents may cause a change in the prefened chair form, and consequently will alter the three-dimensional shape of the polysaccharide (see below). Nevertheless. for most monosaccharides one conformation will predominate. The shape of a polysaccharide will be dictated to a large extent by the conformations of the constituent monosaccharide units. There are two factors to be considered when monosaccharides are linked together. The first is that the type of chair forms of the monosaccharides will affect the orientation of the glycosidic bonds. The second is that the position and orientation of the glycosidic linkage will affect the degree of rotation around the glycosidic bond. These points are best illustrated by looking at some specific examples. The simplest polysaccharide structures to consider initially are homopolymers of o-glucose. f3-o-Glucose exists predominantly in the 'c, form and consequently all of the substituents are equatorial to the plane of the ring. In contrast, the (X-anomer of Il-glucose has an axial substituent at C I

Bacterial polysaccharides HO

35

HO OH OH ((-D-Glucose

Il-lJ-Glucose

Fig.3.3 The predominant ring forms of a-D-glucose and j3-D-glucose. Note that the hydroxyl group at C1 is equatorial (i.e. approximately parallel to the plane of the ring) in j3-D-glucose, but axial (approximately perpendicular to the plane of the ring) in a-D-glucose.

(Fig. 3.3). These relatively small changes in monosaccharide conforIllation have a profound effect on the preferred shape of the polysaccharide. On a theoretical basis it can be predicted that up to eight types of polysaccharide shape ilia\" occur (see section 3.2.1). In essence, however, hOl11opolymers of D-glucose form one of onlv four tvpes of polysaccharide shape (Fig. ;).4). The four types of simple pohsaccharicle shape have been elaborated bv Rees (1977). They are all described mathematically as helices, although only one type has the 'appearance' of a conventional helix. Polyglucans linked either ｾＱＭＴ＠ (cellulose) or 0'1-3 will form an extended ribbon structure in which the plane of the sugar ring is almost parallel to the long axis of the helix. l\'eutral polysaccharides of this type tend to he fairly

/ ｾ＠ '\

I

Extended ribbon 131--4 glucan

Hollow helix (11-4 glucan

Crumpled ribbon (11-2 glucan

Flexible COil (11-6 glucan

(( 1-3 glucan

I) 1--3 glucan

131-2 glucan

131-6 glucan

Fig.3.4 The four basic shapes of simple polysaccharides. The shape of a polysaccharide will depend on both the constituent monosaccharide units and the type of glycosidic linkages. The examples used in this diagram are all polymers of I)-glucose. (Adapted from Powell, 1979.)

36

The structure and properties of alginate

insoluble because the surfaces of the helix are relatively hydrophobic, i.e. it is the hydrophobic hydrogen atoms that are axial and exposed to the solvent. Polyglucans with ul-4 (amylose) or [31-3 (curd Ian) linkages can form hollow helical structures, although in solution they will probably exist as random coils. The u or [31-2 linkage will result in a polysaccharide that forms a rigid structure, which is often described as a crumpled ribbon. The steric hindrance that results from the close proximity of the bonds restricts free rotation around the glycosidic bond. In contrast, polyglucans with u or [31-6 linkages (e.g. dextrans) form flexible coils because of the increased movement that is available around the glycosidic linkage. 3.3 THE STRUCTURE OF ALGI NATES 3.3.1 Discovery and identity of alginates

The polysaccharide alginate was first isolated in 1881 by Stanford from seaweeds, or more accurately brown algae (hence the name alginate), and the initial characterization of this material was published in 1883 (Stanford, 1883). Many of the chemical and physical properties of alginates were described accurately by Stanford during the nineteenth century and still hold true today. Subsequent studies on alginates using the most sophisticated chemical and/or physico-chemical techniques, including nuclear magnetic resonance (see Chapter 10), light scattering, viscometric measurements (see section 3.4.2) and electron microscopy (see section 3.4.2), have refined and extended our knowledge of this polysaccharide but have not altered original tenets. Although it was appreciated from the outset that the polymer consisted of negatively charged material, it was not until many years later that a uronic acid was identified as the major component (Schoeffel and Link, 1933). However, these workers assumed incorrectly that [3-D-mannuronate was the only monosaccharide building block present in alginates. Subsequent work established the presence of variable amounts of a second uronic acid, u-L-guluronate (Fischer and Dorfel, 1955), and it is now realized that alginates are copolymers. The observation that P. aeruginosa was able to synthesize an 'alginatelike polysaccharide' was first reported by Linker and Jones (1964), although mucoid strains of this bacterium had been isolated much earlier (Sonnenschein, 1927; see Chapter 4). In a subsequent more extensive study, Linker and Jones (1966) confirmed that an O-acetylated alginate was the major component of the slime of P. aeruginosa. More recently, other related pseudomonads such as P. fiuorescens, P. mendocina, P. putida (Govan et ai., 1981) and P. syringae pv. giycinea (Fett et ai., 1986) have also

The structure of a/ginates

37

been shown to produce alginate under certain growth conditions. Alginate has been isolated from only one non-pseudomonad species of bacteria, namely AwtobactenJine/anriii (Gorin and Spencer, 19(6), in which synthesis of the exopolvsaccharide is closely associated with the formation of vegetative cysts (Page and Sadoff, 1975), a form of differentiation that does not occur in the pseudomonads.

3.3.2 The chemical composition of algi nates The alginates are best described as a family of related molecules. Most of the information concerning the structure of these polysaccharides has been derived from material extracted from marine brown seaweeds. The bacterial polysaccharides are very similar, with only minor, albeit important, differences in composition and structural detail. Alginates are unbranched (1-4)-linked polysaccharides comprised of !3-11-mannuronate and its C5-epimer a-L-guluronate (Fig. 3.5). The uronic acids are monosaccharides which haye been oxidized at C6 to produce a carboxylate group and therefore are negatively charged. The relative proportion of the two uronic acids \'aries from alginate to alginate and is a major factor in determining the properties of the polysaccharide. In fact the mannuronate: guluronate ratio, and more importantly the arrangement of the uronic acids within the polysaccharide, is an important indicator of the properties of a particular preparation of alginate. The mannuronate: guluronate ratio of alginates derived from the brown seaweeds (Stockton et aI., 1980) and from A. lIilleiwuiii (Larsen and Haug, 1971) depends 011 the growth conditions of the organisms. Most workers agree that alginate obtained from a particular strain of P. aerugillosa has a constant rnannuronate: guluronate ratio, regar'dless of the growth conditions, and this is certainly our own experience (P. Gacesa and N. J. Russell, unpublished results). Bacterial alginates are imariably substituted with O-acetyl groups (Sherbrock-Cox ct ai., 19R4). Initial experiments using selective enzymic COO-

ｾ

HO

ｲＩｾｄｍ｡ｬｮｵｯｴ･＠

ｏｈ＠ OH HO

u-L-Guluronate

Fig.3.5 The structures of the constituent uronic acids of alginates. The two uronic acids are identical in structure, except that they are epimeric at C5.

38

The structure and properties of alginate

degradation procedures and analysis of the products by gel permeation chromatography implied that the O-acetyl groups were associated exclusively with the D-mannuronate residues (Davidson et al., 1977). However, it was clear from other experiments that the molar ratio of O-acetyl groups to ll-mannuronate residues exceeded 1.0 in samples of alginates from some isolates of P. aeruginosa (Sherbrock-Cox et al., 1984). More detailed analysis of bacterial algi nates by I H-N MR revealed that some of the D-mannuronate residues were 2,3-di-O-acetylated, although the mono-Oacetylated forms were more prevalent (Skjak-Braek et al., 1986). The reason for the O-acetylation of bacterial alginates is not entirely clear; it has been suggested that it might be part of a control mechanism which operates during biosynthesis of the polysaccharide (see Chapter 10). The presence of O-acetyl groups may also alter the susceptibility of alginates to degradation by free radicals (see Chapter 8) or their immunogenicity (see Chapter 7). Other implications of the O-acetylation of alginates with regard to the solution properties of this polysaccharide are considered later in this chapter (section 3.4).

3.3.3 The block structure of algi nates It would appear from the Haworth projections of the !3-D-mannuronate and the a-L-guluronate residues (Fig. 3.5) that there is little difference in their structures. However, the epimerization at C5 results in a marked change in conformation of the two monosaccharide units. As the carboxyl group is the most bulky suhstituent on the sugar ring, the most energetically favourable conformation will maintain this group in the equatorial position. Consequently, !3-D-mannuronate will exist preferentially in the'C I chair form, whereas the a-l.-guluronate residues will adopt the IG, conformation (Fig. 3.6). The combination of the two ring forms of the uronic acids will result in polysaccharides with different three-dirnensional structures. With !3-11mannuronate the linkages through the C 1 and C4 positions will both be equatorial to the plane of the sugar rinp-. In contrast the linkages via these

ｈｏｾＸ｜Ｎ＠

Ｍｏｾ＠ ｈｾｏ＠

OH

ｾｈ＠

HO B-LJ-Mannuronate

Fig. 3.6

(H-Guluronate

The preferred conformation of the uronic acids.

The structure of alginates

39

same carbon atoms will be axial for the a-L-guluronate residues. Thus the sequence of ur·onic acids within a particular alginate molecule has profound effects on the polysaccharide structure. The two different uronic acids may be arranged in three different ways within the alginate molecule to form block structures. There may be homopolymeric regions, i.e. poly-!)-IHnannuronate or poly-a-Lguluronate, or heteropolymeric regions in which there is a random arrangement of the monomers. All three types of structure may occur within a single alginate molecule. As the polymannuronate regions contain di-equatorial linkages they will form ribbon-like structures (Fig. 3.7) similar to those of cellulose. However, unlike cellulose, the presence ofthe negative charge on C6 ensures that the polvsaccharide is soluble. In blocks of polyguluronate the ur·onic acids will be linked di-axially and will form buckled ribbons (Fig. 3.7); significantly, this is a structure analogous to that found in another polyuronide, pectate, the polysaccharide largely responsible for the gelling properties of jams, conserves and other fr·uit-derived products. A COil sequence of these differences in the orientation of the glycosidic bonds is that the various block structures have different physical lengths. Data from light-scattering and viscometric experiments (Srnidsr0d 1'1 al., 197,3) show that the relative unperturbed linear length of the blocks increases in the order: GG ｢ｬｯ｣ｫｳｾ＠

MM

｢ｬｯ｣ｫｳｾ＠

MG blocks

This relative order for the length of the blocks correlates well with that calculated by conformational analysis (Srnidsr0d 1'1 Ill., 1973; Whittington,

. . ｯｾＸＬ＠

Fig. 3.7

ｈｾｏＯ＠

ｈｾｏＸＬ＠ ｈｾｏ＠

....

The block structures of alginate. Poly-j3-rJ-mannuronate (above) and poly-a-l-guluronate (below).

40

The structure and properties of alginate

Fig. 3.8 The chelation of calcium by the regions of polyguluronate within the alginate molecule (egg-box structure). The solid circles represent the Ca1+ ions. (Adapted from Powell, 1979.)

1971). An implication of these observations is that algi nates are fairly stiff polymers in solution because of the limited free rotation about the glycosidic bonds; those molecules richest in guluronate blocks will be the least flexible. As indicated above, the mannuronate: guluronate ratios of alginate preparations will affect the physical properties of the polysaccharide. This is due almost entirely to the resultant arrangement of the block structures within the polymer and particularly evident when the polysaccharide interacts with cations. Alginate is a polyanion and as such is able to interact with cations. Like all cation-exchangers the selectivity and strength of binding depends both on the nature of the cation and the properties of the polymer. Divalent and polyvalent cations are bound strongly by all types of alginate and effectively cross-link the polysaccharide to form a gel matrix. However, regions of polysaccharide form particularly strong chelation complexes with divalent cations, especially the calcium ion. The so-called 'egg-box model' has been proposed to explain this specific interaction (Rees, 1972). The buckled chains of polysaccharide form a structure akin to the cross-section of an egg-box in which the calcium ions are the 'eggs'. The binding of the calcium ion is strong because, in addition to the ionic binding to the carboxyl groups, various ring and hydroxyl oxygen atoms are able to chelate the cations (Fig. 3.8). Furthermore, the polyguluronate regions are able to discriminate between cations largely on the basis of hydration volume Ｈｓｭｩ､ｳｲｾ＠ and Grasdalen, 1984). Thus polyguluronate binds calcium ions very strongly and other divalent cations less so, whereas regions rich in

Physical properties of alginates

41

polymannuronate or mixed sequences do not discriminate between the binding of Ca 2+ or Mg2+ (Smidsn.;d, 1974). Therefore, alginates rich in polyguluronate make firm but brittle gels, whereas those in which polymannuronate or mixed sequences predominate are more elastic. For effective gelation to occur it is important that tJ:te blocks of guluronate are at least 20 monomer units long. This is because gelation is dependent on the binding of cations in a cooperative manner and it is essential that junction zones of sufficient length are available for this process to occur. In brown seaweeds and A. vinelandii all three types of block structure may occur within the alginate molecule (Gacesa, 1988), but this does not appear to be the case for pseudomonads. Although a wide spectrum of mannuronate: guluronate ratios has been reported for alginate extracted from various strains of P. aeruginosa, these values are never less than 1.0 (see, for example, Evans and Linker, 1973). A systematic study of clinical isolates using' H-NMR to determine the organization of the monomers demonstrated unequivocally the complete absence of polyguluronate blocks in the alginates of P. aeruginosa (Sherbrock-Cox et al., 1984). Subsequent studies in other laboratories have endorsed these results (Skjak-Braek et aI., 1986). However, there is still some debate as to whether other Pseudomonas spp. produce alginates containing blocks of polyguluronate. Chemical analysis of algi nates from ten pathovars of P. 100000 (i.e. well above the minimum for immunogenicity) and it is acidic due to the presence of carboxyl groups with a pI of 4.2-4.6 (Pier et al., 1983). P. aeruginosa alginate has either poly-mannuronic blocks or alternating mannuronate and guluronate residues (see Chapter 3). This is in contrast to seaweed alginate, in which poly-guluronate blocks are also present. This may have important consequences for the immunodeterminant groups and for immunological cross-reactivity between bacterial and seaweed alginate. There may be many possible oligomeric sequences of mannuronic and guluronic acids which could account for multiple determinants (Vreeland and Chapman, 1978; Grasdalen et al., 1981).

7.3 METHODS FOR DETERMINATION OF ANTIBODIES AGAINST ALGINATE 7.3.1 Introduction A number of different methods have been used to measure the presence or absence of antibodies against alginate. Some methods (immunofluorescence, enzyme-linked immunosorbent assay, radioimmunoassay and immunoblotting) measure the binding of specific antibodies to the antigen, whereas haemagglutination and immunoelectrophoresis are dependent on phenomena such as agglutination and precipitation which are secondary to the antigen-antibody reaction. The former methods are generally more sensitive than the latter methods and in addition yield information on the immunoglobulin class and subclass of the reacting antibody. Common to all the methods (except the electrophoretic

118

Immunology of surface antigens

methods, which can separate antigens) is the requirement for a highly purified alginate preparation. 7.3.2 Immunodiffusion and immunoelectrophoresis ｈｾｩ｢ｹ＠ et at. (1975) attempted immunodiffusion and crossed immunoelectrophoresis using a crude ethanol-precipitated fraction from P. aeruginosa. They were unable to show an immunoprecipitate when using serum from CF patients chronically infected with P. aeruginosa. This may have been due to the inability of an alginate-antibody complex to form precipitates rather than to the absence of antibodies. Later, Vreeland and Chapman (1978) used crossed immunoelectrophoresis with seaweed alginate as antigen and antiserum from immunized rabbits. They had to partially hydrolyse high-molecular-weight alginates prior to electrophoresis and the agarose gel had to contain 0.4 mM CaCl 2 for optimal visualization of precipitates. In this way they observed several precipitin lines. Pier et al. (1983) obtained a single precipitin line with purified mucoid antigen by Ouchterlony immunodiffusion when tested against antisera raised against the whole organism. Likewise a single precipitin arc was obtained with purified mucoid antigen by immunoelectrophoresis. Henderson et al. (1984) used an Ouchterlony double-diffusion electrophoresis technique. The antigens consisted of dialysed antigenic extracts of four seaweed alginates. No details were given on the interpretation of the assay.

7.3.3 Polyacrylamide gel electrophoresis and immunoblotting Bucke (1974), Fettetal. (1986) and Simpson etal. (1988) have used various modifications of tube and slab polyacrylamide gel electrophoresis. The alginate stains with alcian blue and presents as a broad band. Preliminary studies in our laboratory show that by using a high voltage it is possible to blot seaweed alginate to nitrocellulose paper and detect it with antialginate antibodies from hyperimmunized rabbits. 7.3.4 Haemagglutination assay Doggett and Harrison (1972) sensitized human type-O red blood cells with purified capsular polysaccharide. They reported that titres were essentially the same as with an autoclaved serotype pool, but gave no figures. ｈｾｩ｢ｹ＠ et al. (1975) tried the same procedure but were unable to demonstrate antibodies in the serum of CF patients. Pier et al. (1983) used sheep red blood cells coated with crude mucoid antigen. This antigen contains 15-28% protein of the total weight but

Antibodies against alginate

119

sera were adsorbed with whole non-mucoid organisms prior to testing. More recently, Pier et at. (1986) and Speert et at. (1987) have shown that non-mucoid organisms express alginate on the surface. Adsorption with non-mucoid strains may therefore also remove anti-alginate antibodies. 7.3.5 Immunofluorescence antibody test H(iSiby et at. (1975) could not demonstrate alginate antibodies when heat-fixed smears of mucoid substance on microscopy slides were reacted with the serum of patients and examined with fluorescein-conjugated anti-IgG, anti-IgM and anti-IgA. 7.3.6 Enzyme-linked immunosorbent assay The enzyme-linked immunosorbent assay (ELISA) procedure requires that the antigen is efficiently and reproducibly immobilized to a solid phase (e.g. polystyrene or polyvinyl chloride). Generally, this is accomplished easily with protein antigens, but is often unsuccessful using polysaccharide antigens (Sutton et aI., 1985). These substances usually carry a net negative charge due to many acidic groups and adsorb poorly to plastic and other supporting materials. This problem can be circumvented by binding the polysaccharide to immobilized antibodies (Callahan et at., 1979; Kaplan et at., 1983), or coupling to poly-L-lysine (Gray, 1979; Leinonen and Frasch, 1982), or proteins (Soderstrom et al., 1983) or by the biotinylation of the polysaccharide followed by reaction with avidin (Sutton et at., 1985). Bryan et al. (1983) found that good attachment was obtained directly with purified bacterial alginate on polystyrene microtitre plates at pH 7.0 with 0.04 M sodium phosphate buffer. Suspension of the antigen in 0.1 or 0.04 M carbonate buffer at pH 9.8 was found to be much less successful. The use of poly-L-lysine-coated plates were associated with two- or fourfold lower titres of antibody than the direct attachment method. Speert et at. (1984) used polyvinyl chloride microtitre plates and suspended their antigen in carbonate/bicarbonate buffer at pH 9.6. Their antigen preparation contained up to 5% protein and the antibody response was found to be maximal at a dilution of 1 : 32. The result must therefore be interpreted with caution in CF patients as they may react avidly with the protein contamination. Baltimore et at. (1986) also used polyvinyl chloride plates, but did not state the conditions for their coating procedure. However, their soluble extract contained 39% protein, which may well interfere with the measurement of antibodies directed against alginate. The best possible solid support and coating procedure could be

120

Immunology ofsurface antigens

determined by using radiolabelled bacterial alginate, performing blocking and washing procedures, cutting out the wells and counting the radioactivity.

7.3.7 Radioimmunoassay This method does not seem to have been employed in measuring alginate antibodies, but as ELISA (see above) is possible then radioimmunoassay should be possible as well.

7.4 lMMUNOGENlClTY OF ALGINATE 7.4.1 Normal individuals Seaweed alginate is an important additive in a wide variety of foodstuffs and beer, has several applications in industry, is used in implantable biomaterials (Sandford and Baird, 1983) and is prescribed as an anti-ulcer medication. Thus, it must be expected, if alginate is immunogenic, that some 'normal' individuals would have reacted to it. However, it must be borne in mind that serological cross-reactivity may not exist between seaweed and bacterial alginate (Pedersen, et al., 1989), presumably due to the presence of poly-guluronate blocks in the former. Bryan et al. (1983) found that apparently healthy controls had detectable IgG antibodies against both seaweed alginate and bacterial alginate, but that they formed a distinct group from CF patients colonized with P. aeruginosa. Baltimore et at. (1986) also found detectable levels of antibodies belonging to IgG, IgA and IgM classes to sodium alginate and extracts from mucoid P. aeruginosa, whereas Speert et al. (1984) found that adults had negligible anti-alginate antibody levels. Henderson et al. (1984) examined workers in the alginate industry and found that 4.5% of the workforce had precipitating antibody against alginate and found evidence of an association with signs and symptoms of pulmonary hypersensitivity. The data presented are somewhat at variance but it seems reasonable to conclude that anti-alginate antibodies may be found in healthy persons. This reactivity does also seem to include reactivity towards bacterial alginate (Pier et al., 1983; Baltimore et al., 1986).

7.4.2 Animal studies Bryan et al. (1983) claim to be the first to show that alginate is immunogenic, as they were able to raise antibodies in mice and rabbits

Immunogenicity of alginate

121

and also demonstrated their presence in CF patients. The ability of rabbits and mice to produce anti-alginate antibodies has been repeated since then by others (Pier et at., 1983; Irvin and Ceri, 1985). Rats also produce anti-alginate antibodies when immunized with bacterial alginate (Woods and Bryan, 1985). The use of Freund's complete adjuvant did not lead to a better response compared with when the immunizing antigen was only the polysaccharide (Bryan et al., 1983). Larsen et at. (1985) and Irvin and Ceri (1985) have both produced mouse monoclonal antibodies against alginate with a specificity for L-guluronate residues. Speert et al. (1987) have also produced monoclonal antibodies against alginate, but no mention was made of the antigen recognition sequence. 7.4.3 Patients with CF Table 7.4 summarizes the findings of four studies on the prevalence of anti-alginate antibodies in CF patients. In all studies CF patients with P. aeruginosa lung infection had higher antibody levels against the alginate antigen than did non-colonized CF patients and normal controls. Speert et al. (1984) found that disease severity, as assessed by the Schwachman clinical score (Schwachman and Kulczyki, 1958), was not significantly correlated with the level of antibody to algal or bacterial alginates. Furthermore, there was no significant correlation between levels of antibody algal and bacterial alginates. The antibodies measured in these studies were mainly of the IgG class. Baltimore et at. (1986) also detected specific antibodies of the IgA and IgM classes, but the difference in titres between CF patients infected with P. aeruginosa and those of the controls did not reach statistical significance. Bryan et at. (1983) were unable to measure IgA antibodies in sputum because of non-specific binding of the I gA complex to the plates. Using the ELISA technique on fluid obtained from broncho-alveolar lavage Baltimore et al. (1986) could detect both IgA and IgG antibodies. However, these antibodies may have occurred by passive transudation into the inflamed lung from the blood, because the second antibody was specific only for the heavy chain of IgA and not for the secretory component present in secretory IgA. Subclass specificity of antibodies against bacterial alginate has not been measured. However, CF patients who were not colonized with P. aeruginosa responded to P. aeruginosa lipopolysaccharide with subnormal levels of specific IgG 2 antibodies (Moss et at., 1986). Recently, the same group also demonstrated an impaired natural antibody response in CF patients against the capsular polysaccharide from H. injiuenzae compared to normal controls (Moss et al., 1987). These investigators speculate that the

ａ｢ｲ･ｶｩ｡ｴｯｮｾＺ＠

56 16 44 3 28 54 Titre"", 512 in 68.5% Titre"", 256 in 96% CF+Pand9-12% CF + P Titre os:: 128 in 100% CF - P and CF - P controls

26 8 18 In a dilution 1 : 32 serum from CF + P Had higher 00 than CF - P (p < 0.0008)

Speert et at. (1984) Bryan et al. (1983) > 99.99% uronicacirl < 5% protein 1.2 fLg LPS/ml ELISA ELISA rabbit/mouse NO NOll: 1.200 10 8 32 Mean titre of: IgG CF + P 31584-65965 CF - P 110-170 Control 49-521

ELISA NO

Baltimore et al. (1986) 39% protein

IgA 813-9507 6-45 31-58

IgM 96-142 34-44 59-108

LPS, lipopolysaccharide; HA, haemagglutination assay; ELISA, enzyme-linked immunosorbcnt assay; ND, not determined; CF - P, CF patients not colonized wth P. aerug;nosa; ｃｾﾷ＠ + P, Cf patient':> coloniLed with P. aefUg;nosa; 17, number of ｐ､ｴｩ･ｮｾＮ＠

Human studies Controls, n CF - P. aeruginosa, n CF + P. aeruginosa, n Results

HA Rabbit 1 :8-1 : 128

Pier et al. (1983) > 99% uronic acid

Summary of studies determining antibodies against alginate from mucoid P. aeruginosa

Author Pu rity of antigen (% total) Method Animal immunization Titre

Table 7.4

The role of alginate in human disease

123

IgG 2 response to polysaccharide antigens, of potential importance for biological activity, may be impaired in CF. In our clinic we have found that high levels of IgG 2 and IgG:1 correlated with poor clinical condition and high levels of precipitating antibodies against P. aeruginosa (Pressler et al., 1988). Work is currently under way to determine whether anti-alginate antibodies are restricted to the IgG 2 subclass. 7.5 THE ROLE OF ALGINATE IN HUMAN DISEASE 7.5.1 Studies in vitro

Alginate may be an important structure mediating adhesion (see Chapter 6). Adherence can be inhibited by addition of mmlOclonal antibodies specific for bacterial alginate (Baker and Austria, 1987). Cash et al. (1979) have developed a model of chronic P. aeruginosa lung infection in the rat (see Chapter 5) and using this model Woods and Bryan (1985) found that anti-alginate antibodies offered strain-dependent protection against chronic lung infection. They found that in some instances antibodies to alginate may promote clearance of bacteria from the lung. Furthermore, by analysing histological specimens using immunofluorescence microscopy they found an overwhelming reaction to IgG, IgA and IgM when using anti-alginate antibodies as the primary antibody. However, it was readily apparent from their studies that anti-alginate antibodies may be harmful, because antibody deposition was evident in the lung tissue of some of the animals immunized with alginate. They concluded that immune complex formation should be considered as a possible consequence of immunization with alginate. Their concept of the role of immune complexes in the pathogenesis of CF lung disease agrees well with studies from our laboratory Ｈｈｾｩ｢ｹ＠ et al., 1986). 7.5.2 Polyclonal B-cell activation

Alginate may have a role as a polyclonal B-cell activator, since, like other polysaccharides, it is aT-independent antigen (Roitt et al., 1985). Poor clinical status in CF has been associated with high levels of IgG (Turner et at., 1978; Matthews et al., 1980), and a substance which non-specifically induces high levels of IgG may adversely affect the outcome of the lung disease associated with CF. Pier and Elcock (1984) found that rabbits immunized with alginate or whole cells differed, because heterologous antibodies elicited by alginate generally showed high affinities and specificities for heterologous antigens. Kinetic analysis demonstrated that non-specific immunoglobulin synthesis was seen shortly after antibodies to homologous alginate were

124

Immunology of surface antigens

elicited. Alginate is mitogenic for mouse and human B-cells (Ames et at., 1983; Daley et at., 1985), with some enhancement of response after depletion of T -cells (Ames et at., 1983). The mitogenic response was not inhibitable by polymyxin B, which blocks lipopolysaccharide-induced mitogenicity (Daley et at., 1985), suggesting that the alginate was able to activate the B-lymphocytes. However, it is probably better to measure polyclonal B-cell activation using a plaque-forming assay (Roitt et at., 1985), but no such studies have been published using alginate. Furthermore, it has been found that alginate induced the formation of interleukin-l by mouse splenic macro phages (Daley et at., 1985). Ames et at. (1985) showed that the anti-phagocytic properties attributable to alginate could be overcome by specific antibodies to alginate. In a study of older CF patients (mean age 19.5 years, range 12-35 years) who were not colonized with P. aeruginosa, Pier et at. (1987) found that, although colonized CF patients had higher opsonophagocytic killing antibody titres against mucoid P. aeruginosa (p < 0.0(05), the noncolonized CF patients had higher titres of alginate-specific opsonophagocytic killing antibodies. Therefore, they suggested a role for this type of antibody in resistance to P. aeruginosa colonization in CF (Pier et at., 1987). In order to prevent colonization it seems mandatory to elicit a specific response that effectively prevents adhesion of non-mucoid strains (probably the initial colonizers) to tracheal epithelium (see Chapter 6). Although the presence of alginate on the surface of non-mucoid P. aeruginosa has been described (Pier et at., 1986; Speert et at., 1987), it is doubtful whether a vaccine aimed at preventing colonization should be focused solely on alginate. 7.5.3 The role of alginate Bacteria usually produce a capsule when they are in a condition of nutritional starvation. The capsule may then function as a sieve or a trap which concentrates nutrients essential for the existence of the bacteria (Costerton et at., 1981). Mucoid P. aeruginosa usually appears after a period of variable length of non-mucoid colonization. This might imply that at that stage the bacterium experiences a state of nutritional starvation and has to switch to the mucoid phenotype in order to survive. Iron is a necessary element in the daily bacterial diet and may be made available by the action of pseudomonal proteinases on the host ironchelating protein transferrin (Doring et at., 1988). However, the function of these proteinases is neutralized after approximately 6-9 months (Doring and Hli>iby, \983) and therefore the iron source is made less accessible. It is a common clinical observation that mucoid strains appear approximately 6 months after infection with non-mucoid strains, which is

Changes in other surface antigens

125

in accordance with this theory. Pier and Elcock (1984) suggest that alginate may be able to disrupt the normal regulatory immune mechanisms, allowing for the persistence of mucoid P. aeruginusa and elevated immunoglobulin levels. These conditions may then potentiate the progression of lung disease in CF by virtue of a hypersensitivity response to bacterial and tissue antigens resulting in lung tissue destruction (Pier and Elcock, 1984). 7.6 CHANGES IN OTHER SURFACE ANTIGENS IN MUCOID AND NON-MUCOID STRAINS 7.6.1 Lipopolysaccharide A number of somatic and extracellular factors of P. aeruginosa are known to give rise to an antibody response in human infection Ｈｈｾｩ｢ｹＬ＠ 1982; Lam et at., 1983; Cryz, 1984). As far as we are aware no study has shown whether the antibody response to mucoid organisms changes with different sites of infection or with phenotypic differences between mucoid strains (Pugashetti et al., 1982). Lipopolysaccharide is a macromolecule unique to the outer surface of Gram-negative bacteria. The molecule is made up of three parts: lipid A, a core region and O-antigen repeating polysaccharide units. Deficiencies in lipopolysaccharide from the majority of mucoid strains involved in CF have been reported (Hancock I't a!., 19S3; Penketh ptal., 1983; Ojeniyi et rd., 1985; Fomsgaard et al., 1988). These deficiencies involve the loss of all or part of the O-antigen units from the molecule with the result that bacteria become non-typable or polyagglutinable in reaction with standard O-typing antisera (Hancock et al., 1983; Pitt etal., 1986). These alterations in agglutinability correlate with loss of serum resistance (Pitt et aI., 1986). \Ve have shown immunochernical similarities between lipopolysaccharide core/lipid A antigens in typable and polyagglutinable strains from CF lungs, which indicate that this region of the molecule is conserved when O-antigen units are lost (Fomsgaard et al., 1988). Sexton and Reen (1986) showed, using electrophoresis of various P. aeruginosa fractions and immunoblotting with CF serum, that the serum contained anti-lipopolysaccharide antibodies without any obvious difference between mucoid and non-mucoid strains. 7.6.2 Flagella Mucoid strains are also reported to be non-motile (Royce and Miller, 1982a) and this accords with our own findings in stable mucoid strains isolated from CF lungs. However, we have found that half of the strains

126

Immunology of surface antigens

which were non-motile using phase-contrast light microscopy of a suspension of live cells were flagellated when viewed by electron microscopy. Most CF patients infected with P. aeruginosa show antibodies against a flagellar preparation early after onset of infection (Shand et ai., 1988).

7.6.3 Outer membrane proteins

Outer membrane proteins F (porin), H1/H" and I, and possibly pili of P. aeruginosa, have been shown to be antigenic in experimental mouse infections (Hedstrom et ai., 1984). Protein F protects mice challenged intra peritoneally with P. aeruginosa (Gilleland et ai., 1984). The presence of antibodies to protein F correlated well with survival in a group of patients with haematological malignancies who became infected with P. aeruginosa (Matthews et al., 1986). Hancock et al. (1984) have identified antibodies to outer membrane proteins F, E, H" and I in serum from patients with chronic P. aeruginosa lung infection. Fernandes et al. (1981) have reported antibodies to outer membrane proteins with molecular weights of 58000, 37500 and 34 000 in serum from CF patients (Fernandes I't al., 1981). The presence of antibodies in CF serum, as noted earlier, does not confer protection against lung infection (H0iby ft ai., 1987). In a study of P. aeruginosa isolated directly from infected CF lungs (Brown et ai., 1984) bacteria were shown to be growing under conditions of iron limitation, as revealed by the expression of several highmolecular-weight proteins which could also be observed when the same isolate was grown in the laboratory under iron-depleted conditions (Meyer et al., 1979). The patient's serum contained antibodies to the iron-regulated outer membrane proteins (Brown et al., 1984; Anwar et al., 1984). This indicates that P. aeruginosa is growing in the CF lung under conditions of iron starvation. Mucoid strains are more stable under iron-limited conditions (Ombaka et al., 1983; Boyce and Miller, 1982b) and iron deprivation in the lung may be a selective pressure which sustains the mucoid form. An antibody response to outer membrane proteins, including iron-regulated membrane proteins, has also been reported in a rat lung infection model (Cochrane I't al., 1987). In a longitudinal study of antibody response to protein antigens we have found that antibodies to iron-regulated membrane proteins do not appear early in the course of infection, but could be detected in all serum samples showing eight or more precipitin peaks. Antibodies to ironregulated membrane proteins became more pronounced in the later stages of progressively advanced disease (G. H. Shand, S. S. Pedersen, M. R. W. Brown and N. H0iby, unpublished results).

References

127

7.7 CONClUDING REMARKS Infection with mucoid P. aeruginosa in patients with CF gives rise to a more pronounced antibody response than does infection with nonmucoid strains. In CF a strong immune reaction is associated with more severe disease; accordingly, the prognosis in CF is worst for patients with mucoid infection. Alginate is immunogenic in scveral mammalian species, and antibodies against alginate can be detected in patients with CF. However, the functional ability of these antibodies is not known, but dearly they are not sufficient to cause elimination of the bacteria from the lungs. Furthermore, the interaction of alginate with the host defence system involves the ability to adhere to tracheal cells (see Chapter 6), antiphagocytic properties, the ability to scavenge fr'ee oxygen radicals released from stimulated polymorphonuclear leukocytes, and acting as a barrier to antibiotic penetration (see Chapter 8), as well as inducing a pronounced and non-specific humoral response. Biosynthesis of alginate is metabolically expensive for the bacteria (Mian et at., 1978; Jarman and Pace, 1984) and is associated with loss of lipopolysaccharide, motility, serum resistance and with polyagglutinability. By adapting to a deficient phenotypic appearance encased in an alginate glycocalyx in the hostile environment of the human lungs the bacteria may have opted for long-term survival rather than short-term virulence. Therefore, there must be an advantage for mucoid strains in this particular disease, which may be associated with an impaired ability to mount a proper subclass-specific humoral response to polysaccharide antigens. Being a surface-expressed bacterial structure, interest in the immunology of alginate is centred around the possibility of preventing the infection by immullotherapy. It remains to be shown conclusively that anti-alginate antibodies are protective in animal studies and in patients without concomitant immune-complex deposition in the lungs. The whole area of mucosal immunity has received very little attention so far, but induction of a protcctive IgA immune response rrlay well be the way to prevent colonization and to prevent formation of immune complexes.

REFERENCES Ames, P., Eardley, D. and Pier, G. B. (1983) In vitro and in viI,o polyclonal B cell activation by mucoid exopolysaccharide frolll PSI'lUlOIlWflllS (Jl'l"IIgifw.Vl. 2hd Intfncima COnlnfl/{f 01/ Antimicrobial Agent.1 and Chmwthnaj)" American Society for Microbiology, Washington, DC, Abstr. 763. Ames. P., Desjardins, D. and Pier, G. B. (19R5) Opsonophagocytic killing activity of rabbit antibody to Pseudolllollas aerugil!osll mucoid exopolysaccharide. Tnfeel. Tmlflllil. 49, 2R 1-5.

128

Immunology ofsurface antigens

Anwar, H., Brown, M. R. W., Day, A. and Weller, P. H. (1984) Outer membrane antigens of mucoid Pseudomonas aeruginosa isolated directly from the sputum of a cystic fibrosis patient. FEMS Microbiol. Lett. 24, 235-9. Baker, N. and Austria, A. (1987) Inhibition of adherence of mucoid strains of Pseudomonas aeruginosa to tracheal epithelium by antibodies to alginate. A nnu. Mtng. Am. Soc. Microbiol. Abstr. D102. Baltimore, R. S. and Mitchell, M. (1980) Immunologic comparison of mucoid strains of Pseudomonas aeruginosa: comparison of susceptibility to opsonic antibody in mucoid and non-mucoid strains.].lnlect. Dis. 141,238-47. Baltimore, R. S., Fick, R. B.] I' and Fino, L. (1986) Antibody to multiple mucoid strains of Pseudomonas aeruginosa with cystic fibrosis, measured by an enzyme-linked immunosorbent assay. Pediatr. Res. 20,1085-90. Barrett, D. J. and Ayoub, E. M. (1986) IgG e subclass restriction of antibody to pneumococcal polysaccharides. Clin. Exp. hnmunol. 63,127-34. Barrett, D. J., Ammann, A. J., Stenmark, S. and Wara, D. W. (1980) Immunoglobulin G and M antibodies to pneumococcal polysaccharides detected by enzyme-linked immunosorbent assay.lnfect.lmmun. 27, 411-17. Barrett, D. J., Lee, C. G., Ammann, A. J. and Ayoub, E. M. (1984) IgG and IgM pneumococcal polysaccharide antibody responses in infants. Pediatr. Res. 18, 1067-71. Bishop, G. T. and Jennings, H. J. (1982) Immunology of polysaccharides. In: Aspinall, G. O. (ed.) The Polysaccharides, Vol. I, Academic Press, New York, pp.291-330. Borgono,J. M., McLean, A. A., Vella, P. P., Widhour, A. F., Canepa,J., Davidson, W. L. and Hilleman, M. R. (1978) Vaccination and revaccination with polyvalent pneumococcal polysaccharide vaccines in adults and infants. Proc. Soc. Exp. Bioi. Med. 157, 148-54. Borowski, R. S. and Schiller, N. L. (1983) Examination of the bactericidal and opsonic activity of normal human serum for a mucoid and non-mucoid strain of ｐｳ･ｵ､ＰＱｮｬＨｌｾ＠ aeruginosa. CUlT. Microbiol. 9, 25-30. Boyce,]. R. and Miller, R. V. (1982a) Motility as a selective force in the reversion of cystic fibrosis-associated mucoid Pseudomonas aeruginosa to the non-mucoid phenotype in culture. Infect. Immun. 37, 840-4. Boyce, J. R. and Miller, R. V. (1982b) Selection of non-mucoid derivatives of mucoid Pseudomonas aeruginosa is strongly influenced by the level of iron in the culture medium.lnlect. Immun. 37,695-70 I. Brett, M. A., Ghonheim, A. T. M., Littlewood, J. M. and Losowsky, M. S. (1986) Development of enzyme-linked immunosorbent assay (ELISA) to detect antibodies to Pseudomonas aeruginosa cell surface antigens in sera of patients with cystic fibrosis.]. Clin. Patlwl. 39, 1124-9. Brown, M. R. W., Anwar, H. and Lambert, P. A. (1984) Evidence that mucoid . Pseudomonas aeruginosa in the cystic fibrosis lung grows under iron-restricted conditions. FEMS Microbiol. Lett. 21, 113-17. Bryan, L. E., Kureishi, A. and Rabin, H. R. (1983) Detection of antibodies to Pseudomonas aeruginosa alginate extracellular polysaccharides in animals and cystic fibrosis patients by enzyme-linked immunosorbent assay. ]. Clin. Microbiol. 18,276-82. Bucke, C. (1974) Polyacrylamide gel electrophoresis of alginic acid.]. Clmnnatogr. 89,99-102. Burns, M. W. and May,J. R. (1968) Bacterial precipitins in serum of patients with cystic fibrosis. Lancet i, 270-2.

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Cabral, D. A., Loh, B. A. and Speert, D. P. (1987) Mucoid Pseudomonas aeruginosa resists non opsonic phagocytosis by human neutrophils and macrophages. Pediatr. Res. 22,429-31. Callahan, L. T., Woodhour, A. F., Meeker, J. B. and Hilleman, M. R. (1979) Enzyme-linked immunosorbent assay for measurement of antibodies against pneumococcal polysaccharide antigens: comparison with radioimmunoassay.]. Clin. Microbiol. 10,459-63. Cash, H. A., Woods, D. E., McCullough, B.,]ohanson, W. G.,]r and Bass,J. A. (1979) A rat model of chronic respiratory infection with Pseudomonas aeruginosa. Am. Rev. Resp. Dis. 119,453-9. Cochrane, D. M. G., Anwar, H., Brown, M. R. W., Lam, K. and Costerton,J. W. (1987) Iron deprivation ill vivo: surface protein antigens of Pseudomonas aeruginosa in infection. Antibiot. Chemother. 39, 125-35. Costerton,J. W., Irvin, R. T. and Cheng, K.J. (1981) The bacterial glycocalyx in nature and disease. Annu. Rev. Microbiol. 35, 299-324. Cowan, M.J., Amman, A.J., Wara, D. W., Howie, V. M., Schultz, L., Doye, N. and Kaplan, M. (1978) Pneumococcal polysaccharide immunization in infants and children, Pediatrics 62,721-7. Crowder,J. G. and White, A. (1970) A serologic response in human Pseudomonas infection.]. Lab. Clin. Merl. 75, 128-36. Cryz, S. J.,]r (1984) Pseudomonas aeruginosa infections. In: Germanier, R. (ed.) Bacterial Vaccines, Academic Press, New York, pp. 317-51. Daley, L., Pier, G. B., Liporace,J. D. and Eardley, D. D. (1985) Polyclonal B cell stimulation and interleukin I induction by the mucoid exopolysaccharide of Pseudomonas aeruginosa associated with cystic fibrosis. ]. Immunol. 134, 3089-93. Diaz, F., Mosovich, L. L. and Neter, E. (1970) Serogroups Pseudomonas aeruginus!l and the immune \"esponse of patients with cystic fibrosis.]. Infect. Dis. 121, 269-74. Dochez, A. R. and Avery, O. T. (1917) Elaboration of specific soluble substances. ]. Exp. Med. 26,477-93. Doggett, R. G. (1969) Incidence of mucoid Pseuriomullas aeruginosa from clinical sources. Appl. Microbiol. 18,936-7. Doggett, R. G. and Harrison, G. M. (1972) Pseudomonas aemginosa: immune status in patients with cystic fibrosis. InFect. hmnun. 6,628-35. Doring, G. and H¢iby, N. (1983) Longitudinal study of immune response to Psmdomonas aeruginosa antigens in cystic fibrosis. Illlect. Immun. 42, 197-20 I. Doring, G., Pfestorf, M., Botzenhart, K. and Abdallah, M. A. (1988) Impact of proteases on iron uptake of Pseudomonas aerugiuosa pyoverdin from transferrin and lactoferrin.ln/ecl.lmmun. 56,291-3. Fernandes, P. B., Kim, c., Cundy, K. R. and Huang, N. N. (1981) Antibodies to cell envelope proteins of Pseudomonas aeruginusa in cystic fibrosis patients. Inlect. Immun. 33, 527-32. Feu, W. F., Osman, S. F., Fishman, M. L. and Siebles, T. S., III (1986) Alginate production by plant-pathogenic pseudomonads. Appl. Environ. Microbiol. 52, 466-73. Fomsgaard, A., Conrad, R. S., Galanos, c., Shand, G. H. and H¢iby, N. (1988) Comparative chemistry and immunochemistry of lipopolysaccharide from typable and polyagglutinable Pseudomonas aeruginosa strains.]. Clin. M icrobiul. 26,821-6. Friend, P. A. (1986) Pulmonary infection in cystic fibrosis.]. In/ect. 13,55-72.

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Gilleland, I I. E., .II', Parker, M. G., Matthews,J. M. and Berg, R. D. (1984) Use of a purifIed outer membrane protein F (pm'in) preparation of Pseudomonas aeruginosa as a protective vaccine in mice. Injee!. Immun. 44, 49-54. Glode, \1. P., Lewin, E. B., Sutton, A., I.e C. T., Gotschlich, E. C. and Robbins,J. B. (1979) Comparative immunogenicity of vaccines prepared from capsular polysaccharides of group C Neissnia tnfllillgitidis O-acetyl-positive and 0acetyl-negative variants and [;;schericitia coli K92 in adult volunteers. I Infecl. Dis. 139,52-9. Govan,J. R. W. and I Ianis, G. S. (1986) Pseudo/l/onas llPluginosa and cystic fibrosis: unusual bacterial adaptation and pathogenesis. i'vficrobiol. Sci. 3, 302-S. Grasdalen, H. B., Larsen, B. and Smidsr0d, O. (1981) "C-n.m.r. studies of monomeric composition and sequence in alginate. Cal'iJoityr!l'. Rn. 89, 179-91. Gray, B. M. (1979) ELISA methodology for polysaccharide antigens: protein coupling of polysaccharides for adsorption to plastic tubes. I IlilInUlwl. JHel/u){L, 28, IS7-92. Hammarstl'Om, L., Persson, M. A. A. and Smith, C. I. E. (1985) Immunoglobulin subclass distribution of hUlllan anti-carbohydrate antibodies: aberrant pattern of IgA deficient donors. Imlllunology 54: 821-6. Hancock, R. E. W., Mutharia, L. \1., Chan, L., Da\'\'eau, R. P., Speert, D. P. and Pier, G. B. (1983) Pseudolllonas aeillginosa isolates from patients with cystic fibrosis: a class of serum-sensitive nontypable strains deficient in lipopolysaccharide O-side chain.Illjfcl. IIIIIIIUIl. 42, 170-7. Hancock, R. E. W., Mouat, E. C. A. and Speert, D. P. (19S4) Quantitation and identification of antibodies to outer membrane proteins of PSl'lulolllonas anugillo.la in sera of patients with cystic fibrosis.I Infect. Dis. 149,220-6. Hedstl'Olll, R. C., Pa\'lovskis, O. R. and Galloway, D. R. (1984) Antibody response of infected mice to outer membrane proteins of PSelldOlllOI/aS aemgillo.la. Injeci. Immull. 43, 49-53. Heidelberger, M. (1956) Lntures in InwlIuwchelllistrj', Academic Press, New York. Henderson, A. K., Ranger, A. F .. Lloyd . ./ .. \1cShalT\, c., Mills, R . .J. and Moran, F. (1984) Pulmonary hypersensitivity in the alginate industry. Scottish i'vled.J. 29,90-5. H0iby, N. (194 7a) Epidemiological investigations of the rt'spiratory tract bactt'riology in patients with cystic fibrosis. Acta Pat/wi. ,'v[iClobiol. .)mll!/. B 82, 541-50. H0iby, N. (1974b) P,II'udo/flonas atl'l1gillosa infection in cystic fibrosis: relationship between mucoid strains of Pseudomonas a('!'Ugiriosa and the humoral immune response. Ac/a Pat/wi. i'vlicrobiol. S((lllri. B 82, 551-8. H0iby, !\i. (1975) Prevalence of mucoid strains of Pseudoillonas a(,l'I1gillosa in bacteriological specimens from patients with cystic fibrosis and patients with other diseases. /\cla Pat/wi. ,HicroiJiol. Smnd. B 83, 549-52. H0iby, 1'\. (1977a) Antibodies against PII' IU/lllllO lUl,l aelligil/U\a in serum from normal persons and patients colonized with mucoid or non-mucoid PSI'llr/omonas aPl'llgillosa: results obtained by crossed immunoelectrophoresis. At/a Pat/wi. ,'v[icl'OiJiol. Seand. C 85,142-8. H0iby, N. (1977b) PWUdO/l/OIUlS aC/'/Igil/o\{l infection in cystic fihrosis: diagnostic and prognostic significance of P.II'lIdo/flOllaS afrllgillIJsa precipitins determined by means of crossed immunoelectrophoresis. A sun·ey. Acla Pal/wi. MiuoiJiol . .)mlld. C. SUNI. 262. 1-96. H0iby, N. (1979) Immunity: humoral response. In: Doggett, R. G. (ed.) PsCUr!U/l/OIUl.1 1lI'lIIgilllJ,la: ClilliUlI /flllni/i'.I/fl/iolt.\ oj infnlioll lind rllITl'llllhemjiy, Academic Press, New York, pp. 157-89.

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H0iby, N. (1982) Microbiology of lung infections in cystic fibrosis patients. Acla Paedialr. Swnd. SUj,/,i. 301, ,)3-54. H0iby, N. and Axelsen, N. H. (1973) Identificat.ion and quantification of precipitins against Pleudolllollas aerugillosa in patients with cystic fibrosis by means of crossed immunoelectrophoresis with intermediate gel. Aria Pallwi. /HilTobioi. Seal/Ii B 81, 298-308. H0iby, 1'\. and Oiling, S. (1977) PII'llliomollas IIfmgillosa infection in cystic fibrosis. Bactericidal effect of serum from normal individ uals and patients with cystic fibrosis on P. aemgillos!1 strains from patients with cystic fibrosis or other diseases. Acta Palhoi. iHirmbiol. SUllid. C 85,107-14. H0iby, No, Andersen, V. and Bendixen, G. (1975) Pseudomonas anugi/lO.la infection in cystic fibrosis: humoral and cellular immune responses against P.lelido/l/o//a,1 ([i'rugino.l(l. Aclo Patho!. ,\TillObio!' SUllul. C 83, 459-68. H0iby, 1'\., Flensborg, E. W., Beck, B., Friis, B.,Jacobsen, S. V. andJacobsen. L. (1977) P.li'mloll/ollos olTllgillo,la infection in cystic fibrosis. SUllld . .J. Re.lil. Di.l. 58.65-79. H0iby, 1'\., D(iring. G. and ｓ｣ｨｩｾＩｴｺＬ＠ P. O. (1986) The role of immune complexes in the pathogenesis of bacterial infections. /\111111. ReI'. AIicml,ioi. 40, 29-53. H0iby, 1'\., Doring, G. and Schi\1tz. P. O. (1987) Pathogenic mechanisms of chronic PII'IlI!IIJI!O/Ul.I atrl1gillO,l(1 infections in cystic fibrmis patients. Alilibilit. Chell/ollin. 39,60-76. Huang. 1'\. ;\I. and Doggett, R. G. (1979) Antibiotic therapy of PSf//{iOIlIO/WI anllgi//usa. In: Doggett. R. G. (ed.) PI Pl/duII/O/UI.I anl/gilioslI: clillimlll/([lIi/n' tatioll.l 0/ in/ectioll Ilild ClllT(,1I1 thnajly, Academic Press, \lew York. pp. 411-44. Inin, R. T. and Ceri. I I. (191'\5) I IIllllunochemical examination of the PSl'lldolllo/Uis (lemgino.l(l g-Iycocalyx: a monoclonal antibody which recognizes l.·glliuronic acid residues of alginic acid. (;111/.]. ,\licmiJiol. 31,268-73. Jarman, T. R. and Pace, C. W. (1984) Energy requirement for microbial exopolysaccharide svnthesis. ilrrh. iHicmiJilil. 137, 231-:,}. Jennings, H. J (El83) Capsular polysaccharides as human raccines. Adl'. CoriJohwlr. Choll. niochtlll. 41, J55-·208. Johnston. R. B .. Anderson, 1'., Rosen. F. S. and Smith. D. II. (1973) Characteriz· ation of human antibody to polyribophosphate, the capsular antigen of HlltlllujJhilus in/fuellwe. type;\1. Clill. 1111 IJI II/wi. 1lIIlIlIlIIojHlliI. 1,234-40. Kaplan, S. L., Mason, E. O.,Johnson, C .. Btoughton, R. A., Hurley, n. and Parke, J c. (1983) En/yme·linked immunosorbent assay for detection of capsular antibodies ag-ainst 1/(lelllo/,lIi/1/1 il/jll/fIlZI/{' type b: comparison with radio· immunoassay.]. Oill. Alinohio/. 18,1201-4. Kenne. L. and Lindberg, B. (J !J8:) Bacterial polJSaccharides. In: Aspinall, G. O. (eel.) The PO/Y,llIcc/wridl's, \'01. 2, Academic Press, :-.Ie\1 York. pp. ＲＸＷＭＳＶｾＩＮ＠ Klinger, J. D., Straus, D. C .. Hilton, C. B. and Bass, J A. (1978) Antibodies to proteases and exotoxin ,.\ of PWWiOIllOllllS lIerugi II 0.1 II in patients with cystic fibrosis: demonstration bi radioilllmunoassay.]. Ili/ni. [)i.l. 138,19-58. Lam.J. 5., MllIharia, L. :VI., lIancock, R. E. W., lI¢iby, 1'\., l.am, K., Ba:k, L. and Costerton, J. W. (198:\) Immullogenicity of PSl'llIioIllO/WS lIerugillosa outer membrane antigens examined by crossed immunoelectrophoresis. 111/1'1'1. lilli/lim. 55, I

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142

Alginate and evasion of host defence

experiments is a measure of the high-molecular-weight material associated with the macrophage, as well as the low-molecular-weight material released from the bacteria within the macrophage. Degradation is purely a measure of the latter, and so is a measure of the material digested by the macrophage. Uptake and degradation of bacteria parallel each other very closely in these experiments, implying that alginate has a primary effect on the association and ingestion of bacteria, rather than on killing or digestion. The dose-related inhibitory effect on the macrophageassociated high-molecular-weight pool (Fig. 8.lc) reinforces this supposition. Meshulam et al. (1984) also concluded that the reduced neutrophil bactericidal activity for P. aeruginosa was a function of reduced ingestion, since the rate of intracellular killing was similar for mucoid and non-mucoid strains. Mucoid strains from a variety of sites (urinary tract infection, wound, water, and lungs of a CF patient) were examined, and the authors concluded that mucoid P. aeruginosa strains showed significantly reduced phagocytic uptake by leucocytes when compared to non-mucoid strains. They did find that mucoid strains occasionally exhibited normal uptake and their data show some overlap in the comparison of uptake by polymorphs of [:lH]adenine-labelled mucoid and non-mucoid strains. However, bacterial suspensions were washed before use, which may partly account for this finding. Meshulam et al. (1984) state that there was considerable variation in the extent to which their mucoid strains produced extracellular material and, one can possibly conclude, of the degree to which this exopolysaccharide was removed from bacteria by centrifugation. A general feature of all studies of the effect of P. aeruginosa alginate on phagocytosis is that it is usually only significant at high concentrations, suggesting a physical effect on phagocytosis rather than some discrete chemical event. In our hands (Simpson et al., 1988) P. aeruginosa alginate only inhibits uptake and degradation significantly at high concentrations (i.e. 1.0 mg/ml, see Fig. 8.1). In addition, commercial seaweed alginate (similar in constitution to bacterial alginate, but non-acetylated, see Chapter 3) inhibits uptake and degradation of P. aeruginosa by mouse peritoneal macrophages to almost exactly the same degree (Fig. 8.2). Other workers have also found an inhibitory effect only at high levels, even with a variety of cell types and states of activation (Schwarzmann and Boring, 1971; Ruhen et al., 1980; Oliver and Weir, 1983, 1985). The observed variation in the effective concentrations of alginate preparations is probably a function of different isolation and purification techniques leading to different physical states of the polymer. Nonetheless, in vivo in the CF lung, mucoid P. aeruginosa grows as a microcolony surrounded by its viscous alginate matrix (see Chapter 5) that will affect phagocytosis.

Mucoid P. aeruginosa and CF (a)

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Fig.8.2 The uptake (a) and degradation (b) of non-viable non-mucoid radiolabelled P. aeruginosa by murine peritoneal macrophages in the presence of 0 mg/ml (.A.), 0.04 mg/ml (6), 0.2 mg/ml (+) and 1.0 mglml (0) sigma seaweed alginate acid (*p < 0.05; **p < 0.01). Uptake (a) is a reflection of the sum of TCA-soluble material in the supernatant and the TCA-insoluble material associated with the macrophages. Degradation (b) is purely the former.

8.2.3 Phagocytosis of other particles in the presence of P. aeruginosa alginate Bacterial alginate also inhibits the uptake of zymosan and latex particles (Oliver and Weir, 1985; Simpson et al., 1988). Each particle type has different uptake kinetics to bacteria but inhibition is marked. particularly at an alginate concentration of 1 mg/rnl (Fig. 8.3). Grasso f'l al. (1984) have found a similar dose-related inhibition of the phagocytosis of Sacdtaromyces cerevisiar. If a particle can be phagocytosed then bacterial alginate, certainly at a relatively high concentration, can inhibit the process. If bacterial alginate exerts its effect on uptake or events preceding uptake, which properties of the alginate polymer are relevant to the inhibition? There is more D-rnannuronic acid than L-guluronic acid in bacterial alginate and polyguluronate blocks are absent (see Chapter 3). When the mannuronic acid monomer was tested at the same uronic acid

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The zymosan (a) and latex particle (b) association, measured as macrophage-associated 'TCA-precipitable pool, after incubation with murine peritoneal macrophages in the presence of o mg/ml (A), 0.04 mg/ml (6), 0.2 mg/ml (+) and 1.0 mg/ml (0) exogenous purified P. aeruginosa alginate (*p < 0.05; **p < 0.01).

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20

Fig.8.3

, Chichester. McGourty, C. (19R9) CF screening premature? Na/urt', 342, ｾＩＳＴＮ＠ McPherson, M. A. and Dormer, R. L. (1987) 'l'he molecular and biochemical basis of cystic fibrosis. Biosei. Rej). 7, 167-85. McPherson, M. A. and Goodchild, M. C. (1988) The biochemical defect in cystic . fibrosis. Clill. Sri. 74,337-45. Morris, G. and Brown, :VI. R. \\'. (1988) 1\o\'el modes of action of aminoglycoside antibiotics against PSf/lllolnolla.1 aemgium(/. hwett i, 1359-61. Riordan, J. R., Rommens, J. M,. Ke\'em, B-S. ('/ al. (1989) Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Scimre,245, 1066-73. Roberts, C. M., Batten, J and Hodson, :\1. E. (1985) Ciproflaxacin-resistant Pseudomonas. Lal/at i, 1442. Rommens, J. M., Iannuzzi, M. C. Kerem, B-S. et al. (1989) Identification of the cystic fibrosis gene: chromosome walking and jumping. Sciena. 245, 1059-65. Scambler, P. Ramsay, M., Estivill, X., Halford, S., iIo, M.-F., Sutherland, Ii., Wicking, c., Beli, G., Tata, F., Williamson, R. and Wainwright, B. (1988) New markers close to the cystic fibrosis locus. Abstr. I. Cystic Fibrosis ResP!lrth WorknsConf.,1988. Smyth, R. L., Scott,J. P., Higenbottam, T. W., Hodson, M. and Walwork,.J. (198S) Early experiences of heart-lung transplantation in cystic fibrosis. Abstr. 24. Cystic Fibrosis Research Work!'n Conf., 1988. Sokatch,J. R. (cd.) (1986) The bactnia, vol. X: The biology a/Pseudomonas, Academic Press, 1\ew York. Tsang, V., Khagani, A., Fitzgerald, M., Banner, 1\., Hudson, M. E. and Yacoub, M. (1988) Heart and lung transplantation for cystic fibrosis. Abstr. 23. Cystic Fibrosis Research Workers Canf, 19R8. Wainwright, B. J, Scambler, P. J, Schmidtke, J, Watson, E. A., Law, H.-Y., Farrall. M., Cooke, H.J., Eiberg, H. and Williamson, R. (1985) Localization of cystic fibrosis locus to human chromosome 7cen-q22. NatuT(' 318,381-5. Williamson, R., Farrall, M., Lench, 1\;., Stanier, P., Coutelle, C. and Williams, C. (19R8) The use of CF probes for antenatal diagnosis and cancer testing. Abstr. 2. Cystic Fibrosis Rnearch Workers Cout:, 1988.

Index

Acetyl transferase 181-2, 195,224 Acetylation, of alginate 37-8, 42, 45-6,117,151,166-7,198-200, 202,207 N-acetylneuraminic acid, see Sialic acids Adhesins 96-107 Adhesion, of P. aeruginosa 55, 77-8, 95-107,124,136 Adjuvant 115 Aeruginocin, see Pyocin types Aldolase Entner-Doudoroff 6, 184-5 glycolytic 186 Alginate acetylation 37-8,42,45-6, 117, 151, 166-7,198-200,202,207 and antibiotic susceptibility 85, 152--4 as adhesin 100-3, 105 as ion exchanger 40, 78, 80 biofilms containing 77-87 biosynthesis 59, 65-9,181-205 block structure 38-41, 45,117,192, 200 calcium chelation 40-1, 44-6, 59-61 chemical composition 37-8 discovery 36 electron microscopy 44, 78, 86, 91 evasion of host defence 135-59, 215-17 gel formation 40-1, 44-6, 59-61 genetics of biosynthesis 206-20 immunology 117-25 interactions with exoenzymes 160-80 ionization 42-3,166-7 M:Gratio37,54, 165, 192, 199-200, 210 molecular size 43--4 nuclear magnetic resonance 45, 190-2

release of exolipase 162-76 scavenging of free radicals 147-52 seaweed 36, 117, 120, 140, 142 viscosity 43, 45, 60-1, 192 water binding 45-6 Alginate conversion 206, 210-14,224 Aginate lyase 43, 173, 223-4 Alkaline phosphatase, of P. aeruginosa 165,173 Alkaline proteinase, of P. aeruginosa 9, 160,171-2 Aminoglycoside antibiotics 23, 63--4, 153--4,223 Amoebae, phagocytic 81 Animal model infections 89-92, 98, 123,222-3 Antibiotic resistance, of P. aeruginosa 114,223 Antioiotic selection, of mucoid phenotype 58-9 Antibiotic sensitivity, of P. aeruginosa 64-5,85,114,152--4 Antibiotic therapy, in CF 22--4, 223--4 Antibody anti-alginate 65, 96, 100-1, 120--4, 127 anti-polysaccharide 114-17 anti-pseudomonaI52, 88, 90 monoclonal 96, 100-1 opsonizing 89, 113 precipating 109-11, 120 Aspergillus fumigatus I 7-18 Azlocillin 23--4 Azotobacter vinelandii, alginate biosynthesis 37,181-3,193, 199-200,207-8,210 B-Iymphocyte 115, 123--4 Bacteriophage in biofilms 82 of P. aeruginosa 4-6 typing 58

230

Index

Biocides 80, 83-5 Biofilms in acquatic ecosystms 77-82 Pseudomonas in vitro 82-5 see also Microcolony growth Biosynthesis, of alginate genetics 66-8, 206-20 ill I,ivo 65-9 pathway 181-205 regulation 59,66-9 Block structure, of alginate 38-41, 117,192,200 Bronchial epithelium 62, 97-8 Bronchoscopy 86-7 Buccal cells, adhesion of P. aeruginosa 55,96-7 Burns, Pseudomonas infection 76, 87-8, 224 Calcium effect on antibiotics 153-4 gelation of alginate 40-1, 44-6, 59-61,101 in adherence 101 regulation of epimerase 199-200, 210 sputum concentration 44 Carbenicillin 24, 58, 64,154,206 Carbohydrate metabolism, of P. aeruginosa 6, 183-90 Ceftazidime 16,23-4,63 Chemiluminescence 149-51 Chromosome map, of P. aeruginosa 8, 208 Cilia 98-100 Ciprofloxacin 23,63,223 Clinical management 20-4 Clinical monitoring 19-20 Colonization, by P. aeruginosa clinical characteristics 15-17 incidence 51-2 mucoid infections 53-6, 62-4, 96-107,110-14,121-5,173-5 non-mucoid infections 53-6, 96-100, 103, 110-14, 124 of respiratory tract 15-17,53-6, 62-4,96-107 Complement 137 Conjugation, of P. aeruginosa 7 C-reactive protein 63 Crossed electrophoresis 109

Cystic fibrosis clinical management 20-4 clinical monitoring 19-20 discovery of gene 13,222 genetics 14 incidence 14,221 prenatal diagnosis 14-15, 222 symptoms 13 Detachment hypothesis 163 Egg-box model, of alginate 40 Elastase P. aeruginosa 57, 112, 160-2, 171, 216 neutrophil 57, 147 Electrophoresis, crossed I ｏｾｴ＠ Entner-Doudoroff pathway 6, 184-90, 192 Enzyme-linked immunosorbent assay (ELISA) 117, 119, 122 Epimerase 181-3, 194-5, 198--202, 207,209-10,224 Epimerization 38 Epithelium tracheal 62, 96-105 bronchial 62 buccal 96-7 Excision marker rescue, of alg genes 212-13 Exoenzyme S, of P. aeruginosa 9,160-1 Exoenzymes, of P. aeruginosa 9, 160-80 Exopolysaccharide, bacterial chemical structures 30-3 immunology 114-25 in biofilms 77-86 in phagocytosis 139-45 shape 33-6 ExotoxinA9,63, 112, 160-1, 167, 172-3 Fibronectin 55, 96 Flagella 1,52,111-12,125-6 Fouling, bacterial 82-3 Free radicals formation 147-9 scavenging by alginate 149-52 Fructose diphosphatase 188 Fructose, incorporation in alginate 182-92

Index FIlClIS

gfmlilni I RI

231

Haemagglutination 117-18,122 HflPl/wjJhillis illfillPllZtlp 13,56, 111,221

Cangliotetraosylceralllide 102, 104 Gangliotriaosylceralllide 103-4 GDP-lIlannose dehydrogenase fi6, IR2-3,191-202,207-9 GDP-lIlannose pyrophosphorylase 182-3,194-7,207-R Genes, of Esc/zl'I'ic/zio ('uli IIIfll/A 193, EJ5 olfljJC 198 ompF 198

Genes, of P. afl"llgillu.lfl algA 193-7, 20R-9 ([1gB 208-9, 214-15 algD 67, 195, 197-8, 20R-9, 214, 221 algG 195,210 algQ66,214 algR 66, 208-9, 214-15 algS 59, 66, 6R, 20R-9, 211-17

algT 59,66,68,208-9,211-17 flIg 20R-, 22,1 .Ife also Biofilms MOllocytes 135

232

Index

Mucin 56, 62, 97, 101-5 Mucoid P. aeruginosa adhesion 95-107 alginate biosynthesis 181-205 animal model of infection 89-92, 98,123,222-4 antibiotic susceptibility 64-5, 152-4 colony morphology 3, 30, 50-1, 59-61,64 exoenzymes 160-80 genetics 206-20 immunology 109-34 in biofilms 77-87, 223 incidence 51-2 infections 13-28,50-75,87-89,221 phagocytosis of 81,91, 113, 124, 138-43 precipitating antibodies 110-11 stability of phenotype 56-7, 58-60, 68-9,87 Myeloperoxidase 148 Neutrophils 135, 138-9, 142, 148 Nuclear magnetic resonance spectroscopy, of alginate 'H45,192 ':lC 190-2 non-mucoid P. aeruginosa adhesion 95-107 colony morphology 3,50-1,61 immunology 111-14, 125-34 infections 53--6, 206 phagocytosis of 140-5 revertants 53-4,56-7,69,99, 171-7,193-4,206,210-14 O-antigen 30, 53, 90 Opsonization 89, 113, 124, 135-8, 149 Osmolarity, and alginate synthesis 66-8 Outer membrane 4, 30, 85, 90, 126, 160,175-7,198 Oxidative (respiratory) burst 136, 147-9 PAO strains, of P. aeruginosa 7-8, 59-60, 193-4 Peptidoglycan 4, 29, 160 Phagocytosis inhibition by starch 145

of P. aeruginosa 81, 91,113,124, 135-47 of latex particles 143-5 of zymosan 143-54 Phagosome 136-7 Phosphofructokinase 188 Phospholipase C, of P. aeruginosa 63, 160, 165 Phosphomannomutase 182-3, 194-7, 201,207-8 Phospho man nose isomerase 182-3, 193-6,207-9 Physiotherapy, in CF treatment 21-2 Pigments, fluorescent 3-4, 51 Pili 4, 85, 96, 97,102,105,126 Planktonic bacteria 79-82, 85, 87-8 Plasmids, of Pseudomonas 4-5, 211-13 Polyacrylamide gel electrophoresis 118, 176-7 Polyagglutinating antigen 54, 125 Polymerase 182-3, 194-5, 198-201, 207,209,224 Polymorphonuclear leucocytes 57, 135, 138-9 Polysaccharide, see Exopolysaccharide Precipitating antibody 109-11, 120 Precipitins, see Precipitating antibody Prenatal diagnosis, ofCF 14-15 Proteinases of P. aeruginosa 9, 57, 62-3,124, 160,171-2,215-17 neutrophil 55, 57 lysosomal 146-7 salivary 55 Pseudolysogeny 58 Pseudomonas aeruginosa adherence 95- 107, 136 alginate structure 36-41 alginate properties 41-6 antibiotic susceptibility 64-5, 114, 152-4 carbohydrate metabolism 6,183-90 chromosome map 8, 208 colonization in CF 15-17,52,62-4, 124 conjugation 7 digestion of 136-7,140-7 environmental niches 52, 76-82 flagella 1,52,111-12,125-6 general characteristics 1-2

Index hypersusceptible strains 64-5 immunology 109-34 in biofilms in ,!ilro 82-7 infection models 89-92, 98, 123, 222 -3 killing 136 lipopolysaccharide 4, 29--30, 90, 122,125,162,166-8,171, In, 175 metabolism 6 microcolony growth ,16, 62, 65, 76-94,911-100,173-5 morphology 2-3 motility 52 mucoid infections 13-28,50-75, 87-9 oligonucleotide cataloguing I PAO strains 7-8, 59-60, 193-4 phagocytosis 81,91, 113, 135-47 pigment production 3-4, 51 pulmonary infection 15-17 pyocin tv pes 5, 53, FA, 56, 206 reversion 56-7 rRKA of I serotype 53, 168 serum sensitivity 54,111-12 taxonomy 1-2 transduction 7-8 ITP genes 8-9 Pseudomonas cepilcia 103 Pseudomonas ftuorescens, alginate synthesis 36, 59,181 Pseudomunas mflulocina, alginate synthesis 36, .'i9, 181, 1811, 190, 192 Pseudomunas 1m/ida, alginate synthesis 36,59,181

Radioimmunoassay 117, 120 ResistatKe, to antibiotics 4 Restriction map, of (jIg genes 209, 211-12 Reversion, of P. al'mginosa 56-7 Robbins device 83, 85

PSPlldomoll([s PYOCYIlIINl 2, 4 PSl'wlofflona.l syringae, alginate

Uronic acids metabolism 186 structure 32, 37-9

synthesis

36,59,181 Pulmonary infection, in CF 15-18, 61-4 Pyocin types 5, 53-4, 56, 206 Pyocyanin 4 Pyoverdin 57 Pyruvate dehydrogenase 186

233

Serotypes, of P. ([I'ruginosa 53, 168 Serum sentitivity, of P. anuginosa 53-4,111-12,125 Sessile bacteria 81-2,85 Sialic acids 96-7, 101-5 Sputum antibiotics in 153-4 carriage of P. Ilemginosa 55-6, 811-9 production in CF 23 proteinases 55,58 ｓｬ｡ｐｨｾｯｻＢＨＩｕｉ＠ 11 U 1'1' 11.1 13, 15,23, 114, 138,221 Stool, carriage of P. aerugmO.la 55-6 Streptomycin 154 Superoxide 147-9 Surfactant deoxvcholate 56 lung 63 T-Iymphocyte 115, 124 Tobramycin 23, 511, 63-4, 100, 153-4 Tracheal epithelium, 62, 96-105,124 Transduction, of alg genes 211-13 Transduction, of P. aNuginosa 7-11 Transferrin 57 Transposon 211-14 Tricarboxylic acid (TCA) cvcle 6, 192 . I ＱＵＭｾｩＬ＠ Trypsin, treatment of cells 96

Virulence factors 9, 62-3, 87, 137, 215-17 Virus, infection in CF 18 Viscosity, of alginate 43,45,60-1, 192