Biological Properties of Peptidoglycan: Proceedings Second International Workshop, Munich, Federal Republic of Germany, May 20–21, 1985 9783110874297, 9783110107371

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Biological Properties of Peptidoglycan

Biological Properties of Peptidoglycan Proceedings Second International Workshop Munich, Federal Republic of Germany May 20-21,1985

Editors PH. Seidl • K H . Schleifer

W DE G Walter de Gruyter • Berlin • New York 1986

Editors

Peter H. Seidl, Dr. rer. nat. Karl Heinz Schleifer, Prof. Dr. rer. nat. Lehrstuhl für Mikrobiologie Technische Universität München Arcisstraße 21 D-8000 München 2 Federal Republic of Germany

Biological properties of peptidoglycan. „Second International Workshop on the Biological Properties of Peptidoglycan"--Pref. Includes bibliographies and indexes. I. Peptidoglycans-Congresses. I. Seidl, R H. (Peter H.), 1948 II. Schleifer, Karl H. III. International Workshop on the Biological Properties of Peptidoglycan (2nd : 1985 : Munich, Germany) [DNLM: 1. Peptidoglycan-congresses. QW 52 B6138 1985] QP702.P47B56 1986 616.07'9 86-19791 ISBN 0-89925-262-1 (U.S.)

CIP-Kurztitelaufnahme

der Deutschen

Bibliothek

Biological properties of peptidoglycan : proceedings, 2. internat, workshop, Munich, Fed. Republic of Germany, May 20-21,1985 / [2. Internat. Workshop on the Biolog. Properties of Peptidoglycan]. Ed. P H. Seidl ; K. H. Schleifer. Berlin ; New York : de Gruyter, 1986. ISBN 3-11-010737-6 NE: Seidl, Peter H. [Hrsg.]; International Workshop on the Biological Properties of Peptidoglycan

Copyright © 1986 by Walter de Gruyter & Co., Berlin 30. All rights reserved, including those of translation into foreign languages. No part of this book may be reproduced in any form - by photoprint, microfilm or any other means nor transmitted nor translated into a machine language without written permission from the publisher. Printing: Gerike G m b H , Berlin. - Binding: D. Mikolai, Berlin. - Printed in Germany.

PREFACE This book is based on the lectures presented at the "Second International Workshop on the Biological Properties of Peptidoglycan" in Munich from May 20 to 21, 1985. The aim of this workshop was to summarize the current state of knowledge and to demonstrate the manifold progress made since the first workshop held on this topic in 1974. Included in this book are contributions from various scientific disciplines, covering such fields as immunology, pathology, microbiology, medicine, preparative organic chemistry, ultrastructure and tumor research. Some of the data presented at the workshop will indeed result in diagnostic as well as therapeutic application. Determining antibodies to peptidoglycan may be useful in the diagnosis of bacterial infections and 'MDP drugs' could be helpful as adjuvants for veterinary and human vaccines and as anticancer, antitumor and antiparasitic agents. Besides the applied aspect, numerous papers enable us to understand the biology of peptidoglycan in more detail. Such presentations as the description of soluble peptidoglycan as the natural immunogen may lead to a better understanding of the mechanism of immunogenicity of peptidoglycan. The elegant elucidation of the induction of slow wave sleep by muramyl peptides was one of the highlights of this workshop. We would like to express our gratitude to the Deutsche Forschungsgemeinschaft for its financial support and to the Carl Friedrich von Siemens Stiftung for its hospitality. Furthermore, the success of our workshop is mainly due to the excellent presentations of the participants and we thank all authors for their contributions. To the publisher we convey our thanks for good co-operation in publishing these proceedings so rapidly. Finally we are grateful to all our colleagues, in particular to B. Heymer, for their help in organizing the workshop and/or in the realization of the proceedings. Munich, June 1986

P.H. Seidl K.H. Schleifer

SPONSORS

Bayer AG, 5090 Leverkusen, FRG Behringwerke AG, 3550 Marburg, FRG Biotest Pharma GmbH, 6000 Frankfurt, FRG Boehringer Ingelheim Diagnostica, 8046 Garching b. München, FRG Boehringer Mannheim GmbH, 6800 Mannheim, FRG Carl Friedrich von Siemens Stiftung, 8000 München, FRG Ciba-Geigy GmbH, 7867 Wehr/Baden, FRG Deutsche Forschungsgemeinschaft, 5300 Bonn, FRG Deutsche Gesellschaft für Hygiene und Mikrobiologie e.V., FRG Dr. Karl Thomae GmbH, 7 950 Biberach an der Riss, FRG Heinrich Mack Nachf., 7918 Illertissen, FRG Luitpold-Werk München, 8000 München, FRG

CONTENTS

I. STRUCTURAL ASPECTS, IMMUNOCHEMISTRY OF PEPTIDOGLYCAN AND ANTIBODIES TO PEPTIDOGLYCAN Structure and Immunochemistry of Peptidoglycan P.H. Seidl, K.H. Schleifer The Diagnostic Value of Antibodies to Peptidoglycan and other Staphylococcus aureus Antigens in Serious Staphylococcal Infections B. Christensson

21

On the Relationships between Conformational and Biological Properties of Murein H. Labischinski, L. Johannsen

37

A Three-Dimensional Model of Peptidoglycan Corresponding with the Results of X-Ray and Electron Diffraction, High Resolution Electron Microscopy and the Properties of Peptidoglycan H. Formanek

43

Structure Determination of Peptidoglycans by Fast Atom Bombardment Mass Spectrometry S.A. Martin, K. Biemann, R.S. Rosenthal

49

The Peptidoglycan and Linked Polymers of the Unicellular Cyanobacterium Synechocystis PCC 6714 U.J. Jürgens, J. Weckesser

55

Chemical and Immunochemical Studies on the in vivo and in vitro Degradation of Peptidoglycan Subunits and Cell Wall Complexes in Relation to Inflammatory Diseases A. Fox, J. Gilbart, J. Harrison, G. Pararajasegaram, A. Wells, M. Hammer, C.H. Yang, Y. Ishikawa, R. Whiton, S.L. Morgan

61

Specific Immunoglobulin E Antibodies to Peptidoglycan? J. Seidl, P.H. Seidl, K.H. Schleifer

67

Specific Immunoglobulin G Antibodies in Man against the Glycan Strand of Peptidoglycan E. Zauner, S. Reissenweber, G. Leitherer, K.H. Schleifer P.H. Seidl

75

X Release of Penicillin-Binding Proteins from ß-Lactam Treated Bacteria: Determination by Anti-ß-Lactam Antibodies R. Hakenbeck, H. Ellerbrok, T. Briese, N.F. Adkinson

83

Anti-Peptidoglycan Serology in Patient Sera and Experimental Production of Anti-Peptidoglycan Antibody by Immunisation with Rheumatoid Factor H.B. Evans, K.K. Phua, P.M. Johnson

89

Antibodies against a Synthetic Peptidoglycan-Precursor Pentapeptide Containing Lysine Cross-React with Soluble Peptidoglycan Containing Diaminopimelic Acid A.R. Zeiger

95

Antibodies to Various Bacterial Cell Wall Peptidoglycans in Patient Sera and in Rabbit Sera H.I. Wergeland, C. Endresen

99

Antibodies to the Pentapeptide Subunit of Peptidoglycan in Human Sera T. Kuchenbauer, K.-D. Tympner, P.H. Seidl, K.H. Schleifer

105

Problems with 'Teichoic Acid" Antigen Used in Staphylococcus aureus Serology Ch. Herzog, V. Just, R. Berger, M. Just

113

Lipid A Antibodies in Man W. Marget, P. Mar, H. Haslberger, M. Huber

121

Electron Microscopic Localization of Peptidoglycan in the Cell Wall of Streptococcus pyogenes by Means of Labelled Antibodies and Lysozyme M. Wagner, M. Rye, B. Wagner

129

Immunoelectron' Microscopic Studies on Peptidoglycan from Gram Positive Bacteria: Specific Reactions with the Glycan Moiety, the Pentapeptide Subunit and the Interpeptide Bridge N. Franken, J.R. Golecki, P.H. Seidl, P. Zwerenz K.H. Schleifer

135

XI

II. DEGRADATION OF PEPTIDOGLYCAN, PEPTIDOGLYCANLIKE PRODUCTS IN THE HOST Soluble Peptidoglycans: Lymphocyte-Activating Bacterial Products Found in Man A.R. Zeiger

145

How Are Cell-Wall Components of Pathogenic Microorganisms Degraded in Infectious and Inflammatory Sites?: Facts and Myths I. Ginsburg

167

State of Peptidoglycan in Spheroplasts of Proteus mirabilis grown in the Presence of Different BLactam-Antibiotics K. Huber, H.H. Martin

187

Degradability by Lysozyme of Staphylococcal Cell Walls as a Function of O-Acetyl Groups H. Labischinski, P. Giesbrecht

191

O-Acetylation of Staphylococcal Cell Walls as an Important Factor for their Degradability Within Bone Marrow-Derived Macrophages J. Wecke, E. Kwa, L. Johannsen, P. Giesbrecht, M. Lahav, I. Ginsburg

197

Metabolic Fate of Peptidoglycan Monomer from Brevibacterium divaricatum and Biological Activity of its Metabolites J. Tomasic, Z. Valinger, I. Hrsak, B. Ladesife

203

Determination of the Size Distribution of the Glycan Strands Released from Murein of E. coli by Human Serum Amidase H. Ludewitz, J.-V. Holtje

209

Endogenous Induction of Bacterial Lysis by Cloned PhiX17 4 Gene E Product U. Blasi, R.E. Harkness, A. Witte, G. Halfmann, W. Lubitz

215

Radioimmunoassays for the Selective Detection of the Glycan Moiety and the Pentapeptide Subunit in Secreted Peptidoglycans P. Zwerenz, F. Hagen, G. Leitherer, P.H. Seidl, K.H. Schleifer

221

XII

III. ACTION OF PEPTIDOGLYCAN ON CELLS Effects of Peptidoglycan on the Cellular Components of the Immune System R. Dziarski Suboptimal Concentrations of LPS are Necessary for in vitro Activation of Rat Alveolar Macrophages by Muramylpeptides J.-P. Tenu, J.-F. Petit, D. Nolibe Antiviral and Antitumor Effects of Liposome-Entrapped MTP-PE, a Lipophilic Muramylpeptide G. Schumann Effects of Staphylococcal Peptidoglycan on Phagocytic Cells and Host Mediation Systems A. Fleer, F.C.A. Jaspers, J. Verhoef Cellular Response to Streptococcal Peptidoglycan and Tetanus Toxoid in Glomerulonephritis-Patients: Correlation to Complement Genotype H.E. Feucht, G.J. O'Neill, G. Riethmüller, A. Brase, P.H. Seidl IV. DIVERSE BIOLOGICAL ACTIVITIES OF PEPTIDOGLYCAN Acute and Chronic Inflammation Induced by Peptidoglycan Structures and Polysaccharide Complexes S.A. Stimpson, J.H. Schwab, M.J. Janusz, S.K. Anderle R.R-. Brown, W.J. Cromartie Biological Activity of Peptidoglycan in Man B. Heymer, J. v. Mayenburg Pathomechanisms of Peptidoglycan in Man J. v. Mayenburg, B. Heymer Role of Peptidoglycan in Gonococcal Arthritis R.S. Rosenthal, F.R. Montoya, W. Nogami, T.J. Fleming Teichoic Acid-Free Peptidoglycan of Staphylococcus aureus RM 5 9 Does Not Activate Complement M. Loos, P.H. Seidl, K.H. Schleifer

XIII

Somnogenic Muramyl Peptides J.M. Krueger

329

Synthetic Lipopeptide Analogues of PeptidoglycanAssociated Lipoprotein Are Potent Novel B-Lymphocyte Mitogens W.O. Bessler, B. Kleine, M. Cox, A. Lex, B. Suhr, K.-H. Wiesmüller, G. Jung, C. Martinez-Alonso

335

GUEST LECTURE Lipid A, the Endotoxic Principle of Bacterial Lipopolysaccharides: Chemical Structure and Biological Activity E.Th. Rietschel, H. Brade, L. Brade, K. Kawahara, Th. Liideritz, U. Schade, A. Tacken, U. Zahringer

341

V. ADJUVANT ACTIVITY AND IMMUNOMODULATION BY PEPTIDOGLYCAN Macrophage Activation by Muramyl Peptides M. Parant, L. Chedid ~

353

Immunomodulation by Muramyldipeptide (MDP) H. Finger, C.H. Wirsing von Koenig

371

Disaccharide MDP Analogs, Preparation and Some Biological Properties M. Zaoral, J. Farkas, M. Ledvina, J. Jezek, J. Rotta, M. Rye

379

Biological Activity of Synthetic Disaccharide-Peptide Analogues of Peptidoglycan M. Rye, J. Rotta,^R. Straka, M. Zaoral, J. Farkas, M. Ledvina, J. Jezek, J. Pekarek

383

Enzymatic Obtention of Biologically Active Glycopeptides from Actinomadura R 39 M. Guinand, P. Morel, M.J. Vacheron, G. Michel, P. Dupassieu, D. Yavordios

389

Adjuvant Active Peptidoglycans Induce the Secretion of a Cytotoxic Factor by Macrophages F. Vacheron, M. Guenounou, C. Nauciel

395

Stimulation of Nonspecific Resistance Against Aerogenic Viral and Bacterial Infections by Muramyl Dipeptide Combined with Trehalose Dimycolate K.H. Masihi, W. Brehmer, W. Lange

401

XIV Increased Adjuvant Activity of MDP by Direct Coupling of MDP to the Immunogen M. Jolivet, F. Audibert, L. Chedid, E.R. Clough

407

Contributors and Participants

413

Author Index

421

Subject Index

423

STRUCTURE AND IMMUNOCHEMISTRY OF PEPTIDOGLYCAN

Peter H. Seidl, Karl Heinz Schleifer Lehrstuhl für Mikrobiologie, Technische Universität München, D-8000 München 2, FRG

I. Structure of Peptidoglycan Peptidoglycan (murein, mucopeptide), the main cell wall polymer of eubacteria, is common to both Gram-positive and Gram-negative bacteria. Only a few prokaryotic organisms such as the Mycoplasmatales and archaebacteria lack peptidoglycan. Chemically, it is a heteropolymer consisting of glycan strands cross-linked through short peptides (Fig. 1). Investigations of the chemical structure of peptidoglycans from numerous bacterial strains demonstrated the

GlcNAc -p-1,4—Mur NAc-p -1,4 Fig. 1. Scheme of the primary structure of peptidoglycan. Unusual abbreviations: MurNAc, N-acetylmuramic acid; DA, diamino acid. Substituents in brackets may be missing.

Biological Properties of Peptidoglycan © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

2 existence of almost 100 different peptidoglycan types (1,2). The glycan moiety of the various peptidoglycans is rather uniform and reveals only a few variations such as acetylation or phosphorylation of the muramyl-6-hydroxyl groups, the occasional absence of N-acetyl substitution or lacking of part of the peptide substituents. In some mycobacteria and nocardiae, the N-acetyl group of muramic acid is oxidized to give an N-glycolyl-residue. The number of chemically different amino acids occurring in a particular peptidoglycan is restricted to three to six. Peptidoglycan does not contain branched amino acids, aromatic amino acids, cysteine, methionine, arginine, histidine and proline. The stem peptide or peptide subunit linked to the carboxyl group of muramic acid always consists of alternating L- and D-amino acids. Depending on the mode of crosslinking, two main groups of peptidoglycan named A and B, have been distinguished (1). The crosslinkage of Group A peptidoglycans extends from the distal amino group of the diamino acid in position 3 of one stem peptide to the carboxyl group of D-alanine in position 4' of an adjacent stem peptide either directly or via an interpeptide bridge (Fig. 1). Peptidoglycans of Group B are characterized by a crosslinkage between the «.-carboxyl group of D-glutamic acid in position 2 in one stem peptide and the C-terminal D-alanine in position 4' of an adjacent stem peptide. There is always a diamino acid necessary in the interpeptide bridge since two carboxyl groups must be crosslinked. Group B peptidoglycans are rather rare and are only found in some coryneform bacteria and in a few anaerobic organisms (2). The peptidoglycan of Gram-negative bacteria is rather uniform. It usually forms a monomolecular layer and is directly crosslinked. In most cases meso-diaminopimelic acid is present as the diamino acid in position 3 of the stem peptide, but a few exceptions are known. The peptidoglycan of spirochaetes often contains ornithine instead of diaminopimelic acid (3,4) among some fusobacteria, mesodiaminopimelic acid is replaced by its sulphur analogue meso-lanthionine (5). Gram-positive bacteria contain a multilayered peptidoglycan and reveal a great variation in the composition and primary structure of the constituent peptide moiety. Various peptidoglycan types can be distinguished, depending on the different amino acid sequence of the stem peptide and/or the interpeptide bridge (for review, see 1,2).

3 The amino acid sequence of the stem peptide shows some variation among the different peptidoglycans (Fig. 2). The amino acids in parantheses may replace the corresponding amino acids or substituents. The greatest variation is found in position 3. To date, 11 different amino acids have been detected in this position, mesodiaminopimelic acid and lysine are the most common ones (for review, see 1,2). Position 4 is occupied by D-alanine. The carboxyl group of this D-alanine is often blocked by crosslinkage to an adjacent stem peptide. In non-crosslinked stem peptides the D-alanine in position 4 may be split off or substituted by another D-alanine; the latter D-alanine represents a remnant of the peptidoglycan precursor. Regarding the biological properties of peptidoglycan, one should keep in mind that the structure of peptidoglycan is not as regularly as depicted in the scheme of Fig. 1. Several enzymes of the bacterial cell may split bonds in the peptidoglycan (6). Glucosaminidases and muraminidases split glycosidic linkages in the glycan strand; amidases may split the linkage between muramic acid and L-alanine in position 1 of the peptide subunit, thus leading to muramic acid residues with a free carboxyl group; endopeptidases may split linkages in the stem peptide or in the interpeptide

IJUR 1 2 3

L-ALA (3-HYG)

(GLY,L-SER)

D - G L U — * N H M-A2PM |

A

D-ALA

5

D-ALA

2

(GLY,GLYNH2,D-ALANH2,

(L-LYS,L-0RN,LL-A2PM,

CADAVERINE)

M-HYA2PM

L-DAB,L-HYL,L-HSR,L-ALA,L-GLU,L-LAN)

Fig. 2. Variations of the stem peptide (peptide subunit). Amino acids in parentheses may replace the corresponding amino acids or substituents. Unusual abbreviations: A2pm, 2,6-diaminopimelic acid; Dab, diaminobutyric acid; HyA2pm, 2,6-diamino-3-hydroxypimelic acid; Hyg, threo-B-hydroxyglutamic acid; L-Hyl, L-hydroxylysine; Hsr, homoserine; Lan, lanthionine.

4 bridge, causing additional free amino groups and carboxyl groups; D,D-carboxypeptidases may split off the D-alanine in position 5 of the peptide subunit and L,D-carboxypeptidases may split off the D-alanine in position 4. Strains lacking carboxypeptidases, such as many staphylococci and streptococci, reveal up to 20 per cent of the stem peptides as non-crosslinked pentapeptide subunits with a C-terminal D-alanyl-D-alanine.

II. Immunochemistry of Peptidoglycan Cell wall peptidoglycan is immunogenic. Up to date, at least five main antigenic epitopes (Fig. 3) of peptidoglycan have been discovered (7) that is a) the glycan strand, b) and c) N-terminal and

Fig. 3. Schematic representation of the five antigenic epitopes of peptidoglycan: a) the glycan strand; b) N-terminal and c) Cterminal sequences of the interpeptide bridge; d) the noncrosslinked peptide subunit tetrapeptide; e) the non-crosslinked peptide subunit pentapeptide.JT: endopeptidases splitting glycyl-glycine bindings.

5

C-terminal sequences of the interpeptide bridge, d) the non-crosslinked tetrapeptide subunit, and e) the non-crosslinked pentapeptide subunit. In man, specific antibodies to the glycan moiety, to the tetrapeptide subunit and to the pentapeptide subunit have been hitherto detected. 1. Antigenic properties of the glycan moiety The findings on the immunological properties of the glycan strand are summarized in Table 1. Early studies by Karakawa and Krause (8) on the antigenic properties of peptidoglycan indicated that the hexosamine polymer of peptidoglycan was a main antigenic epitope. Rolicka and Park (9) demonstrated that antibodies against the glycan strand recognized acetylglucosamine and not acetylmuramic acid, whereas Wikler (10), investigating antisera to Micrococcus luteus, claimed that acetylmuramic acid and not acetylglucosamine was the immunodominant sugar. Schleifer and Seidl (11) were finally able to resolve this controversy. A glycan strand specific antibody population directed to acetylglucosamine and a po-

ANTIGENIC PROPERTIES OF THE GLYCAN STRAND

GLCNAC

IMMUNODOMINANT;

CROSS-REACTIVITY

BE-

KARAKAWA ET AL.

1967

SPECIFIC ANTIBODIES TO MDP:

TWEEN A-POLYSACCHARIDE AND

REICHERT ET AL.

1980

A) MDP CONJUGATED TO

PEPTIDOGLYCAN

CARRIERS VIA GLUTAMIC GLCNAC AND NOT MURNAC

ROLICKA AND PARK.

IMMUNODOMINANT

1969

MURNAC AND NOT GLCNAC

WIKLER

IMMUNODOMINANT

1975

ANTIBODY

POPULATIONS

TO GLCNAC AND MURNAC

ACID B) MDP CONJUGATED TO CARRIERS VIA ANOMERIC C-L CARBON ATOM OF MURNAC

SCHLEIFER AND SEIDL,

1977

AS DETERMINANT SUGARS

SPECIFIC ANTIBODIES TO

SEIDL ET AL.,

THE GLYCAN MOIETY IN

ZAUNER ET AL.

MDP DOES NOT BIND TO

AUDIBERT ET AL.

HUMAN SERA; ANTIBODY

ANTIBODIES AGAINST

1978

COMBINING SITE AT LEAST

PEPTIDOGLYCAN

(GLCNAC-MURNAC), 2

Table 1. Antigenic properties of the glycan strand

.1985

6 pulation directed to acetylmuramic acid were found in the same antiserum . The occurrence of antibodies to the glycan strand led to the question whether the synthetic immunoadjuvant MDP would bind to such antibodies or not. An answer to this question might indicate whether reactivity of MDP with natural occurring peptidoglycan antibodies could induce clinical hazards, preventing the application of MDP or its derivatives in man. No binding of radiolabelled MDP to naturally occurring or experimentally induced antibodies was detected (12). This lack of reactivity was surprising since MDP represents part of the monomeric subunit of the peptidoglycan backbone. Although MDP in isolated form was found not to elicit an immune response, it became immunogenic when it war coupled to an appropriate carrier. Conjugates, carrying MDP substituents via the T-carboxyl group of glutamic acid of MDP, or via the anomeric C-1 carbon atom of the N-acetylmuramyl portion of MDP, were prepared (13). Both conjugates were immunogenic and the antibodies reacted with free synthetic MDP. Recently, we developed a Farr-type radioactive hapten binding assay for detecting glycan moiety specific antibodies. As an antigen, we employed low-molecular weight glycopeptides from Bacillus subtilis peptidoglycan. The structural features of the glycopeptides are presented in detail by Zauner et al., this book. It is important that the glycopeptides isolated did not reveal any pentapeptide subunits with C-terminal D-alanyl-D-alanine, and did not contain non-crosslinked tetrapeptide subunits. Thus, it was excluded that antibodies to the tetra- or pentapeptide subunit were detected employing the glycopeptide as an antigen. The glycopeptides were radiolabelled with iodine and binding of the radioactive glycopeptide hapten to animal hyperimmune sera, to human normal sera and to sera from patients was measured. High binding (70-80%) of radioactively labelled glycopeptides was obtained with rabbit reference hyperimmune sera (11) containing high levels of antibodies to the glycan moiety. No binding was achieved, however, to specific antibodies against the peptide moiety of peptidoglycan (14-18), and to antisera against the A-carbohydrate of streptococci, known to be specifically directed against N-acetylglucosamine (19). Specificity of the test system was further examined in appropriate hapten binding inhibition studies (Fig. 4). Besides the

7

homologous glycopeptide, soluble peptidoglycans from various bacterial strains revealing variations in the glycan moiety as O-acetylation (staphylococci) , partial lack of N-acetvlation (bacilli) or peptide substitution (Micrococcus luteus), gave a strong inhibitory effect. The size of the antibody combining site was at least

Fig. 4. Binding inhibition reaction of radio-iodinated glycoDeptides to peptidoglycan antiserum (A-variant streptococcal antiserum. (•) Bacillus subtilis W23 glycopeptide; (o) acetvlchitotetraose; (e) glycan oligosaccharide (GlcNAc-MurNAc)2. Soluble peptidoglycans (MW >10 000) from (•) Bacillus subtilis, (Q} Bacillus licheniformis, Micrococcus luteus, Staphylococcus epidermidis. The following compounds gave no significant inhibition (umin with peptidoglvcans from (•) Micrococcus roseus ATCC 418, (o) M. varians C C M 8 8 3 , and (x) S. aureus H or S. epidermidis ATCC 14990.

10

ration of staphylococci from micrococci (21). Carboxyl terminal oligoglycine sequences of the interpeptide bridges from staphylococci further reveal an independent antigenic epitope of peptidoglycan as was proved using appropriate synthetic protein-peptide conjugates (Fig. 5d). Oligoglycine peptides revealing free carboxyl groups are the product of endopeptidases splitting glycylglycine linkages (22). 3. Antigenic properties of the pentapeptide subunit a) Characterization of animal hyperimmune sera The stem peptide was presumed to be an antigenic epitope of peptidoglycan already in 1968 by Karakawa and Krause (23). Employing a series of synthetic peptides as inhibitors in the quantitative peptidoglycan precipitin reaction, Schleifer and Krause (1971) demonstrated an antibody population with specificity for the pentapeptide subunit in A-variant streptococcal antisera (24). The conclusions drawn from their results are summarized in Table 2. The pentapeptide subunit being an antigenic epitope was consequently corroborated by several approaches using synthetic antigens. Zeiger and Maurer (1973) coupled synthetic pentapeptide subunits to a random alanine/glutamic acid copolymer (25). Schleifer and Seidl 1.

THE

PENTAPEPTIDE

TETRAPEPTIDE TIDE 2.

THE A N T I B O D I E S

THE NANT THE

AND NOT T H E

PREDOMINANTLY

THE A N T I G E N I C

DETERMINANT

OCCURRING OF T H E

PEP-

SUBUNIT

PORTION 3.

IS

OF T H E

C-TERMINAL SITE

ARE

GLUTAMIC

SITE

IS

ACID)

AGAINST

D-ALANYL-D-ALANINE

OF T H E

THE

C-TERMINAL

IS

THE

IMMUNODOMI-

ANTIGEN

CONTRIBUTION

TIGENIC

DIRECTED

PEPTIDE

OF T H E OF

OR

OTHER

LESSER IS

AMINO A C I D S

IMPORTANCE

INSIGNIFICANT

TO T H E

(LYSINE

AN-

AND

(L-ALANINE)

Table 2. Characterization of the pentapeptide subunit as an antigenic epitope of peptidoglycan (24)

11

(1974) synthesized the protein peptide conjugate albumin-(CH2COGly-L-Ala 2 -D-Ala 2 -OH) 39 , carrying peptide substituents with C-terminal D-alanyl-D-alanine; the amino acids L-alanine, D-glutamic acid and L-lysine from the stem peptide were replaced (14). Employing the conjugates synthesized by Zeiger and Maurer (1973) and by Schleifer and Seidl (1974) as an immunogen specific antibodies directed against the peptide substituents were obtained. Monospecific antibodies to the C-terminal D-alanyl-D-alanine moiety of the pentapeptide subunit (14) allowed demonstrating the distribution of pentapeptide subunits in the cell walls of several Gram-positive bacteria (26,27, Franken et al., this book). b) Characterization of human sera Antibodies directed to the pentapeptide subunit of peptidoglycan are frequent in sera from normal blood donors or from patients (28,29). Recently, Franken et al. (1984, 1985) linked tri-D-alanine covalently to albumin via the amino group (18,30) and employed this protein peptide conjugate as an antigen in an ELISA. Specific IgG (18) and IgA class antibodies (30) against the immunodominant D-alanyl-D-alanine moiety of the pentapeptide subunit were detected in human sera. It was furthermore clear from appropriate inhibition studies in the ELISA (Figure 7) or from inhibition of radiolabeled H-L-Ala-D-Glu(L-Lys-D-Ala2-OH)-OH hapten binding (18, 31, Figures 8,9) that human sera also may contain antibodies with exclusive or predominant specificity for the carboxyl-terminal Dalanyl-D-alanine sequence of the pentapeptide subunit, similar as shown for streptococcal hyperimmune sera (24, compare Table 2). The results from the radioactive hapten binding inhibition studies with specific antibodies in human serum 004, depicted in Fig. 9, are of interest for several reasons: 1. In contrast to the strong inhibitory effect of Ac2-L-Lys-D-AlaD-Ala-OH or of tri-D-alanine, oligoglycine peptides or D-AlaGly-OH did not inhibit, thus excluding cross-reactivity between the C-terminal sequences of the interpeptide bridge (16) and the pentapeptide subunit. 2. Ac2~L-Lys-D-Ala-D-Ala-OH and Ac-j-L-Lys-D-Ala-D-lactic acid are both substrates for the penicillin-sensitive D-alanine carboxypeptidases of Bacillus subtilis, Escherichia coli and Staphylo-

12 Gly2-L-Ala-0-Ala2 L-Ala-O-duL-Lys-O-Alaj)

üSerum058

m L-Ala-O-GlulL-Lys-D-Ala) Gly2-L-Ala-D-Ala2 L-Ala-D-öu(U.ys^O-Ala2)

L-Alo-D-GlulL-Lys-D-Alo) ay2-L-Ala-D-Ala2 L-Ala-O-GML-Lys-D-Ala^

L-Ala-O-Glu(L-Lys-O-Ala) 1 10 100 1Ö00 Inhibitor Concentration (umoles/l)

J

o;33 3.3 33 Inhibitor Concentration (umoles/l)

Fig. 7. Inhibition of binding of specific IgG in human sera nos. 004, 058 and 262 to albumin-(D-Ala-D-Ala-D-Ala-OH)g in the ELISA technique, using as inhibitors several peptides: (o) HD-Ala-D-Ala-D-Ala-OH, (x) H-L-Ala-L-Ala-L-Ala-OH, (+) H-L-AlaL-Ala-D-Ala-OH (18). Fig. 8. Inhibition of radiolabelled H-L-Ala-D-Glu(L-Lys-D-Ala-DAla-OH)-OH hapten binding (human sera nos. 004, 058 and 262) by various synthetic inhibitors: (•) H-Gly-Gly-L-Ala-D-Ala-DAla-OH), O H-L-Ala-D-Glu(L-Lvs-D-Ala-D-Ala-OH)-OH, M H-LAla-D-Glu(L-Lys-D-Ala-OH)-OH (18,31) .

Ac2-Lys-D-Ala2 •°D-Ala3 •Penicillin G •Ampicillin

0,33

i-Glv- MDP 3.3 33 Inhibitor Concentration lumoles/l)

Fig. 9. Inhibition of radiolabelled H-L-Ala-D-Glu(LLys-D-Ala-D-Ala-OH)-OH hapten binding (human serum no. 004) by various inhibitors.

13

coccus aureus (32). The ester substrate was hydrolysed faster than the peptide analogue, diacetyl-L-Lys-D-Ala-D-Ala-OH, by the B. subtilis (15-fold) and E. coli (4-fold) carboxypeptidases, no rate acceleration was observed for the S. aureus carboxypeptidase (32). However, in contrast to the strong inhibition found with Ac2-L-Lys-D-Ala-D-Ala-OH, Ac2~L-Lys-D-Ala-Dlactic acid did not react as hapten in the radioimmuno-assay. 3. Due to proposed structural analogy between acyl-D-alanyl-D-alanine and penicillin (32) , we investigated a possible crossreactivity between E-lactam antibiotics and R-D-alanyl-D-alanine and came to the following conclusions: - All 22 penicillins and cephalosporins investigated (Penicillin G and Ampicillin depicted in Fig. 9) bound to antibodies with specificity against D-alanyl-D-alanine. - In general, cephalosporins bound less than penicillins. - An intact B-lactam ring was absolutely necessary for the interaction of the antibiotic with the antibody. A failing or very weak inhibitory effect of penicillin or tri-Dalanine on the binding of radioactive H-L-Ala-D-Glu (L-Lys-D-Ala,,OH)-OH to several human sera led us to studying in detail specificity of human antibodies to the pentapeptide subunit and investigating binding of several iodine labelled synthetic peptides to human sera. Fig. 10 gives some typical examples. As depicted in Fig. 10a, all radioactively labelled peptides employed bound quite similarly to human serum 004. As was already shown from the previous studies (compare Figs. 7-9), this human serum revealed specific antibodies exclusively directed against D-alanyl-D-alanine, similar as described for A-variant streptococcal antisera by Schleifer and Krause, 1971 (24). In contrast, it is evident from Fig. 10b that serum 426 strongly bound the pentapeptide subunit HL-Ala-D-Glu(L-Lys-D-Ala2-OH)-OH and the peptide H-Gly-Gly-L-Lvs-DAla 2 -OH; the peptide H-L-Ala-D-Glu(L-Ala-D-Ala2~OH)-OH revealing L-alanine instead of lysine, or tri-D-alanine inhibited less, thus obviously indicating a dominant role of lysine. On the other hand, serum 019 bound radioactively labelled H-L-AlaD-Glu(L-Lys-D-Ala2-OH)-OH or the peptide L-Ala-Glu(L-Ala-D-Ala2OH)-OH to the same extent, peptides lacking D-glutamic acid inhibited less. The results regarding specificity of antibodies direc-

14

•Ala-D-Glutys-D-Ala +Ala-D-Glu(Ala-D-Ala

O) o c o

1:5-

u)

R120

1:400

dSerum 004 20

40

60

b) Serum 426

c)Senjm019

0 20 40 60 0 20 Hapten Binding Capacity (%)

40

60

Fig. 10. Binding of radioactively labelled synthetic peptides with structural similarity to the pentapeptide subunit to human sera nos. 004, 426 and 019.

ted against the pentapeptide subunit in human sera are summarized in Table 3.

1. FOR SOME HUMAN SERA, ANTIBODIES DIRECTED AGAINST THE PENTAPEPTIDE SUBUNIT REVEAL PREDOMINANT SPECIFICITY FOR D ALANINE AS THE IMMUNODOMINANT GROUP, L-LYSINE AND/OR D GLUTAMIC ACID PLAYING A MINOR ROLE, SIMILAR AS DEMONSTRATED FOR ANIMAL HYPERIMMUNE SERA BY SCHLEIFER AND KRAUSE, 1971 (24). 2. FOR SOME HUMAN SERA, L-LYSINE AND/OR D-GLUTAMIC ACID SIGNIFICANTLY CONTRIBUTE TO THE ANTIGENIC SITE, THE SEQUENCE D-ALANYL-D-ALANINE PLAYING A MINOR ROLE.

Table 3. Specificity of human antibodies directed against the pentapeptide subunit.

15

4. Antigenic properties of the tetrapeptide subunit The tetrapeptide subunit further represents an independent antigenic epitope of peptidoglycan. This was clear from serial radioactive hapten binding studies employing as haptens the radioactivelv labelled tetrapeptide subunit or the pentapeptide subunit. It is obvious from Fig. 11 that serum 033 strongly bound the radiolab e l e d pentapeptide subunit and lacked specific antibodies to the tetrapeptide subunit. Serum 327, on the contrary, strongly bound the radioactively labelled tetrapeptide subunit whereas the pentapeptide subunit was bound less. The final proof for the occurrence of specific antibodies to the tetrapeptide subunit in serum 327 was furnished by appropriate inhibition studies (Fig. 12). The tetrapeptide subunit and C-terminal sequences of the tetrapeptide inhibited strongly, whereas the sequences H-L—Lys-L-Ala or C-terminal sequences of the pentapeptide subunit did not inhibit or only insignificantly. The tetrapeptide from the pseudomurein, H-L-Ala-L-Lys-L-Glu-L-Ala-OH did also not inhibit. Fig. 11

Fig. 12

L-Ala-D-GlulL-Lys-D-Ala) o D-Glu(Lys-D-Ala) a Ac 2 -L-Lys-D-Ala

L-Ala-D-Glu(L-Lys-D-Ala2) ACt-L-Lvs-D-AI»,

0.33

3i3

33

Inhibitor Concentration l(jmoles/l)

0 20 ¿0 60 0 20 40 60 Hapten Binding Capacityfii)

Fig. 11. Binding of radioactively labelled synthetic tetrapeptide subunit (•) or pentapeptide subunit (•) to human sera nos. 033 and 32 7. Fig. 12. Inhibition of radiolabelled tetrapeptide subunit H-L-AlaD-Glu(L-Lys-D-Ala-OH)-OH hapten binding (human serum no. 327) with several synthetic inhibitors.

16

In conclusion, the stem peptide of peptidoglycan reveals at least two main antigenic epitopes, i.e. the pentapeptide subunit and the tetrapeptide subunit. With few exceptions, no cross-reactivity was observed between the tetrapeptide and the pentapeptide of the stem peptide. 5. Immunoglobulin classes involved in the immune response to peptidoglycan Specific antibodies to peptidoglycan have been reported of the IgG

20

30

Elution Volume (ml) Fig. 13. Separation of IgG, IgM and IgA antibodies by the Pharmacia FPLC-system. Two 1-ml samples of serum no. 449, revealing specific immunoglobulins to peptidoglycan sequence R-D-Ala-DAla-OH of the IgG, IgA and IgM classes, respectively, were separated on a Mono Q anion-exchange column (for detail, see (30)). Specific antibodies of the particular immunoglobulin classes were detected by ELISA (18,30).

17

class (18,29,33,37), of the IgA class (29,30,33,36), of the IgM class (29,35-37) and of the IgE class (38). However, only Zeiger et al. (29) and Franken et al. (18,30) employed chemically welldefined antigens thus clearly proving that the immunoglobulin class identified was directed to a sequence of peptidoglycan. Recently, separating the particular immunoglobulin classes by the Pharmacia FPLC chromatography system we could demonstrate in human sera the occurrence of specific IgG, IgM and IgA class antibodies to the same epitope of peptidoglycan (Fig. 13, (30)). Regarding specific immunoglobulin M antibodies reported (35-37) , one should be aware of the fact that unless carefully excluded, all indirect immunoassays may be severely disturbed by the presence of rheumatoid factor. This holds particularly true for sera exhibiting both specific IgG and IgM antibodies to the same antigen. Similarly, reported IgE antibodies with specificity for peptidoglycan may not yet be accepted (Seidl et al., this book). Conclusion The immunochemistry of peptidoglycan in man is rather complex. At least 3 main classes of immunoglobulins may be directed to at least five main antigenic epitopes, with a broad spectrum in particular antigenic specificity. Studying the immunochemistry of peptidoglycan may, however, be useful for understanding the immune response to peptidoglycan in man.

References 1. Schleifer, K.H. and 0. Kandier. 1972. Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol. Rev. 36, 407-477. 2. Schleifer, K.H. and P.H. Seidl. 1985. In: Chemical Methods in Bacterial Systematics (M. Goodfellow and D.E. Minnikin, eds). Academic Press, London, pp. 201-219. 3. Schleifer, K.H. and R. Joseph. 1973. A directly cross-linked L-ornithine containing peptidoglycan in cell walls of Spirochaeta stenostrepta. FEBS Letters 36, 83-86.

18

4. Yanagihara, Y., K. Kamisango, S. Yasuda, S. Kobayashi, I. Mifuchi, I. Azuma, Y. Yamamura and R.C. Johnson. 1984. Chemical compositions of cell walls and polysaccharide fractions of spirochetes. Microbiol. Immunol. 2_8, 535-544. 5. Vasstrand, E.N., T. Hofstad, C. Endresen and H.B. Jensen. 1979. Demonstration of lanthionine as a natural constituent of the peptidoglycan of Fusobacterium nucleatum Fevl. Infection and Immunity 2J5, 775-780. 6. Rogers, H.J., H.R. Perkins and J.B. Ward. 1980. Microbial cell walls and membranes. Chapman and Hall, London, U.K. 7. Heymer, B., P.H. Seidl and K.H. Schleifer. 1985. Immunochemistry and biological activity of peptidoglycan. In: Immunology of the bacterial cell envelope (Stewart-Tull DES, ed). John Wiley, Chichester, U.K. 8. Karakawa, W.W., H. Lackland and R.M. Krause. 1967. Antigenic properties of the hexosamine polymer of streptococcal mucooeptides. J. Immunol. 99_, 1179-1182. 9. Rolicka, M. and J.D. Park. 1969. Antimucopeptide antibodies and their specificity. J. Immunol. 103, 196-203. 10. Wikler, M. 1975. Isolation and characterization of homogenous rabbit antibodies to Micrococcus lysodeikticus with specificity to the peptidoglycan and to the glucose-N-acetylaminomannuronic polymer. Z. Immun.-Forsch. 148, 193-200. 11. Schleifer, K.H. and P.H. Seidl. 1977. Structure and immunological aspects of peptidoglycans. In: Microbiology (D. Schlesinger, ed). American Society for Microbiology, Washington, 12. Audibert, F., B. Heymer, C. Gross, K.H. Schleifer, P.H. Seidl and L. Chedid. 1978. Absence of binding of MDP, a svnthetic immunoadjuvant, to antipeptidoglycan antibodies. J. Immunol. 121, 1219-1222. 13. Reichert, C.M., C. Carelli, M. Jolivet, F. Audibert, P. Lefrancier and L. Chedid. 1980. Synthesis of conjugates containing N-acetylmuramyl-L-alanyl-D-isoglutaminyl (MDP). Their use as hapten-carrier systems. Mol. Immunol. V7, 357-363. 14. Schleifer, K.H. and P.H. Seidl. 1974. The immunochemistry of peptidoglycan. Antibodies against a synthetic immunogen crossreacting with peptidoglycan. Eur. J. Biochem. 43, 509-519. 15. Seidl, P.H. and K.H. Schleifer. 1975. Immunochemical studies with synthetic immunogens chemically related to peptidoglycan. Z. Immun.-Forsch. 149, 157-164. 16. Seidl, P.H. and K.H. Schleifer. 1977. The immunochemistry of peptidoglycan. Antibodies against a synthetic immunogen crossreacting with an interpeptide bridge of peptidoglycan. Eur. J. Biochem. 74, 353-363.

19

17. Seidl, P.H. and K.H. Schleifer. 1978a. Specific antibodies to the N-termini of the interpeptide bridges of peptidoglycan. Arch. Microbiol. 118, 185-192. 18. Franken, N., P.H. Seidl, E. Zauner, H.J. Kolb, K.H. Schleifer, L. Weiss. 1985. Quantitative determination of human IgG antibodies to the peptide subunit determinant of peptidoglycan by an enzyme-linked immunosorbent assay. Mol. Immunol. 22, 573579. 19. Eichmann, K., D. G. Braun, T. Feizi and R.M. Krause. 1970. The emergence of antibodies with either identical or unrelated individual antigenic specificity during repeated immunizations with streptococcal vaccines. J. Exp. Med. 131, 1169-1189. 20. Seidl, P.H. and K.H. Schleifer. 1979. The immunochemistry of peptidoglycan. Serological detection of a difference in a single N-terminal amino acid. Mol. Immunol. 1_6, 385-388. 21. Seidl, P.H. and K.H. Schleifer. 1978b. Rapid test for the serological separation of staphylococci from micrococci. Appl. Environ. Microbiol. 35_, 479-482. 22. Wadström, T. 1973. Bacteriolytic enzymes from Staphylococcus aureus. Contrib. Microbiol. Immunol. 397-405. 23. Karakawa, W.W., D.G. Braun, H. Lackland and R.M. Krause. 1968. Immunochemical studies on the cross-reactivity between streptococcal and staphylococcal mucopeptide. J. Exp. Med. 128, 325340. 24. Schleifer, K.H. and R.M. Krause. 1971. The immunochemistry of peptidoglycan. The immunodominant site of the peptide subunit and the contribution of each of the amino acids to the binding properties of the peptides. J. Biol. Chem. 246, 986-993. 25. Zeiger, A.R. and P.H. Maurer. 1973. Immunochemistry of a synthetic peptidoglycan-precursor pentapeptide. Biochemistry 12, 3387.-3394 . 26. Seidl, P.H., J.R. Golecki, N. Franken and K.H. Schleifer. 1985. Immunoelectron microscopic studies on the localization of peptidoglycan peptide subunit pentapeptides in bacterial cell walls. Arch. Microbiol. 142, 121-127. 27. Seidl, P.H., P. Zwerenz, J.R. Golecki and K.H. Schleifer. 1985. Streptococcus pyogenes grown under sublethal concentrations of penicillin G accumulates close to the septum pentapeptide subunits of peptidoglycan. FEMS Microbiol. Lett. 30^, 325-329. 28. Heymer, B., K..H. Schleifer, S. Read, J.D. Zabriskie and R.M. Krause. 1976. Detection of antibodies to bacterial cell wall peptidoglycan in human sera. J. Immunol. 117, 23-26. 29. Zeiger, A.R., C.U. Tuazon and J.N. Sheagren. 1981. Antibody levels to bacterial peptidoglycan in human sera during the time course of endocarditis and bacteremic infections caused by Staphylococcus aureus. Infect. Immun. 33^, 795-800.

20 30. Franken, N., P.H. Seidl, T. Kuchenbauer, H.J. Kolb, K.H. Schleifer, L. Weiss and K.-D. Tympner. 1984. Specific immunoglobulin A antibodies to a peptide subunit sequence of bacterial cell wall peptidoglycan. Infect. Immun. £4, 182-187. 31. Heymer, B., D. Bernstein, K.H. Schleifer and R.M. Krause. 1975. A radioactive hapten-binding assay for measuring antibodies to the pentapeptide determinant of peptidoglvcan. J. Immunol. 1J_4, 1191-1196. 32. Rasmussen, J.R. and J.L. Strominger. 1978. Utilization of a depsipeptide substrate for trapping acyl-enzyme intermediates of penicillin-sensitive D-alanine carboxypeptidases. Proc. Natl. Acad. Sei. USA 7j>, 84-88. 33. Helgeland, S.M. and A. Grov. 1971. Immunochemical characterization of staphylococcal and micrococcal mucopeptides. Acta Pathol. Microbiol. Scand. Sect. B, 79, 819-826. 34. Heymer, B., W. Schachenmayr, B. Bültmann, R. Spanel, 0. Haferkamp and W.C. Schmidt. 1973. A latex agglutination test for measuring antibodies to streptococcal mucopeptides. J. Immunol. m , 478-484. 35. Verbrugh, H.H., R. Peters, M. Rozenberg-Arska, P.K. Peterson and J. Verhoef. 1981. Antibodies to cell wall peptidoglycan of Staphylococcus aureus in patients with serious staphylococcal infections. J. Infect. Dis. 144, 1-9. 36. Wilhelm, J.A., L. Matter and K. Schopfer. 1982. IgG, IgA and IgM antibodies to S. aureus purified cell walls (PCW) in normal and infected individuals. Experientia ^8, 1375-1376. 37. Wheat, L.J., B.J. Wilkinson, R.B. Kohler and A.C. White. 1983. Antibody response to peptidoglycan during staphylococcal infections. J. Infect. Dis. 147, 16-22. 38. Schopfer, K., S.D. Douglas and B. Wilkinson. 1980. Immunoglobulin E antibodies against Staphylococcus aureus cell walls in the sera of patients with hyperimmunoglobulinemia E and recurrent staphylococcal infection. Infect. Immun. 27_, 563-568.

THE

DIAGNOSTIC

VALUE

STAPHYLOCOCCUS

OF A N T I B O D I E S

AUREUS

ANTIGENS

TO

PEPTIDOGLYCAN

IN S E R I O U S

AND

OTHER

STAPHYLOCOCCAL

INFEC-

TIONS

B.

Christensson

D e p a r t m e n t of Lund, Sweden

Infectious

Diseases,

University

Hospital,

S-221

85

Introduction Staphylococcus in m o d e r n

aureus

medicine.

ill p a t i e n t s , infections. well

are

The

use

there S.aureus

primary

foci,

to e s t a b l i s h

The

the

When

blood

of S . a u r e u s can be

cultures

antibiotic

treatment,

tic

as w e l l

value,

bacteriological gested,

that

in a n t i b o d y

this

has been

These

and

used for

in

problem

seriously

staphylococcal

joint

prostheses

predisposing

factors

infections

number

patients

of t h e s e

isolation

a valuable

are

negative,

of

infections

as for

(1-4).

community-ac-

often

lack

may

isolation,

are

response

levels by

to

against others

of d i f f e r e n t

have

obvious

be

difficult

complicated

by

still

of t h e b a c t e r i a ,

which

in

can

many

be

serology

seated

due

treatment S.aureus

but

be

to

previous diagnos-

also

been

reflected

teichoic

S.au-

unavailable

It h a s

could

mainly

instances.

can be of

infections,

suspected.

acid

to

sugby

(5),

a but

(6-8). focused

serological

an u n c o m p l i c a t e d

is c e r t a i n l y

complement

staphylococcal

investigations

between

septicemia

deep

opposed

a combination a septicemia

often

staphylococcal

as w h e n

the

fall

present

other

an i n c r e a s i n g

origin

upon bacteriological serology

guish

been

increasing

of e n t r y

vascular

are

bacteremic

septicemia.

and

reus

The

abuse

an

(2).

diagnosis

based

drug

has

quired

devices,

portals

of v a l v u l a r ,

serious

However,

constitute

Intravenous well-known

as i n t r a v e n o u s

achieving

infections

course

upon assays

the

question

can help

of S . a u r e u s

endocarditis

distin-

septicemia

or m e t a s t a t i c

Biological Properties of Peptidoglycan © 1986 Walter de Gruyter & Co., Berlin • New Y o r k - Printed in Germany

to

whether and

abscesses.

22 This

distinction

cations

require

treatment

and

replacement

Materials Patients

and

important,

often

and

and

is

a more

also

drainage

since

aggressive surgical of d e e p

and

patients

with

these

long-standing

intervention

like

compli-

antibiotic heart

valve

abscesses.

Methods controls

S.aureus endocarditis. Twenty-two to 25 p a t i e n t s w i t h S.aureus The diagendocarditis w e r e i n v e s t i g a t e d in t h e v a r i o u s a s s a y s . nostic c r i t e r i a w e r e e l e v a t e d b o d y t e m p e r a t u r e ( > 3 8 . 5 C) for at l e a s t two d a y s t o g e t h e r w i t h t w o or m o r e p o s i t i v e b l o o d c u l t u r e s . Additionally, the p a t i e n t s d e v e l o p e d c h a n g e s in heart murmurs (n = 1 3 ) , t y p i c a l u l t r a s o u n d e c h o c a r d i o g r a p h i c s i g n s (n = 1 3 ) , s e c o n d a r y m a n i f e s t a t i o n s ( n = 1 6 ) or d i a g n o s i s w a s m a d e at s u r g e r y ( n = 2 ) tricuspid valve or autopsy (n=6). Six of 10 d r u g a d d i c t s h a d i n v o l v e m e n t ; no p a t i e n t s h a d i n f e c t e d p r o s t h e t i c v a l v e s . S.aureus complicated septicemia. The c r i t e r i a of e l e v a t e d body temperature and p o s i t i v e blood cultures among the 10-23 p a t i e n t s i n v e s t i g a t e d w e r e t h e s a m e as for t h e e n d o c a r d i t i s p a t i e n t s , but there w e r e o t h e r w i s e no s i g n s of endocarditis. These patients generally developed s e c o n d a r y i n f e c t i o u s f o c i in the skeleton ( n = 7 ) b u t a l s o d e e p s e a t e d a b s c e s s e s in i n t e r n a l o r g a n s ( n = 5 ) a n d meningitis (n=4). Patients with persistently infected joint p r o s t h e s e s ( n = 7 ) t h a t for s o m e r e a s o n w e r e n o t r e m o v e d or d r a i n e d within a few d a y s w e r e c o n s i d e r e d c o m p l i c a t e d e v e n if m e t a s t a t i c infections did not d e v e l o p . 5.aureus uncomplicated septicemia. This group comprised 14-23 p a t i e n t s and c r i t e r i a of body t e m p e r a t u r e and b l o o d c u l t u r e s w e r e as m e n t i o n e d a b o v e . No i n f e c t i o u s c o m p l i c a t i o n s d e v e l o p e d . When primary foci were p r e s e n t (n=19) they were easily eradicated w i t h i n a few d a y s . T h e s e f o c i w e r e s k i n i n f e c t i o n s ( n = 1 0 ) , i n t r a v e n o u s d e v i c e s (n = 6) a n d o s t e o s y n t h e t i c m a t e r i a l ( n = 3 ) . N o n - S . a u r e u s e n d o c a r d i t i s and s e p t i c e m i a . The d i a g n o s t i c c r i t e r i a of these i n f e c t i o n s w e r e t h e s a m e as for S . a u r e u s endocarditis and septicemia. M o s t of t h e 27 e n d o c a r d i t i s a n d 39 septicemia patients investigated were infected with gram-positive bacteria, mainly s t r e p t o c o c c i and S . e p i d e r m i d i s . In t h e a n t i - a l p h a toxin a s s a y ( s e e b e l o w ) o n l y f o u r a n d 18 p a t i e n t s , respectively, were tested. Febrile controls. When these 63 p a t i e n t s w e r e a d m i t t e d to the hospital, s e p t i c e m i a of u n k n o w n o r i g i n w a s i n i t i a l l y suspected. They w e r e e v e n t u a l l y c o n s i d e r e d as n o n - s e p t i c e m i c w h e n at least two b l o o d c u l t u r e s w e r e n e g a t i v e . Their s u b s e q u e n t d i a g n o s e s w e r e pneumonia (n=23), viral infection (n=18), urinary tract infection (n=8), f e v e r of u n k n o w n o r i g i n ( n = 5 ) , s k i n a b s c e s s ( n = 3 ) a n d o n e p a t i e n t e a c h p r o v e d to h a v e t o x o p l a s m o s i s , m a s t i t i s , c h o l a n g i t i s , pleural empyema, brain abscess and gas g a n g r e n e .

23 H e a l t h y c o n t r o l s I. T h i s g r o u p c o n s i s t e d of 1 6 0 i n d i v i d u a l s w h i c h were divided into d i f f e r e n t age groups. T h e y w e r e a l l h e a l t h y at t h e t i m e of s e r u m s a m p l i n g a n d h a d no p r e v i o u s h i s t o r y of s t a p h y lococcal infection. Healthy monthly

c o n t r o l s II. These 11 individuals s e r u m s a m p l i n g for six m o n t h s .

were

followed

with

Antigens Alpha toxin. S.aureus a l p h a t o x i n f r o m s t r a i n W o o d 46 w a s p u r i f i e d by i o n e x c h a n g e c h r o m a t o g r a p h y , i s o e l e c t r i c f o c u s i n g a n d g e l c h r o m a t o g r a p h y a c c o r d i n g to T h e l e s t a m et al (9) a n d W a d s t r o m and Mollby (10). T h e p u r i t y of t h e p r e p a r a t i o n w a s v e r i f i e d by m e a n s of p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s and I m m u n o e l e c t r o p h o r e s i s . Peptidoglycan. Peptidoglycan (PG) from two d i f f e r e n t strains, E2371, r i c h in p r o t e i n A ( 1 1 ) , and E1369, protein A deficient (12), was p r e p a r e d by a m o d i f i c a t i o n of t h e m e t h o d of P a r k and Hancock (13) u s i n g t r i c h l o r a c e t i c a c i d and t r y p s i n t r e a t m e n t of whole S.aureus cells. This insoluble preparation was solubilized in three different ways; by u l t r a s o n i c a t i o n (SO-PG), lysozyme t r e a t m e n t ( L Z - P G ) or l y s o s t a p h i n t r e a t m e n t ( L S - P G ) . A l l PG p r e p a rations were investigated in crossed Immunoelectrophoresis against polyspecific rabbit hyperimmune serum against S.aureus c e l l w a l l s . M o r e o v e r , t h e d i f f e r e n t PG p r e p a r a t i o n s w e r e c o m p a r e d by c r o s s e d - l i n e i m m u n o e l e c t r o p h o r e s i s . Crude staphylococcal antigen. Ultrasonication of t h e p r o t e i n A negative s t r a i n W o o d 46 w a s u s e d to p r e p a r e t h e c r u d e staphylococcal a n t i g e n (SA) (14). This preparation was investigated in crossed immunoelectrophoresis against both rabbit hyperimmune serum and h u m a n s e r u m a n d m o r e t h a n 50 different precipitates were obtained. A similar preparation has also been proposed to c o n t a i n t e i c h o i c a c i d , l i p o t e i c h o i c a c i d as w e l l as p e p t i d o g l y c a n (15). Lipase. Purified lipase from S . a u r e u s s t r a i n TEN 5 (16) was p r e p a r e d a c c o r d i n g to t h e m e t h o d s d e s c r i b e d by J u r g e n s et al (17) and J u r g e n s and H u s e r (18) u s i n g a m m o n i u m s u l p h a t e p r e c i p i t a t i o n of t h e c u l t u r e s u p e r n a t a n t , u l t r a f i l t r a t i o n , a d s o r p t i o n on O c t y l Sepharose CL-4B and finally elution with a linear Triton X-100 gradient. T h e p r e p a r a t i o n c o n t a i n e d b o t h t h e 44 kD a n d t h e 4 3 kD lipase, where the l a t t e r p r o b a b l y r e p r e s e n t s the n i c k e d 44 kD e n z y m e . T h e p u r i t y o f t h e l i p a s e p r e p a r a t i o n w a s v e r i f i e d by S D S p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s , d e t e r m i n a t i o n of e n z y m e a c t i vity and lack of h e m o l y t i c a c t i v i t y , inhibition of catalytic a c t i v i t y by c o r r e s p o n d i n g a n t i s e r u m a n d i m m u n o d i f f u s i o n ( 1 7 - 1 9 ) .

Serological

methods

Radioimmunoassay (RIA). Purified alpha toxin was iodinelabe11ed. d i l u t e d s e r u m a n d l a b e l l e d a n t i g e n in plastic After incubating t u b e s t h e a n t i g e n - a n t i b o d y c o m p l e x e s w e r e p r e c i p i t a t e d by adding an e x c e s s of a S . a u r e u s C o w a n I s u s p e n s i o n , thus utilizing the non-specific ability of p r o t e i n A to b i n d to t h e F c p o r t i o n of

24 IgG. After centrifugation, the r a d i o a c t i v i t y of the bacterial pellet was calculated as c o r r e s p o n d i n g to t h e amount of IgG antibodies against alpha toxin (20). Solid-phase radioimmunoasay (SPRIA). The SA- and the v a r i o u s P G p r e p a r a t i o n s w e r e c o a t e d on p l a s t i c t u b e s , and remaining b i n d i n g sites were b l o c k e d by b o v i n e s e r u m albumin. After incubating d i l u t e d s e r u m , b o u n d s e r u m IgG a n t i b o d i e s w e r e d e t e c t e d by a d d i n g iodinated protein A (14,21,22). Enzyme-linked immunosorbent assay (ELISA). The same p r e p a r a t i o n s as in S P R I A t o g e t h e r w i t h t h e l i p a s e p r e p a r a t i o n w e r e p l a c e d i n t o t h e w e l l s of m i c r o t i t e r p l a t e s . The following s t e p s w e r e according to a m o d i f i c a t i o n of t h e E L I S A of E n g v a l l a n d P e r l m a n n ( 2 3 ) with the a d d i t i o n of b o v i n e s e r u m a l b u m i n , d i l u t e d p a t i e n t s e r u m , a l k a l i n e - p h o s p h a t a s e c o n j u g a t e d a n t i - h u m a n IgG a n d p - n i t r o p h e n y 1 phosphate substrate. T h e a m o u n t of s p e c i f i c s e r u m IgG antibody b o u n d c o r r e s p o n d e d to t h e o p t i c a l d e n s i t y v a l u e m e a s u r e d s p e c t r o p h o t o m e t r i c a l l y after the e n z y m e - s u b s t r a t e reaction (22,24,25). Statistical

analysis

The reproducibility error of t h e m e t h o d single determination Upper limit

o f a n a s s a y w a s e x p r e s s e d as t h e analytical c a l c u l a t e d as t h e s t a n d a r d d e v i a t i o n o f the (22).

normal antibody l e v e l s w e r e s e t at t h e 99 % of t h e v a l u e s in t h e f e b r i l e c o n t r o l g r o u p .

confidence

A s i g n i f i c a n t c h a n g e in a n t i b o d y l e v e l w a s b a s e d u p o n t w o standard deviations of b o t h t h e a n a l y t i c a l e r r o r of t h e m e t h o d and the mean v a r i a t i o n of a n t i b o d y l e v e l s in the healthy control g r o u p II ( 2 4 , 2 5 ) . Predictive values test results were vall (27).

for p o s i t i v e (PV p o s ) a n d n e g a t i v e c a l c u l a t e d a c c o r d i n g to V e c c h i o (26)

(PV and

neg) Kron-

Results There

w a s no

patients

with

single

serological

S.aureus

capable

of

diagnosing

endocarditis/septicemia

or

excluding

patients

with

antibody

levels

during

one

determined

(Table

1).

were

an u n c o m p l i c a t e d to

assay course

four

of

weeks

infection, after

onset

when of

all those peak

infection

25 Table 1. Number a n d P e r c e n t a g e of P a t i e n t s a n d Controls with Positive P e a k A n t i b o d y L e v e l s in S t a p h y l o c o c c u s a u r e u s S e r o l o g y

NONS.

AUREUS COMPL

Alphatoxin

RIA

PG-ELISA SA-ELISA

ENDO

SEPT

S.

AUREUS

UNC0MPL

FEBRILE

HEALTHY

SEPT

ENDO

SEPT

NON-SEPT

CONTROLS

N.D.

4/160 N.D.

14/22

4/10

1/14

0/4

0/18

64 %

40

7 %

0%

0 %

20/25

7/23

6/23

10/27

1/39

0/63

80 %

30 %

26 %

37 %

3 %

0 %

21/25

17/23

6/23

3/27

3/39

1/63

11 %

Q O

1 .6 %

84

74 %

26 %

Lipase-

19/24

14/23

3/23

0/27

ELISA

79

61

13 %

•

Alpha

toxin

The

alpha

were

seen

mia

(Table

%

%

%

2.5

0/ /O

%

N.D.

0/39

0/63

0/157

0 %

0 %

0 %

antibody toxin

RIA

was

in p a t i e n t s

endocarditis cated

%

%

1).

highly

with

However,

was

only

the

64 % ,

and u n c o m p l i c a t e d

specific,

non-S.aureus

and

sensitivity

and only

septicemia

no

cross-reactions

endocarditis

or

septice-

in d i a g n o s i n g

40 % a n d

groups,

S.aureus

7 % in t h e

compli-

respectively.

PG-antibody When

the

solubilized

PG-preparations

investigated

in c r o s s e d

precipitates

but with

(22).

No t r a c e s

preparations crossed-line influenzed optimal lysozyme ring

cross-reacted

while

SO-PG.

variations acid

were

serological two

the

hours

seen.

with

sonication

all

showed

of

the

lysostaphin

continued

Moreover,

when

The time

activity

they

and LS-PG

in e l e c t r o p h o r e t i c

serologically

Immunoelectrophoresis.

time was

LZ-PG

Immunoelectrophoresis

some

of t e i c h o i c

the

SO-PG,

for

enzyme

all

and

four

PGin

treatment

preparations. hours

30 m i n u t e s

two

mobility

investigated

for

were

in

The with

prepa-

26 No

differences

were

seen whether

negative

S.aureus

striking

differences

preparations

when

The

sensitivity

was

impossible

slightly

higher

tory

assays better

could

used

with

patient

shown

sonicated sera

in T a b l e

1.

and

and

56 %,

respectively,

also

test

the

complicated

considered

(Table

positive,

and the some

2).

%)

non-S.aureus

The

However, were

The

with

a

labora-

were

dilutions vs.

1/40).

determined remained

in un-

SPRIA.

was

chosen

of p e a k could

for

antibody be

peak

levels

are

specificity

not

uncommon

endocar-

to 94 as a

density

the

and

all

%

antibody

was considered in o p t i c a l

testing

S.aureus

raised

a positive

endocarditis

it

reproducibility serum

antibodies

in d i a g n o s i n g

only

SPRIA

sera.

(1/3000

IgG s u b c l a s s e s

in t i t e r

using

ELISA.

routine

and higher

subclass

A 50 % r i s e

antibodies

as

LS-PG

or

negative

specificity

results

not

and

A

most

LZ- and

satisfactory

advantages.

18.1

The

in S P R I A

PG-SPRIA

as a n t i g e n

rise

significant.

cross-reacting

SO-PG.

septicemia

when

a significant

result

the

The s e n s i t i v i t y

ditis but

using

A in t h e

PG

from

or p r o t e i n

PG.

SO-,

low,

were

IgG3

protein

too

SO-PG

of a l l

possible

using

much

improved

antibodies

when

The ELISA the

with

the

as a n t i g e n s

positive

+/-

A rich

preparing

and

had

12.0 % vs.

whereas

detected

was

PG-ELISA

the P G - E L I S A

Furthermore, ELISA,

the

(+/-

be

used

LZ-PG

sensitivity

comparing

protein

for

seen between

were LS-PG

with

the

used

to d i s t i n g u i s h

obtained

was

were

were

they

of t h e

results

When

strain

was

in,

and level

positive

values

low,

was

because

mainly

gram-

septicemia.

SA-antibody The of

results the

SPRIA

of

PG-assays. regarding

laboratory.

The

using

peak

ELISA

but

often

positive

Similar

the

to

SA-assays Again,

the

sensitivity

antibody

levels

in

the

was

in d i a g n o s i n g was

SA-ELISA

ways

similar

superior

and c o n v e n i e n c e

S.aureus

PG-ELISA,

in m a n y

SA-ELISA

reproducibility

complicated the

were

for

S.aureus

comparable

to t h a t

septicemia

patients

than

serial

to

those

to t h e the

SA-

routine

endocarditis of

the were

PGmore

in t h e

PG-ELISA

(Table

1).

serum

sampling

raised

the

27 sensitivity,

because

(> 50 % r i s e a positive high,

but

patients

Lipase

significant

in o p t i c a l level

some

was

density

not

reached

cross-reactions

(Table

changes

values)

in

could

(Table

2).

antibody

be

levels

detected

The

even

specificity

were

seen

in n o n - S . a u r e u s

were

only

investigated

if was

infected

1,2).

antibody

Antibodies

to

S.aureus

This

was

ditis

and c o m p l i c a t e d

with

repeated

advantage reaching

the most

sensitive

100 %

assay

septicemia

serum

with

lipase

sampling

this

assay

(Table

1,2).

diagnosing

in 100 % a n d

(Table

was

the

2).

by

S.aureus 89 % ,

endocar-

respectively

Moreover,

extremely

ELISA.

high

the

major

specificity

Table 2. Number and P e r c e n t a g e of P a t i e n t s a n d C o n t r o l s with Positive Peak Antibody L e v e l s or Significant Titer Rises in Staphylococcus aureus Serology N0NS.

AUREUS C0MPL

ENDO PG-ELISA S A - E L ISA Lipase-ELISA Lipase- + P G - or

SA-ELISA

Combined The with

either

AUREUS

UNC0MPL

FEBRILE

SEPT

SEPT

ENDO

SEPT

NON-SEPT

15/16

10/18

7/18

3/11

4/20

3/63

94

56 %

39 %

27 %

20 %

5 %

14/16

15/18

8/18

0/11

2/20

2/63

88

0 %

1055

3 %

0/11

1/25

0/63

%

83 %

44

16/16

%

16/18

3/18

%

100

89 %

17

0 %

4 %

0 %

16/16

14/18

0/18

0/11

0/25

0/63

100

78 %

0 %

0 %

0 %

0 %

% %

%

serology

problem the

S.

PG-

w i t h c r o s s -• r e a c t i n g and

SA-ELISAs.

antibodiesi could not

Instead,

the

three

very

be o v e r c o m e sensitive

28 assays

(PG,

specific, well

SA a n d

were

be u s e d the

positive

peak

well

highly

as in e i t h e r

clinical should

tive

(PV

PG-

all

pos) PV

neg

of

2).

rise

for

could

correlation

positivity

in t h e

This

highly

assays

of a b s o l u t e

lipase

proved

combination

to

(Table

septicemia

course

were ELISA be

2)

patients

an u n c o m p l i c a t e d

upper

as p e r

infection.

the three

only

may

a

where could

or

non-

of e a c h

of 99.5 assays which

be

estimated, University

different as w e l l

variables

group to be

will

test

febrile

%.

The

true

then

be

influenzed a s by

upon

least be

but

was

will

as n e g a t i v e

10 % a m o n g

all

1982.

These

result

in PV

higher

test

results

(Table

%

but 99 %

corresof

99.5

rather

in

88

the

endocarditis/complicated

Hospital

= 2),

controls

at

the

S.aureus

30/34

specificity

must

is

indivi-

(Table

based

the

in r e a l i t y

pos

the

be

posi-

individuals be

in

will 100 %

was

PV

as w e l l

from would

of S . a u r e u s

PV

combinafor

positive

sensitivity

seem

results

at L u n d

positive

The

three

can

all

of t h e t e s t

septicemia

100 %,

mia

results.

The

levels

The p r e v a l e n c e

test among

(26,27).

specificity

of t h e

ly.

neg)

individuals

to a s p e c i f i c i t y

septicemia

(PV

patients

test

values

non-diseased

normal

as

or

negative

and

of the

test

of p r e d i c t i v e

cent

specificity

limit

as m u c h

of a l a b o r a t o r y

positive

the

The

combination

for

also

three

serology

and negative cent

negative

confidence

hardly

with

in t e r m s

endocarditis/complicated

ponding

titer

and c o m p l i c a t e d

significance

as p e r

prevalence

the

lack

o_r S A - E L I S A .

of c o m b i n e d

the s e n s i t i v i t y

(Table

the

The c r i t e r i a

specific

those

be e v a l u a t e d

and

among

from

values

The

by

to

(lipase)

These

infection.

tion

duals

the

endocarditis

Predictive

defined

due

assays.

and highly

differentiated

S.aureus

of t h e m

value £ £ significant

sensitive

S.aureus be

individual

one

in c o m b i n a t i o n .

in p a r a l l e l

between as

lipase),

evaluated

the %

but

unlikeseptice-

diagnosed values than

3).

for 95

%

29 T a b l e 3. P r e d i c t i v e V a l u e s for P o s i t i v e (PV p o s ) a n d N e g a t i v e (PV neg) Results of t h e C o m b i n e d U s e of the PG-, SA- and Lipase E L I S A s in D i a g n o s i n g S . a u r e u s E n d o c a r d i t i s / C o m p l i c a t e d S e p t i c e m i a Sensitivity

88 Si

Specificity

99.5 %

PV

Prevalence

10 %

PV

P°s

95

"1

S

98

'7

*

Discussion When

estimating

proposed which to

to b e u s e d

need

often

to b e

obtained

be

Clinical

assays

a patient between

of i n f e c t i o n .

with

endocarditis

long

persistence

require

a more

from

often

have

a

serology,

probably

serological

there

investigated.

are many

The

often

due

aspects

various

between

what

to

assay results

is

supposed

variations

in

characteristics.

the

with those

a

The higher

of the

antibody

treatment focus

difficult,

diagnostic

that

diagnosis which

criteria

is

seen

These

it

in

can

of

be

of e . g . between

to

to

early the

the also

separate

infection and

S.aureus

underlines

patients

due

patients

is n a t u r a l

course

important

uncomplicated

are probably

foci. and

it and

levels

abscesses

infectious

clinical

often

bacteremia

a complicated

an u n c o m p l i c a t e d

primary

same

S.aureus with

or m e t a s t a t i c

with

The

however,

using

of

laboratories

are

aggressive

those

eradicated. is,

routine

value

characteristics

defining

them

in

thoroughly

and patient

to d i s t i n g u i s h course

diagnostic

in d i f f e r e n t

similar

methodology

When

the

which easily

endocarditis importance

different

of

investiga-

tors. Positive the

relate symptoms day

IgG

onset

for

antibody

levels

of b a c t e r e m i c

the

time

(like the

for

fever,

first

can be e x p e c t e d

infections. serum chills,

positive

sampling general

blood

7-14

days

after

T h e r e f o r e , .it is i m p o r t a n t to

the

day

disability)

culture

or

for and

admission

onset not to

to

the to of the

hospital

30 Adequate All

control

patients

tectable when

as

extremely upper

similar

well

antibody

Therefore, the

groups

the

normal

antibody

patient

present

investigations

tially

was

well

ting this

the change

must not

are

Septicemia evaluated. positive,

patients

the

be

possible

due

to

other

The

cell

bacteria

bacteria

walls

share

of

antibodies

cross-reacting

streptococcal and

need

S. a u r e u s choic

acid

for show

for (15).

has

the ini-

serology evalua-

antibody

level,

group.

This

from

is

healthy

concentrations assay

are

used

important

found

measuring

preparation

been

not

and

gram-

the

pep-

surprising, and

against

lipase

alpha

toxin

assay

assay

exo-

are

not

and

li-

and

technique

preparations

used

regarding

preparation,

purified

when

and

emphasized

of a n t i g e n using

data

mainly

in e . g .

be

in S . e p i d e r m i d i s

toxin

of a n t i g e n

in t h e

other,

also

specificity.

previously when

should

cross-reactions alpha

assays

antigen

The m e t h o d

antigens

the

These

of

be

like

standardization

gen

different

and

in

when

It is t h e r e f o r e

can

the h i g h e s t

aspects

serology

contaminating

S.aureus

and

determinants

However,

consequently

antibodies

Methodological The

sex

used

samples

S.aureus

S.aureus

complex.

infections.

unique

than

antigenic

that

expected,

serum

as

used.

acid

pase

in

be

septicemia

in t h e c o n t r o l

if s i n g l e

should

Moreover,

of a c h a n g e

used.

defining

staphylococcal

clinician.

evaluated

tidoglycan/teichoic

proteins

routine

where

are

age,

controls

de-

antigens

when

group

regarding

represent by

and ELISA

The control febrile

have

S.aureus

important

The

significance

also

is

as p o s s i b l e

and where

used

clinical

certainly controls

been

generally

as S P R I A

group

levels.

symptoms.

suspected,

have

methods

group

clinical

controls

the d i f f e r e n t

of c o n t r o l

initial

may

against

sensitive choice

to t h e

as h e a l t h y

levels

the

lack

preparations,the the

comparing

results

of

anti-

reproducibility the

in tei-

of from

laboratories.

aspects

are

illustrated

in t h e c o m p a r i s o n s

between

the

31 different reacted

PG-preparations.

in c r o s s e d

Although

all

recommended

as a n t i g e n

in S P R I A

antibodies

due

its

poor

sensitivity.

with

both

lysostaphin

enzyme

in r e a c h i n g of

to

treatment

an o p t i m a l

different

batches

solubilization standardize,

the

or E L I S A

sensitivity. may

of P G by and

PG-preparations

immunoelectrophoresis,

also

and

serum

not

be

anti-PG

the

lysozyme

time

for

was

crucial

in e n z y m e

activity

a disturbing

chosen

cross-

could

Moreover,

Variations

have

was

LS-PG

measuring

u1tra-sonication

SO-PG

the

influenze.

is p r o b a b l y

as a n t i g e n

more

The

easy

in t h e

to

present

PG-ELISA. Although lacked

the

a great

problem

tivity.

One

unexpected ticemia alpha

use

of

Single

point

titrations

antibody amount

of

dilution

infection as

to

may

wide. be

reached.

sensitivity

peak

found

serum

reached

generally

had

study

a low

been

sensi-

findings

of

an

S.aureus

sep-

maintained,

that

95 % of

of S . a u r e u s

dilutions

generally the

show

all

S.aureus

Christensson septicemia

is

in T a b l e

a good only

antibody

of a n t i b o d y

and

strains

compared

levels

against

also

by

2 as c o m p a r e d

titer

the

the

serum

antigens

level cut rises

higher

to T a b l e

the

laboratories.

S.aureus

in a n t i b o d y

end-

antibody

influence

the p o s i t i v e

illustrated

with

laborato

absolute

different

of e v a l u a t i n g

the

correlation the

Therefore,

a change

for

affinity

between

although

advantage

serology convenient

not

(32-34).

Therefore,

in

more

However,

standardized

values

RIA

recent

and

has

(30).

significant The

purified

previously

toxin producing

in a r e c e n t

is m u c h

also

the

in a b o u t

(51/88)

antibody be

variation

very

positive

58 %

(31,32). but

bound

needs

normal

is o f t e n

is

highly

toxin

be

is p r e v i o u s l y

dilutions

level

could

of a l p h a

titrations

tory.

toxin,

anti-alpha

producers

single

serum

which

However,

only

toxin

were

delta

the

production

found

to b e a l p h a

is n o t

It

preparations

reason

low p r e v a l e n c e

(28,29).

serial

with

(9,10),

strains.

Hedstrom

The

toxin

possible

toxin

strains

The

alpha

contaminations

1.

during

off

level

as

well

values

for

32 Predictive When

values

a new

the

serological

clinical

significant if

these

sions

value

differences assay.

calculated. level

levels

very far

standing pected,

in the

front

assay

will

large

1 and

reach

use

city

100 % in t h e

cated

septicemia

quite

satisfactory

will

where

were

patient 99.5

made

is m u c h

with

used

no

will

Both

PV

than

95 %

serologi-

a

a n d PV

(Table

the

specifi-

material

pos

In of

However,

give

endocarditis

serological

sus-

higher.

single

and control

!£.

is

a prevalence

100 % .

ELISAs

S.aureus

be h i g h e r

routine

specificity

is to b e

septicemia

of t h e d i s e a s e

present

then

for

if

when

patient

to

least

test

is

important

rates

close

in d i a g n o s i n g

high

situation,

lipase

of at

a

specificity

prevalence

SA- and

specificity

reaching

be

with

a specificity

combination

should

in t h e c l i n i c a l

that

this

on

(PV)

Therefore,

if t h e

seen,

true

concluroutine

high

2 it c a n b e

of P G - ,

no

is e s p e c i a l l y

(27).

Even

a

as

values

populations

However,

calculations

combined of

low

groups.

(pr serum no. F.H. (specific IgE to wheat) as determined by RASTw(Pharmacia, Uppsala, Sweden). It was, however, most surprising that in contrast specific IgE to S. aureus cells or cell walls in serum F.H. reported by Schopfer et al. (1,2), was not affected at all. Removal of IgE antibodies from serum F.H. by affinity chromatography at solid-phase bound anti-human IgE further demonstrated that reported binding of radioactivity in the SPRIA (2) was not due to IgE antibodies. It is obvious from Table 4 that the concentration of total IgE antibodies in serum F.H. was reduced to zero level after affinity chromatography of this serum to matrix-bound anti-IgE. The same was true for allergen (wheat)-specific IgE in this serum (internal control). However, "specific IgE to S. aureus cell walls" was not affected thus demonstrating that the binding reported (2) was not due to IgE antibodies. Fractionation of serum no. F.H. according to molecular weight by HPLC (TSK 4000 column, Pharmacia, Uppsala, Sweden) revealed, that the elution patterns of total IgE and of "specific IgE to S. aureus cell walls" were not identical with respect to molecular weight, thus again supporting the finding that binding obtained (2) was not due to IgE antibodies (Fig. 2). It was of interest that heat inactivation of IgE antibodies in the serum fraction revealing maximal binding in the SPRIA (2) again did not affect binding.

B E F O R E TOTAL

IGE

(IU/ML)

SPECIFIC

IGE

TO WHEAT

(CPM)

"SPECIFIC

IGE

TO S .

A F T E R

AUREUS

CELL WALLS"

7350

40

4470

80

9701

9630

(CPM)

Table 4. Affinity chromatography of serum F.H. to matrix-bound antihuman IgE.

72

Fig. 2. Fractionation of serum F.H. by HPLC on a TSK 4000 column. For details, see materials and methods section.

Fig. 3. Binding of serum F.H. in the SPRIA described by Schopfer et al. (1).

20 o

x

TD C D O JD

i

15

10

o a o TJ a cr

0

1:10

1:100

Dilution of serum

In the present report we demonstrated that reported "specific IgE to S. aureus cells or cell walls" was antibodies. The nature of the binding caused in the unknown, but binding is dependent on serum dilution is noteworthy that "specific IgE to S. aureus cells

binding of not due to IgE SPRIA is yet (Fig. 3). It or cell walls

73 was hitherto only described for patients with hyperimmunoglobulinemia E (1,2). Recently, nonspecific adsorption of IgM from certain pathological sera with raised IgM was reported (9) and parallelity may be discussed. Further detailed studies are, however, necessary to elucidate the nature of the reported binding (1,2).

Acknowledgement This work was supported by DFG grant Se 427/1-1.

References 1. Schopfer, K., K. Baerlocher, P. Price, U. Krech, P.G. Quie and S.D. Douglas. 1979. Staphylococcal IgE antibodies, hyperimmunglobulinemia E and Staphylococcus aureus infections. N. Engl. J. Med. 300, 835-838. 2. Schopfer, K., S.D. Douglas and B.J. Wilkinson. 1980. Immunoglobulin E antibodies against Staphylococcus aureus cell walls in the sera with hyperimmunoglobulinemia E and recurrent staphylococcal infection. Infect. Immun. 2^7, 563-568. 3. Schleifer, K.H. and 0. Kandier. 1972. Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bact. Rev. 36., 407-477. 4. Seidl. P.H., P. Zwerenz, J.R. Golecki, N. Franken and K.H. Schleifer. 1986. Isolation of specific antibodies to the glycan moiety of peptidoglycan and their application in the indirect immunoferritin technique. FEMS Microbiol. Letters, in press. 5. Hartree, E.F. 1972. Determination of protein: a modification of the Lowry method that gives a linear photometric response. Analyt.Biochem. 48, 422-427. 6. Bennich, H. and K. Darrington. 1972. Structure and Conformation of the Immunglobulin E System. In: The Biological Role of the Immunglobulin E System (K. Ishizaka and D.H. Dayton, eds.). National Institute of Child Health and Human Development, Bethesda, MD. p. 19. 7. Johansson, S.G.O., T. Berg and T. Foucard. 1972. Circulating IgE antibodies measured by RAST and their significance in allergic diseases. In: The Biological Role of the Immunglobulin E System (K. Ishizaka and D.H. Dayton, eds.). National Institute of Child Health and Human Development, Bethesda, MD. pp. 211219.

74 8. Killingworth, L.M. and Savory J. 1972. Manual nephelometric methods for immunochemical determination of immunoglobulins IgG, IgA and IgM in human serum. Clin. Chem. J_8, 335-342. 9. Palusuo, T. and K. Aho. 1983. Technical falsely positive rheumatoid factor by ELISA in sera with elevated IgM levels. Med. Biol. 61, 203-207.

SPECIFIC IMMUNOGLOBULIN G ANTIBODIES IN MAN AGAINST THE GLYCAN STRAND OF PEPTIDOGLYCAN

E. Zauner, S. Reissenweber, G. Leitherer, K.H. Schleifer, and P.H. Seidl Lehrstuhl für Mikrobiologie, Technische Universität München, Arcisstr. 21, D-8000 München 2, FRG

Introduction The peptidoglycan molecule reveals at least five antigenic epitopes (for review see Seidl and Schleifer, this book). In man, specific antibodies to the non-crosslinked pentapeptide subunit with C-terminal R-D-Ala-D-Ala-OH and to the glycan strand have been hitherto detected, and several studies reported on a diagnostic role of antibodies against the pentapeptide subunit (1,2) . The applicability of these assay systems is restricted, however, since many eubacteria including almost all gram-negative organisms lack such pentapeptides. On the other hand, the glycan strand of peptidoglycan is a rather uniform chemical component of all eubacteria. Therefore, an enzyme-linked immunosorbent assay for routine quantification of glycan specific antibodies should be useful.

Materials and Methods Antigen albumin-(glycopeptide)n. Glycopeptides were isolated from peptidoglycan of Bacillus subtilis NCIB 8060 by treatment with hen egg-white lysozyme (3), dialysis and fractionation of the dialysable material on a Sephadex G-25 column (compare Ref. 4, Fig. 2 and Results section). Glycopeptides were covalently coupled to albumin as a carrier (Sigma A-1887) by reacting 7.17 mg protein and 21 mg glycopeptides in 1.5 ml 0.1 M sodium carbonate buffer pH 9.6 with 100 ul 25% glutardialdehyde for 18 hours at 4°C. After addition of 0.2 moles lysine, the reaction mixture was dialysed and lyophilized. ELISA• The ELISA was performed according to published procedure (5) with the following modifications: Polystyrene microtiter wells were coated with 50 p.1 of the antigen (2 ug/ml) ; 0.02 M phosphatebuffered saline pH 7.2 containing 0.2% Tween 20 was employed for

Biological Properties of Peptidoglycan © 1986 Walter de Gruyter & Co., Berlin New York - Printed in Germany

76

dilution of sera and washings. Inhibition of ELISA. Glycan strands, unsubstituted by peptides, were obtained by action of thiophenol (6) on peptidoglycan of S." aureus 52A5 (glycan I). Aliquots were N-acetylated (glycan II) and, in addition, the carboxyl groups of muramic acid were reduced (glycan III) (6). Glycans I, II, III, diacetyl-chitobiose, triacetyl-chitotriose, MDP (Sigma), diacetyl-L-Lys-D-Ala2~OH (Serva) and diacetyl-L-Lys-D-Ala-OH (UCB-Bioproducts, Brussels, Belgium) were employed as inhibitors according to published procedures (7), the methodology for preadsorption of sera to peptidoglycans was recently reported (8).

Results and Discussion Peptidoglycan from Bacillus subtilis NCIB 8060 was used for isolation of glycopeptides because of its particular primary structure (Fig. 1). Special emphasis was put on complete removal of protein impurities and of teichoic acids from the peptidoglycan employed for isolation of glycopeptides. After extensive incubation of cell walls with trypsin, subtilisin, proteinase K and protease Type V

PEPTIDOGLYCAN OF BACILLUS S U B T I L I S NCIB

8060:

-

LACKS AN INTERPEPTIDE

BRIDGE

-

LACKS NON-CROSSLINKED PENTAPEPTIDE SUBUNITS WITH C-TERMINAL R - D - A L A - D - A L A - O H

PEPTIDOGLYCAN

GLYCOPEPTIDES

MURAMIC ACID

1.06

0.97

GLUCOSAMINE

1.08

1.0

L-ALANINE

1.0

1.0

N-TERMINAL L-ALANINE

0

N.D.

-

LACKS NON-CROSSLINKED TETRAPEPTIDE

1.0

1.0

-

REVEALS 62% OF PEPTIDE SUBUNITS AS TRIPEPTIDES WITH C-TERMINAL DIAMINOPIMELIC ACID

MESO-DIAMINOPIMELIC ACID

1.05

0,98 N.D.

THE HIGH PERCENTAGE (55%) OF MESODIAMINOPIMELIC ACID RESIDUES WITH FREE «•-AMINO GROUPS IS IMPORTANT FOR COVALENT LINKAGE TO THE CARRIER

C-TERMINAL MESODIAMINOPIMELIC ACID

0.62

-

N-TERMINAL MESODIAMONOPIMELIC ACID

0.55

N.D.

-

SUBUNITS

AMINO A C I D / AMINO SUGAR

REVEALS CARBOXYL GROUPS OF MURAMIC ACID COMPLETELY SUBSTITUTED BY PEPTIDE SUBUNITS REVEALS A MINOR CONTENT OF DE-N-ACETYLATED GLUCOSAMINE RESIDUES

D-GLUTAMIC

ACID

D-ALANINE C-TERMINAL

ALANINE

UNSUBSTITUTED AMINO GROUP OF GLUCOSAMINE

0.38

0.22

0

N.D.

0.09

N.D.

Fig. 1. Structural features of peptidoglycan from Bacillus subtilis NCIB 8060.

77

from Streptomyces griseus, the protein content was reduced to 0.7%. Teichoic acids were quantitatively removed by 7 0% hydrogen fluoride (9), as was demonstrated by the phosphate content of 0.05% and not detectable polyols by GLC (compare Fig. 2). Detection of Glycan Specific IgG Antibodies Employing albumin-(glycopeptide) as an antigen in the ELISA described above, specific IgG antibodies to the glycan moiety could be detected in human sera. Fig. 3a shows the binding curves of Fig. 2

Fig. 3

©

PEPTIDOGLYCAN FROM BACILLUS S U B T I L I S

PROTEIN

NC IB 8060

: 0 . 7 % •, PHOSPHATE

GLYCEROL,

RIBITOL

E 1.2c C N 1.0CD

: 0.05 X

: NOT D E T E C T A B L E

BY

GLC

-3 0.8'

\

Í 0,6-

o. •>

\

508 3063

D I G E S T I O N WITH LYSOZYME

SOLUBLE

FRACTION

DIALYSABLE

Optical „o => ro

& 0,4+.

1:40

i Dilution of Serum

FRACTION,

BW< 6000-8000 F R A C T I O N A T I O N ON SEPHADEX G 25

GLYCOPEPTIDE

FRACTION

COVALENT

LINKAGE

TO ALBUMIN

ALBUMIN-(GLYCOPEPTIDE)N ANTIGEN

1:40

1:80

1:160

1:320

1.640

Dilution of Serum

Fig. 2. Scheme of the preparation of albumin-(glycopeptide) as an antigen in the ELISA.

used

Fig. 3. Binding of specific IgG antibodies in an ELISA a) to the glycan strand of peptidoglycan. Albumin-(glycopeptide)n was adsorbed to polystyrene microtiter plates, and specific IgG antibodies to the glycan were detected with goat anti-human IgG conjugated to peroxidase; b) to the peptidoglycan sequence R-D-Ala-D-Ala-OH (7).

78 three human sera to albumin-(glycopeptide) (Fig. 3b), to albumin-(D-Ala 3 ) g

, and in comparison

(7). Serum 508 revealed specific

antibodies to the glycan strand and lacked antibodies to the pentapeptide subunit. In contrast, serum 195 contained only specific antibodies against C—terminal R—D—Ala—D—Ala—OH, whereas serum 3063 revealed antibodies to both, the glycan moiety and the pentapeptide subunit. Thus, it was evident that the glycan moiety and the pentapeptide subunit represent independent antigenic epitopes of peptidoglycan in man. Antigenic Specificity of the ELISA Various glycan strands no

more substituted by peptide subunits

were prepared by treatment of peptidoglycan with thiophenol

(6),

compare Fig. 4; Glycan I, completely de-N-acetylated; Glycan II, completely N-acetylated; and completely N-acetylated Glycan III with free carboxyl groups of N-acetyl muramic acid reduced to -CI^OH groups. It was important that binding of glycan strand specific human antibodies to the albumin-(glycopeptide)

antigen was

completely inhibited by oligosaccharides composed of alternating 81,4-linked N-acetylglucosamine and N-acetyl muramic acid residues (Glycan II). The specificity of the ELISA for detecting antibodies directed against the glycan moiety of peptidoglycan was thus clearly proved. It was obvious from comparing the nearly identical inhibition curves obtained with Glycan II and Glycan III

(carboxyl

groups of N-acetyl muramic acid reduced to the -CH^OH group) that

100-

Serum 197

*

Glycan II, N-acetylated

Concentration of InNbitor l u g / m l )

Fig. 4 . Binding inhibition in the ELISA for measuring glycan strand specific antibodies, employing as inhibitors several glycan strand preparations no more substituted by peptides. For details, see text.

79

free carboxyl groups of N-acetyl muramic acid did not contribute to the antibody combining site. Complete removal of N-acetyl groups (Glycan I) affects binding of glycan strand specific antibodies, whereas in contrast, partial de-N-acetylation has no effect (Seidl and Schleifer, this book). Synthetic peptides with structural similarity to the C-terminal sequences of the stem peptide (Ac2-L-Lys-D-Ala-OH, Ac2-L-Lys-D-Ala2-OH) or MDP did not inhibit, thus excluding binding specificity for these structures. Further proof for the specificity of the ELISA was furnished by appropriate inhibition studies, employing as inhibitors culture filtrates from bacterial cells. It is well established that growing bacteria secrete peptidoglycan fragments into the medium (1013; Rosenthal and Fleming, this book; Zwerenz et al., this book). According to this, the ELISA for detecting glycan specific antibodies was specifically inhibited by bacterial culture filtrates taken from the logarithmic phase of growth (e.g. from Staphylococcus aureus, Streptococcus pyogenes, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, Edwardsiella spec., Enterobacter aerogenes, Shigella spec. , Proteus mirabilis; data not depicted). Culture filtrates from yeasts as well as uninoculated nutrient broth did not inhibit.

®

®

Serum 059 Seaim 059. adsorbed to peptidoglycan from Bacillus subtilis E coli Staphylococcus

1.0

c0,8 aureus

•50.6-

0

• Serum 004 Serum 004. adsorbed to peptidoglycan from

\ \ °

\

\

° Bacillus subtilis * E.coti • Staphylococcus aureus

\ 1:20 1:40 1:80 1:160 1:320 Dilution of Serum

1:1601:320 1.6401:12801:2560 Dilution of Serum

Fig. 5. Preadsorption of several peptidoglycans a) to serum 059 revealing specific antibodies to the glycan moiety of peptidoglycan and b) to serum 004 revealing specific antibodies to the pentapeptide subunit of peptidoglycan, prior to subsequent performance of the ELISA a) for detecting glycan specific antibodies b) for detecting specific antibodies against the pentapeptide subunit (7).

80 Finally, the specificity of the ELISA for detecting antibodies against the glvcan strand of peptidoglycan was again corroborated by preadsorption studies (Fig. 5). Human serum 05 9 revealing glycan moiety specific antibodies was preadsorbed to bacterial peptidoglycan, prior to performance of the ELISA for measuring glycan specific antibodies (Fig. 5a). All peptidoglycans examined (several species of staphylococci and streptococci, E. coli, Enterobacter aerogenes, Bordetella pertussis, Klebsiella pneumoniae) markedly reduced subsequent binding in the ELISA for detecting glycan moiety specific antibodies. Cell walls from yeasts (e.g. Candida albicans, Saccharomyces spec.) or pseudomurein did not affect binding. In comparison, specific antibodies to the pentapeptide subunit (serum 004) detected by an appropriate ELISA (7) were only adsorbed by peptidoglycans revealing such peptides (e.g. Staphylococcus aureus). Peptidoglycans from Bacillus subtilis or E. coli lacking pentapeptide subunits, or cell walls from yeasts did not affect binding (compare Fig. 5b). In conclusion, due to its relative structural uniformity (14) and regarding the immunological data reported here, the glycan moiety of peptidoglycan may be considered an antigenic epitope shared by all eubacteria.

Acknowledgement This research was supported by grant 01 ZR 112 from the Bundesminister für Forschung und Technologie (BMFT).

References 1. Heymer, B., K.H. Schleifer, S. Read, J.B. Zabriskie, R.M. Krause. 1976. J. Immunol. 117, 23 2. Zeiger, A.R., C.U. Tuazon, J.N. Sheagren. 1981. Infect. Immun. 33, 795. 3. Schleifer, K.H., L. Huss, O. Kandier. 1969. Arch. Microbiol. 68, 387.

81

4. Seidl, P.H., J.R. Golecki, N. Franken, P. Zwerenz, K.H. Schleifer. FEMS Microbiol. Lett, (in press). 5. Engvall, E., P. Perlmann. 1971. Immunochemistry 8, 871. 6. Kawagishi, S., Y. Araki, E. Ito. 1980. Eur. J. Biochem. 112, 273. 7. Franken, N., P.H. Seidl, E. Zauner, H.J. Kolb, K.H. Schleifer, L. Weiss. 1985. Mol. Immunol. 22, 573. 8. Franken, N., P.H. Seidl, T. Kuchenbauer, H.J. Kolb, K.H. Schleifer, L. Weiss.K.-D. Tympner. 1984. Infect. Immun. 44, 182. 9. Fiedler, F., M.J. Schäffler, E. Stackebrandt. 1981. Arch. Microbiol. 129, 85. 10. Glaser, L., B. Lindsay. 1977. J. Bacteriol. 130, 610. 11. Sinha, R.K., R.S. Rosenthal. 1980. Infect. Immun. 29.•

914

-

12. Mychajlonka, M., T.D. McDowell, G.D. Shockman. 1980. Infect. Immun. 2J3, 65. 13. Doyle, R.J., M.A. Motley, P.H.B. Carstens. 1982. Carbohydrate Res. 104, 147-152. 14. Schleifer, K.H., P.H. Seidl. 1985. Chemical Methods in Bacterial Systematics (M. Goodfellow, D.E. Minnikin, eds). Academic Press, London, p. 201.

RELEASE OF PENICILLIN-BINDING PROTEINS FRCM ß-LACTAM TREATED BACTERIA: DETERMINATION BY ANTI-ß-LACTAM ANTIBODIES

R. Hakenbeck, H. Ellerbrok, Th. Briese Max-Planck Institut für molekulare Genetik, Ihnestr. 63-73, D 1000 Berlin 33 N.F.Adkinson The Good Samaritan Hospital, 5601 Loch Raven Bld., Baltimore, MD 21239

Introduction Antibiotics that interfer with murein biosynthesis belong to the class of lytic antibiotics, i.e. they cause cellular lysis when added to a growing bacterial culture. However, in lysis-defective mutants or in bacteria with a suppressed autolytic systan, it is possible to study biochanical events that are induced in the cells upon treatment with these drugs, since they respond only by grcwth inhibition without acccmpanied lysis. Tcmasz and coworkers have documented in several gram-positive bacteria, that treatment with these antibiotics (under non-lytic conditions) leads to secretion of several cell wall ccmoonents (murein and teichoif acid precursors, as well as lipids and lipoteichoic acid) (1-4) . Recently, we have analyzed also proteins that are released during bacitracin or penicillin treatment of autolysin-defective pneumococci. The proteins were mainly membrane proteins, and they were associated with membrane vesicles which could easily be recovered frcm the culture medium of the drug treated bacteria by high speed centrifugation. Among the membrane proteins, penicillin binding proteins (PBP) could be identified (5). These minor membrane canponents occur in all penicillin-sensitive bacteria. By definition, they are able to bind 13lactams covalently, and it is this feature by which they can be visualized after incubation with radioactive penicillin, SDS-nolyacryl-amide-gel-electrophoresis (PAGE) and fluorography (for review, see 6,7). At the same time, it is this very property which makes it difficult to quantitate than after the cells have been treated with B-lactams. They do bind the (nonradioactive) antibiotics already in vivo by forming a PBP-penicilloyl-(BP0-)connlex (Fig.1), are then partly released associated to the manbrane vesicles. Postincubation of either cells or vesicles with radioactive penicillin, followed by PAGE and fluorography as described above, will then reveal only those PBP that had not reacted

Biological Properties of Peptidoglycan © 1986 Walter de Gruyter & Co., Berlin New York - Printed in Germany

84

COOH

+ E-Ser-OH Ser-E

Penicillin

+

PBP --•> — > (fast)

penicilloyl-PBP

>• pen-derivative (slow)+ p B p

Fig.1: Interaction of PBP with B-lactam antibiotics with the nonradioactive 6-lactam in vivo, or have lost it during the isolation or postincubation period. Thus, PBP could be studied only when cell wall inhibitors others than B-lactams were used for induction of menbrane secretion. Recently we discovered that B-lactam/PBP-ccmplexes can also be dsnonstrated after SDS-PAGE, blotting onto nitrocellulose membrane and immunostaining with anti-6-lactam antibodies (submitted for publication; details of materials and methods are described in that manuscript). The method allows direct determination of penicilloyl-PBP without the need of radioactive penicillin. In the present canmunication, the release of PBP during penicillin- and aztreonam (AZ)treatment of Streptococcus pneumoniae will be documented using the corresponding anti-B-lactam antisera.

Results and discussion 1. Bacterial growth under non-lytic conditions. S. pneumoniae, either an autolysin defective mutant (cwl) or the wildtype (R6) grown under lysis-nonDermissive conditions, were used. It has been described, that the autolysin of oneumococci (an N-acetyl-L-alanine-amidase) (8) which is the only autolysin described in this species can be inhibited by growth in medium suoplanented with 2% choline (9,10). This way, the amidase is obviously no longer capable of interaction with its proper substrate (11,12) and the cells do not lyse. Growth in 2% choline also interfers with cell separation (although proper cell division still occurs), and chain formation is the consequence. It has been argued, that this demonstrates a participation of the amidase in cell separation. Hcwever, the autolytic defective mutant cwl does not grow in chains under normal grcwth conditions, but it did so in 2% choline (Fig. 2B). This could mean, that either the mutant still contains residual amidase activity which is sufficient for cell separation, or that growth in 2% choline interfers also with other, still

85

A

N 100-

50-

¿t o

10 0

r1

"T

2

o

3

h

Fig.2: Growth of pneumoniae under non-lytic conditions. A. At time=0, benzylpenicillin (0.1 ,ug/ml) was added to exponentially growing cultures of cwl , . The control cwl grown with 2% choline (•), and R6 grown with 2% choline culture ranained without antibiotic (9) • Growth was monitored by nephelcmetry (N) B. Strain cwl grown with 2% choline for several generation. undetected enzymes which participate in cell separation. In any case, wild type R6 as well as the mutant grown in 2% choline do not lyse upon treatment with 13-lactam antibiotics (Fig. 2A) . 2. Determination of penicilloyl-PBP during penicillin treatment. S. pneumoniae contains six PBP ranging frcm a molecular weight of 94 kDa to 43 kDa (PBP 1a, 1b, 2x, 2a, 2b and 3). Strains cwl and R6 were grown in the presence of 2% choline, and cwl also without further choline-addition to the medium .Cells were treated with 0.1. ^ug/ml benzylpenicillin corresponding to approximately 10 x MIC, which is sufficient to saturate all PBP with the antibiotic in 5 min at 37°C. After 80 min, the cells were centrifuged and the supernatant (medium) recovered. PBP in the medium were measured either after acetone precipitation (which denatures) the BP0- PBP complex inmediately) , or alternatively, matibrane vesicles were collected frail the medium after high speed centrifugation. Although this step is carried out at 4°C, binding and release of penicillin could occur during this period. This method might therefore not be as accurate as the precipitation procedure, although larger quantities of the vesicles can be collected this way. Fig. 3A shows the outcome of this experiment. One can see, that under all conditions equivalent amounts of PBP are released fran the various bacterial cultures. Judging frcm the staining intensities, approximately 25% of the total cellular PBP ends up outside the cell after penicillin treatment, which corresponds fairly well with values obtained by determination of penicillin-binding

5

6

3 2

3

Fig. 3A. PBP in supernatants of penicillin- treated S. pneumoniae detected on inmunoblots with anti-BPO-antibodies. Cultures of the mutant cwl (1,4), cwl grcwn with 2% choline (2,5), and wild type R6 grown with 2% choline (3,6) received 0.1 ,ug/ml benzylpenicillin for 80 min. Cells were then centrifuged. Either 0.5ml of the supernatant were mixed with 2ml acetone in order to precipitate proteins (1-3), or manbrane vesicles fran 4nl supernatant were recovered by centrifugation for 14h, 48,000 ran, 50 Ti rotor (4-6). An R6 control culture received no drug during growth; after lysis of 1ml culture, PBP were labeled in vitro with penicillin (0). Proteins were separated on SDS-PAGE, blotted onto nitrocellulose and stained after incubation with anti-BPO-antibodies using phosphataseconjugated anti-IgG. PEP are indicated. B. PBP in cells and supernatant of penicillin treated R6. Strain R6 was.grown in the presence of 2% choline, and 0.1 ,ug/ml penicillin was added for 90 min. PBP were separated and visualized as described above. 1: PBP in 5ml medium after 90 min Pen-treatment of the cells; 2: PBP in cells (1ml culture equivalent) after Pen-treatment for 10 min; 3: PBP in control cells (1ml culture equivalent) after labeling with Pen of cell-lysate. activity (manuscript in preparation). Fig. 3B shows another aspect of the protein release, that is a quantitative difference of the PBP in the vesicles canpared to those found in whole cell lysates. PBP 2a is present in larger amounts in the medium than PBP 2x and 2b, whereas the opposite is true in the lysed cell. Anti-BPO-antibodies do not react with PBP 3-ccmplex for still unknown reasons, therefore the amount of PBP 3 can not be accurately determined with this method. Hcwever, it can be determined using anti-aztreonam-antibodies (13) as the AZPBP ccmplex shown below. 3. Determination of aztreonam-PBP during aztreonam treatment. Fig. 4a shows PBP in the medium of AZ-treated cultures of cwl. The MIC for AZ is 50 ^ug/ml. PBP are saturated under these conditions with the exception of PBP 2b, which binds AZ only at 0.5 mg/ml and higher. Nevertheless, one can see that the higher the concentration of AZ the more PBP appear in the medium.

87

'f 1

2

3

4

5

1

2

3

1'

2'

3'

4

5

6

Fig. 4. PEP in cells and medium of aztreonam-treated pneumococci. A. To aliquots of an exponentially growing culture of cwl, different amounts of AZ were added. After 25 min, cells frcm 1ml culture were ranoved by centrifugation and proteins of 0.5ml supernatant acetone precipitated. PBP in the samples were revealed after SDS-PAGE and imnunoblotting using anti-AZ-antibodies. Concentrations of AZ used (mg/ml): 1-0.05; 2-0.2; 3-0.8; 4-2.0; 5-0. B. Strain R6 was grown in the presence of 2% choline and various concentrations of AZ for 5h. Cells were recovered by centrifugation and lysed with detergent. One part was incubated with radioactive penicillin and radioactive BPO-PBP detected after fluorography (1'-3'). The other part was directly prepared for SDSPAGE (0.2ml culture equivalents. AZ-PBP complexes in the cells (1-3) and in 0.5 ml medium (4-6) were visualized after blotting with anti-AT-antiserum. Concentrations of AZ used ( ,ug/ml) : 1,1',4:10; 2,2',5:1; 3,3',6:0.1. Fig. 4B shows PBP in cells and supernatants of R6 grcwn with 2% choline after treatment for several hours with subinhibitory concentrations of f-Z. Binding of AZ to the cell-associated PBP was determined either directly with anti-AZ-antiserum (Fig.4B, 1-3) , or indirectly by postlabeling PBP in cell-lysates with radioactive Pen (1'-3'). After SDS-PAGE and autoradiography, only those PEP show up that have not reacted with AZ during growth. Even at these low concentrations PBP appear to be labeled with AZ, and secretion of PBP occurs although at a much slower rate when canpared to growth in the presence of high concentrations of AZ. Therefore, the samples were taken after several hours of AZ-treatment. Samples 4-6 show AZ-PBP secreted into the medium. It is again striking as has been pointed out already in Fig. 3B, that PBP 2x and 2b are preferentially associated with the cells, whereas almost all of PBP 2a appears in the medium. That finding confirms former results, in which the total protein composition of the secreted material was compared to manbrane and mesoscmal proteins (the term mesosames refers to a manbrane fraction which can be recovered frcm the medium after spheroplasting of the cells). In summary it appears that only a subtraction

88 of the manbrane proteins, which might correspond to a subfraction of the manbrane itself, participate in the secretion process which is induced by antibiotics that interfere with muréin biosynthesis (5). Another aspect concerns the situation in a patient whose infection is treated with ß-lactam antibiotics. Our results show, that massive amounts of proteins (BPO-PBP) that contain the antigenic site for anti-ß-lactam antibodies are released from the pathogenic bacteria, not only during cellular lysis but also frcm tolerant, non-lytic bacteria. Production of anti-ß-lactam antibodies is associated with the antibody-dependent penicillin-allergic reaction against ßlactams (see 14 for review). In which way the BPO-PBP as penicilloyl-antigens influence this reaction ranains to be clarified.

Acknowledgement

The technical assistance of Spassena Tomette is greatfully acknowledged.

References 1. 2. 3. 4.

Home, D., A. Tanasz. 1977. Antimicrob. Agents Chemother. Y\_, 888. Hörne, D., R. Hakenbeck, A. Tcmasz. 1977. J. Bacterid. 132, 704. Waks, S. A. Tcmasz. 1978. Antimicrob. Agents Chanother. 39, 293. Hakenbeck, R., S. Waks, A. Tanasz. 1978. Antimicrob. Agents Chanother. 13, 302. 5. Hakenbeck, R., C. Martin, G. Morelli. 1983. J. Bacteriol. 155, 1372. 6. 7. 8. 9.

Waxman, D.J., J.L. Straninger. 1983. Ann. Rev. Biochan. 52, 825. Frère, J.-M., B. Joris. 1985. CRC Crit. Rev. in Microbiol. 1_1_, 299. Mosser, J.L., A. Tcmasz. 1970. J. Biol. Chan. 245, 287. Briese, Th., R. Hakenbeck. 1983. In: The target of penicillin (R. Hakenbeck, J.-V. Höltje, H. Labischinski, eds.). DeGruyter, Berlin, p.173. 10. Giudicelli, S., A. Tcmasz. 1984. J. Bacteriol. J58, 1188. 11. Höltje, J.-V., A. Tanasz. 1975. J. Biol. Chan. 250, 6072. 12. Briese, Th., R. Hakenbeck. 1985. Eur. J. Biochan. ¿46, 417. 13. Adkinson, N.F., E.A.Swabb, A.A. Sugerman. 1984. Antimicrob. Agents Chanother. 25, 93. 14. Dewdney, J.M. 1977. In: The antigens (M. Sela, ed.). Acadanic Press New York, p. 82.

ANTI-PEPTIDOGLYCAN

SEROLOGY

IN PATIENT SERA AND EXPERIMENTAL PRODUCTION

OF

ANTI-PEPTIDOGLYCAN ANTIBODY BY IMMUNISATION WITH RHEUMATOID FACTOR

H.B. Evans, K.K. Phua, P.M. Johnson Department of Immunology, University of Liverpool, P.O. Box 147, Liverpool, L69 3BX, U.K.

Introduc tion

There

is some association between bacterial immunity and the genesis of

rheumatoid factor (RF) autoantibody in man (1), ditis; a

e.g.

in bacterial

endocar-

RF being characterised in the laboratory by its weak interaction with

determinant in the Fc region of IgG immunoglobulin (2).

reactive

with

Gram-positive

peptidoglycan-polysaccharide

bacterial (PG-PS),

cell

wall

Serum antibodies

structures,

including

are elevated in rheumatoid

arthritis

(RA) and there is no direct cross-reaction of RF as anti-PG-PS,

i.e. RF does

not itself act as an anti-PG-PS antibody (3).

investigated

the of

We have further

anti-PG-PS response in man with respect to the predominant IgG anti-PG-PS

subclass

antibody and the nature of the PG-PS antigen with which

such

antibodies interact. Experimental

animals

substantial

amounts

possibility

of

some

of

immunised with streptococcal cell serum RF-like

idiotypic

anti-IgG(Fc)

complementarity

walls

antibodies

between

RF

produce

(4).

and

The

antibody

reactive with streptococcal PG-PS has therefore been investigated.

Should RF

represent an auto-anti-idiotypic response to a common idiotype of

anti-PG-PS

antibodies, responses, reverse,

as then

i.e.

themselves antibodies.

bear

part it some

of ought

the to

antibodies

idiotypic

normal

immunoregulation

of

be possible experimentally raised against a common RF

activity related to

those

of

This concept is represented in Fig. 1.

Biological Properties of P e p t i d o g l y c a n © 1986 Walter d e Gruyter & Co., Berlin • New York - Printed in G e r m a n y

such to

antibody

produce

idiotype some

the could

anti-PG-PS

90

RF

a-PG-PS

a-idiotype idiotype Fig. 1

» « »

idiotype a-idiotype

Possible idiotypic interrelationship between RF and anti-PG-PS

Materials and Methods

Cell

wall

PG-PS

Streptococcus pyogenes preparation sulphate

was

(SDS)

polymer as

further at

100°C

preparations

were

described previously purified for

(3).

by extraction

15 minutes.

derived

with

PG-PS

at 2 g/ml.

Group

aliquot 1%

This antigen

modification of the anti-PG-PS ELISA technique (3). SDS-treated

An

from

of

sodium was

this

dodecyl

used

in

Wells were coated

The IgG subclass distribution of

A

a

with

anti-PG-PS

antibodies was assessed using monoclonal anti-human IgG1, IgG2, IgG3 and IgG4 reagents

(Unipath)

at 1/1000 dilution,

followed

by

rabbit anti- mouse Ig (Dakopatts) at 1/1000 dilution.

peroxidase-conjugated Results were expressed

as binding indices (5). Details

of

repeated immunisation of Balb/c mice with purified

non-RF immunoglobulin preparations are given elsewhere (6). experiment, components

a on

G-200

(Pharmacia) using

0.1M

and

In an additional

purified RF preparation was separated into IgG-RF and Sephadex

RF

citric

IgM-RF

acid/sodium

citrate buffer,

pH 3.6.

intraperitoneal

immunisations

preparations

Groups of Balb/c mice were given repeated with

either IgM-RF,

in Freund's incomplete adjuvant.

IgG-RF

or

sterile

non-RF

IgG

Anti-PG-PS antibody activity

in sera from these mice was measured by ELISA.

Results

Our juvenile

previous

studies

have shown anti-PG-PS levels

to

be

raised

in

chronic arthritis and both seronegative and seropositive rheumatoid

arthritis (3).

We have now shown that,

activity,

36%

subclass.

In addition, of 10 tuberculosis patients, all with raised anti-PG-

PS,

of

these

of such sera with raised anti-PG-PS

exhibit an IgG isotype

restriction

to

the

IgG2

2 showed restriction to IgG2 antibody (Evans et al., in preparation). In

contrast,

in a study of serial samples from rheumatic fever patients

(n=10),

all of whom had high anti-PG-PS levels, 20% of patients exhibited restriction of antibody to the IgG3 subclass.

However, preliminary experiments comparing

PG-PS

the presence of

preparations

antibody

levels

preparation and,

isolated

consistently

in

appear

with this antigen,

SDS

higher using

have

the

revealed

SDS-treated

that PG-PS

anti-PG-PS antibodies of the IgGI, IgG2

and IgG3 subclasses could be detected in rheumatic fever patient sera. Detailed isolated given

results of anti PG-PS antibody levels in mice

polyclonal

elsewhere

(6).

RF,

immunised

non-RF IgG and non-RF IgM preparations Anti-PG-PS levels for mice immunised with

IgM-RF and IgG-RF preparations are shown in Table 1 . of >3.0 are considered positive.

have

with been

separated

Binding indices (B.I.)

92 TABLE I

Anti-PG-PS Levels in Mice Immunised With IgM-RF, IgG-RF or Non-RF Ig B I.

Immunogen

Mouse No.

3 02 1 87 3 74 4 70 2 45 1 39 1 0 80 0 74

IgM-RF IgM-RF IgG-RF IgG-RF non-RF IgG non-RF IgG PG-PS

1 2 3 4 5 6 7 Pre-immune mouse sera (n=6)

-

Discussion Anti-PG-PS

levels

rheumatoid arthritis,

have

been shown to be raised

in

rheumatic

juvenile chronic arthritis and tuberculosis,

fever, although

it is not certain exactly with which antigenic components of PG-PS that these antibodies react.

In our hands,

SDS-treated

PG-PS,

density

relevant antigenic epitopes than an eguivalent

of

treated with SDS. responses

to

suggesting

higher antibody levels were detected using that this preparation may contain

a

higher

preparation

not

IgG2 antibodies are known freguently to be associated with

carbohydrate antigens (7),

and this may well be the case

for

those human anti-PG-PS antibodies showing IgG-isotype restriction to the IgG2 subclass. an

The

antibody

possibility of

an idiotypic

reactive with streptococcal PG-PS has also

Repeated

immunisation

produces

an anti-PG-PS antibody response (6).

can

complementarity between RF and

of mice

with human RF,

be absorbed by either PG-PS or RF,

been

investigated.

of either IgG

or IgM class,

As this anti-PG-PS

but not non-RF Ig,

activity

a cross-reactive

antibody which reacts with both PG-PS and with a RF idiotype appears to been

produced.

This interpretation is consistent with the view that RF from

unrelated RA patients share common idiotypic determinants. the PG-PS

internal image of a streptococcal antigenic epitope, appear

have

to react with anti-PG-PS antibody.

Such RF may carry since both RF

and

As this structure is

not

present on non-RF Ig, it is assumed to be an idiotypic determinant of RF; the

fact

that an anti-PG-PS response can be raised with either IgM-RF or

IgG-RF

would further support this supposition. This

data

adds

immunoregulatory

weight to the concept that RF is part

of

the

normal

system for modulating certain anti-bacterial responses

and

would put forward an explanation, other than autoreactivity with IgG(Fc), for the genetic conservation of common RF idiotypes.

Uncontrolled RF production

in

perhaps due to

RA may represent an imbalance in this system,

infection

or

continued

presence

of

peptidoglycan.

peptidoglycans exhibit some common antigenicity,

Since

persistent

Gram-positive

this could explain the lack

of association of any single bacterial agent with RF production in RA (8).

Acknowledgements This also

work

thank

was supported by the Arthritis and Rheumatism

Professor

H.R.

Perkins for advice in

the

Council.

production

We

of

streptococcal PG-PS preparations.

References 1.

Williams, R.C. Jr. 1977. In: Autoimmunity: Genetic, Immunologic, Virologie and Clinical Aspects (Talal N. ed.), New York, Academic Press, pp. 457.

2.

Johnson, P.M. 1981.

3.

Johnson, P.M., Phua, K.K., Perkins, H.R., Hart, C.A., Bucknall, R.C. 1984. Clin. exp. Immunol., 55, 115.

4.

Bokisch, V.A., Chio, Med. , 1_38, 1 1 84.

5.

Barnes, R.M.R., Barton, P.G., Doig, J.E., Finn, R., Harvey, M.M. and Johnson, P.M. 1983. J. Clin. Lab. Immunol., 12, 175.

6.

Johnson, 373.

7.

Hammarstrom, L., Granstr'om, M. , Oxelius, C.I.E. 1984. Clin. exp. Immunol. 55^, 593.

8.

Bennett, J.C. 1978.

P.M.,

Clin. Immunol. Allergy 1, 103.

J.W., Bernstein, D., Krause, R.M. 1973.

Phua, K.K., Evans, H.B. 1985.

Arthr. Rheum., 21, 531.

J. exp.

Clin. exp. Immunol., 61, V., Person, M.A.A., Smith,

ANTIBODIES AGAINST A SYNTHETIC PEPTIDOGLYCAN-PRECURSOR PENTAPEPTIDE CONTAINING LYSINE CROSS-REACT WITH SOLUBLE PEPTIDOGLYCAN CONTAINING DIAMI NOPIMELIC ACID

Allen

R.

Zeiger

Department of P h i l a d e l p h i a , PA

Biochemistry, 19107

Thomas

Jefferson

University,

Int r o d u c t i o n Antibodies

have

D-Ala-D-Ala found

In

glycans grown

been

In

that

the

are

titers

elevated

are

juvenile

arthritis

been

employed reagents

an

SPG

from

glycans the

or

could

which

narrow

presence

search The

rheumatic

fever

and

spondyl-

evidence

assay

in

C6).

the The

distinguish

SPG

bacterial by

bacteremia Impli-

urine ELISA

in

like

next

of

for

cell

these the

the

presence

antibiotic

such

as t h e

penultimate

bacteria

peptldonature

differences In t h i s

Biological Properties of Peptidoglycan © 1986 Walter de Gruyter & Co., Berlin New York - Printed in Germany

(8).

D-alanlne

responsible

answered

(7)

synthetic

wall-linked

structural

to b e

man

utilized

the

completely

differences to

question

the

has

p e p t I d o g 1 y e a n - 1 I ke

weight

a

(ELISA)

(A1 a - y - D - G 1 u - L y s - D - A l a - D - A l a ) 5 ' 1

distinguish

the

in m a n .

SPG

V

elicited

structural

present

their

(5).

low m o l e c u l a r

bacteria,

contain

diaminoacid

Reagents

A1 a 4 0

different C9),

a

population

(Gl'u b 0

to

of

transpeptidated

vancomycin,

antibody

Immunogen,

presence

These

since

ankylosing

immunosorbent

penicillin

able

with

and

Immunogen

of

(5).

Importance

serological

the

were

peptidobacteria

antibiotics

Cl)

been

soluble

endocarditis

mounting

natural

the

towards

have

gram-positive

patients

arthritis

Is

dose

which

e.g.,

of

reactive

sequences

in

some

aureau-caused

show

oral

hydrolyzed

sera

are

medical

enzyme-linked

to

an

and

beta-lactam

in t h e

There

a unique

products; and

of

that latter

by

considerable

S P G as t h e m a j o r

following of

of

rheumatoid

(3).

Recently,

two

be

Staphylococcus

cating

man The

secreted

presence

may

(2),

in

(1-4).

peptI dog 1 y c a n - p r e c u r s o r s (SPG)

antibodies

(1),

found

sequences

for

of

(5).

in

SPG

their

manuscript

96 Is w h e t h e r SPG t h a t

the

reagents

in

Materials

and

ELISA.

The

described

previously

ELISA

are

(GIu60

capable

(A2pm)

of

recognizing

in p l a c e of

ELISA

for

the

(5-6)

rabbit

detection

has

of

SPG

been modified

antibody

to

the

that

However,

instead

of

antibody

was

alkaline

an

immunoglobulin a

using

synthetic

purchased

1:500

biotIn-avIdIn phosphatase

Co.) was

The

(Sigma

Chem.

buffer,

pH

9.8,

containing

Absorbances

were

read

Multlskan MC

SPG.

The

(Flow

SPG

(10)

and

the

were

prepared

Included

In

added

polypeptide,

at

1 mg 0.01%

p e r ml

goat

(St.

p-nItropheny1

periodically

was

used.

the

second

at

anti-rabbit

Louis,

phosphate

of

10%

MgCl2 1 GrOUp

0-5 Years

IgM Titer > 3

6-17 Years

0-5 years

6-17 Years

I. LUTI

1/6

6/18

1/6

9/18

II.PN

3/6

4/7

3/6

6/7

2/22

0/3

7/22

IV.Control 0/3

A more precise differentiation of UTI and PN in infants led to the same results as before, although the differences were no longer so pronounced in the case of the lower limit. The titer-limits seem to be a little bit too low and the titer level seems also to depend on the age. Table 9:

ELISA OF IgG AND IgM ANTIBODY TO LIPID A IN HEALTHY ADULTS AND PATIENTS WITH GRAM-NEGATIVE SEPSIS IgG titer 0 1 Patients(adults) (n=6) Healthy adults (n=113)

3

3

100

13

0

IgM titer 1 2 3

1 2 95

0

18> 1

4

1 2

128 Table 9

shows the lipid A antibody titers in healthy individuals.

The determinations were made only a short time ago.

In order to investigate the antitoxic effect of the anti-lipid A titers, we are now performing a double-blind study im Klinikum Großhadern. This is being carried out with high-titered anti-lipid A sera. We were able to show that this effect is pharmacokinetically possible using lipid A antibody sera in sepsis patients and controls - one case with placebo and the other a healthy individual. By means of this study we hope to be able to show the same improved antitoxic effect which is being shown in the USA and Switzerland using the J5 mutant of E.coli.

References

1. Galanos, C., Lüderitz, 0., Westphal 0. 1971. Preparation and properties of antisera against the lipid A component of bacterial lipopolysaccharides. Eur.J.Biochem. 24, 116-122 2. Simon, G., Reindke, B., Marget, W.: Lipoid-A-Antikörpertiter bei Pyelonephritis und anderen Infektionen mit gram-negativen Bakterien. Infection 2 (174) 178-184 3. Marget, W., Schüßler, P., Kruis, W., Weinzierl, M., Rindfleisch, G.: Is the pathogenesis of Crohn's disease similar to that of juvenile recurrent pyelonephritis? Infection 4 (1976) 2-4. 4. Westenfelder, M., Galanos, C., Madsen, P.O., Marget, W.: Pathological activities of lipid A: Experimental studies in relation to chronic pyelongephritis. In: Schlesinger D. (ed.): Microbiology. American Society for Microbiology, Washington, D.C.1977, pp.277-279 5. Schüßler, P., Kruis,W. Marget, W.: Lipoid-A-Antikörper bei Morbus Crohn. Klin.Wschr.54 (1976) 1055 6. Schüßler, P., Kruis, W., Marget, W.: Lipoid-A-Antikörpertiter und O-Antikörpertiter bei Enterocolitis Croh, Colitis ulcerosa und akuter Enteritis. Med.Klin.71(1976, 1898-1902 7. Marget, W., Weiß, M., Ruhland, B.: Lipid A antibody determinations using ELISA on patients at a children's hospital: a preliminary report.Infectior 11 (1983) 82-84

ELECTRON MICROSCOPIC LOCALIZATION OP PEPTIDOGLYCAN IN THE CELL WALL OP STREPTOCOCCUS PYOGENES BY MEANS OP LABELLED ANTIBODIES AND LYSOZYME M. Wagner Central Institute of Microbiology and Experimental Therapy, Jena German Democratic Republic M. Rye Institute of Hygiene and Epidemiology, Prague, Czechoslovakia B. Wagner Central Institute of Microbiology and Experimental Therapy, Jena German Democratic Republic

Introduction Based on the morphological appearance of the cell wall in ultrathin sections and some other evidences it has been believed for a long time that the cell wall of streptococci, as other Grampositive bacteria too, is constructed from several layers, each of them consisting of one defined chemical component. The peptidoglycan was supposed to be the innermost layer forming the rigid basal structure of the wall (1 - 3)« However, phage absorption experiments have shown, that at least parts of the peptidoglycan macromolecule should be accessible on the cell surface (4). The aim of our investigations was to localize the peptidoglycan in the cell wall of group A streptococci by electron microscopic labelling methods. The present review summarizes the results of our previous papers (5 - 8). Materials and Methods Bacteria. Group A Streptococcus strain NY5 (M type 12), lacking the IgG*Pc-binding receptor (9), from the collection of the Central Institute of Microbiology and Experimental Therapy, Jena was used for all experiments. Cell Walls and Peptidoglycan. Cell walls were prepared by mechanical disintegration and differential centrifugation (6). In some experiments the walls were also treated with trypsin. Peptidoglycan was isolated from cell walls by extraction with 10% trichloroacetic acid (4h at 60 C) or by hot forraamide extraction. Peptidoglycan antibodies and ferritin conjugates. Peptidoglycan antibodies from antisera raised against Group A variant streptococci (strains T 12 and T 50) were isolated by absorption to group A peptidoglycan followed by extraction with 0.1M glycine-

Biological Properties of Peptidoglycan © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

130 H C 1 buffer, pH 2.5, and n e u t r a l i z a t i o n (6). F e r r i t i n c o n j u g a t i o n of the a n t i b o d i e s was performed w i t h g l u t a r a l d e h y d e (6). L y s o z y m e - p e r o x i d a s e c o n j u g a t e s . Lysozyrae was conjugated w i t h h o r se-radish p e r o x i d a s e by g l u t a r a l d e h y d e . R e s i d u e s of uncoupled lys o z y m e w e r e separated by c h r o m a t o g r a p h y on C o n A - S e p h a r o s e (Pharm a c i a , U p p s a l a ) . The conjugate showed both the a c t i v i t y of lysozyme (lysis of M i c r o c o c c u s f l a v u s ) and of the p e r o x i d a s e . Inhibition E x p e r i m e n t s . The f o l l o w i n g peptides w e r e used f o r a n t i b o d y i n h i b i t i o n e x p e r i m e n t s : D i a l a n i n e ( D - A l a - D - A l a ) , trialanine (D-Ala-D-Ala-D-Ala), t e t r a p e p t i d e ( L - A l a - D - i G l n - L - L y s - D A l a ) , p e n t a p e p t i d e ( L - A l a - D - i G l n - L - L y s - D - A l a - D - A l a ) and the M 24 p r o t e i n p e p t i d e L - A l a - L - G l u - L - L y s - L - A l a - L - A l a (10). F e r r i t i n conjugated a n t i b o d i e s against p e p t i d o g l y c a n w e r e incubated w i t h the peptides f o r 30 m i n at 37 C followed by 18 h at 4 C, and slight p r e c i p i t a t e s w e r e removed by c e n t r i f u g a t i o n . I n h i b i t o r y concentrations of the synthetic p e p t i d e s w e r e calculated as double those causing 5 0 % i n h i b i t i o n of the p r e c i p i t a t i o n w i t h p e p t i d o g l y c a n (11). Incubation of Cells and Cell F r a g m e n t s w i t h C o n j u g a t e s . To about 0.1 ml of sedimented cells, cell w a l l s or p e p t i d o g l y c a n inhibited or n o n i n h i b i t e d f e r r i t i n - c o n j u g a t e s (0.2 - 0.3 m l ) w e r e added and incubated at 37 C w i t h shaking f o r 1 h . Incubation w i t h l y s o z y m e - p e r o x i d a s e conjugates was done at 0 C i n an ice bath overnight. A f t e r w a s h i n g w i t h ice-cooled v e r o n a l a c e tate b u f f e r the s a m p l e s w e r e fixed w i t h g l u t a r a l d e h y d e , incubated w i t h 3 , 3 ' - d i a m i n o b e n z i d i n e and H ^ O p , and processed f o r t r a n s m i s s i o n electron m i c r o s c o p y (6). A s controls cells and cell f r a g m e n t s w e r e incubated w i t h p e r o x i d a s e and f u r t h e r processed in the same w a y . E l e c t r o n M i c r o s c o p y . U l t r a t h i n sections w e r e examined without p o s t - s t a i n i n g . F o r the e v a l u a t i o n of the inhibition experiments on w h o l e cells, w a l l s and p e p t i d o g l y c a n f r a g m e n t s one h u n d r e d segments of 0 . 2 ;um l e n g t h w e r e selected f o r counting the n u m b e r of f e r r i t i n particles using parts of a n t i t a n g e n t i a l l y sectioned objects. I n h i b i t i o n was calculated in percent of the counts on n o n i n h i b i t e d controls. Results Localization

of p e p t i d o g l y c a n

by f e r r i t i n - c o n j u g a t e d

A l l investigations w e r e carried walls

out on w h o l e cells,

(treated w i t h t r y p s i n and R N a s e ) and isolated

can. W h o l e cells displayed f e r r i t i n particles cell s u r f a c e as w e l l as specificity

of the l a b e l l i n g was

confirmed

2, 3). T h e f e r r i t i n p a r t i c l e s

peptidoglythe

(Fig. 1). The

by n e g a t i v e

results

n o r m a l IgG. Isolated w a l l s

p e p t i d o g l y c a n bound labelled antibodies res p r o t r u d i n g from both sides

isolated

directly on

on f i l a m e n t o u s p r o t r u s i o n s

obtained w i t h f e r r i t i n - c o n j u g a t e d

antibodies

on both surfaces

labelled short f i l a m e n t o u s of the

fragments.

and

(Figs. structu-

131

Pigs. 1-3. Strept.pyogenes, strain NY5, labelled with ferritinconjugated peptidoglycan antibodies. All bar markers represent 0.2 jam. (1) "/hole cell. (2) Cell wall. (3) Peptidoglycan Characterization of the peptidoglycan immunodeterminants The nature of the peptidoglycan immunodeterminants demonstrated by ferritin-conjugated antibodies was investigated in inhibition experiments (Table 1). Table 1: Inhibition of Ferritin-Conjugated Peptidoglycan Antibodies Demonstrated on Group A Streptococcus Cells, Cell Walls and Peptidoglycan Inhibition by Reduction of Particle Count (%) in Comparison to Noninhibited Controls Whole Cells Cell Walls Peptidoglycan Dialanine

76.6

83.8

Trialanine Tetrapeptide Pentapeptide

49.0 41.2 92.6

77.7

87.7 77.0

64.5 98.2 11.2

44.7 97.1 7.6

M Protein Sequence

7.3

The results of these studies clearly show that the pentapeptide exhibited the highest inhibition activity. In the experiments with cell walls and peptidoglycan it inhibited practically all antibody activity. The tetrapeptide had only the half of this activity. The M protein sequence used in the same concentration as the pentapeptide was practically ineffective. The figs. 4-6 show that after inhibition by synthetic peptides labelling was abolished or reduced on both sides of isolated cell walls.

132

Figs. 4-6. Strept.pyogenes, strain NY5• Labelling of cell walls w i t h antibody-ferritin conjugate was inhibited by synthetic peptides. Bar markers represent 0.2 ¿im. (4) N o inhibition. (5) Inhibition by tetrapeptide. (6) Inhibitor) by dialanine Localization of peptidoglycan by peroxidase-conjugated

lysozyme

Incubation of isolated peptidoglycan w i t h lysozyme-peroxidase followed by the reaction w i t h 3,3'-diaminobenzidine and HgC^ led to a heavy labelling on both sides of the fragments (Pig. 7). Similarly, isolated trypsinized cell walls were also labelled to a lesser extent on both aides (Pig. 8). Incubation of peptidoglycan or walls w i t h peroxidase alone h a d no such effect

(Pig.9).

On the other hand, whole cells exhibited only a weak labelling after incubation w i t h lysozyme-peroxidase (Pig. 10).

Pigs. 7-10. Strept.pyogenes, strain NY5. Labelling w i t h lysozymeperoxidase. Bar markers represent 0.2 ^pm. (7) Peptidoglycan. (8) Cell wall. (9) Cell wall, control w i t h peroxidase alone. (10) Whole cell.

133

Discussion The use of iramunoelectron microscopic methods allows a direct proof of the location of antigenic cell components. In our investigations two approaches have been applied to study the location of peptidoglycan in the cell wall of group A streptococci: The accessible immunodeterminant groups were detected by antibodies raised in rabbits against group A variant streptococci and purified by absorption to group A streptococcal peptidoglycan. The location of the glycan portion of the molecule was demonstrated by means of lysozyme conjugated to peroxidase. Ferritin-conjugated peptidoglycan antibodies labelled isolated cell walls and peptidoglycan fragments on both sides. The ferritin particles were mainly bound to filamentous structures. Whole cells were also labelled on their surface. These results contradict models in which peptidoglycan forms the innermost layer of the cell wall (1-3) and support concepts of a more complicated mosaic structure. Inhibition experiments with synthetic peptides demonstrated that the immunodeterminant groups demonstrated in this investigation are predominantly of pentapeptide nature. Interestingly, the distribution of these determinants was found to be similar on both sides of walls and peptidoglycan fragments. Recently, Seidl et al. (12) using antibodies against the D-alanyl-D-alanine moiety of the non-crosslinked pentapeptide also localized pentapeptide subunits of peptidoglycan on the surface of whole cells of Streptococcus pyogenes and some other Gram-positive bacteria. These results confirm our observation that the peptidoglycan of group A streptococci is accessible from the cell surface. The use of lysozyme-peroxidase conjugate based on the finding that conjugates of lysozyme with peroxidase or fluorescent dyes (5, 13) remain the properties of lysozyme to bind to the glycan strand and to lyse the peptidoglycan. Near 0°C, however, only binding to the peptidoglycan takes place. Using this probe both peptidoglycan and trypsinized cell walls were heavily labelled on both sides whereas the surface of whole cells seems to be lesser accessible for the lysozyme. Summarizing, our results show that the peptidoglycan of Streptococcus pyogenes does not form a separate inner layer of the cell

134

wall but must be present throughout the whole wall as a network. This conception is in agreement with contemporary models of the architecture of peptidoglycan of Gram-positive bacteria. Acknowledgement We thank Dr. R. Straka, Prague, for the synthesis of peptides and Dr.E.H.Beachey, Memphis, for providing us the M protein peptide. References 1. Krause, R.M. 1972. In: Streptococci and Streptococcal Diseases. Recognition, Understanding and Management (L.W.Wannamaker and J.M.Mats en, eds.) Academic Press, Hew York, p.3. 2. Davis, B.D., R.Dulbecco, H.N.Eisen, H.S.Ginsburg, W.B.Wood, M.McCarty. 1973. Microbiology, 2nd edn., Harper and Row, Hagerstown, Maryland, p. 708. 3. Kasper, Group B sen and Karger,

D.L., C.J.Baker, H.J.Jennings, 1985. In: Neonatal Streptococcal Infections (K.K.Christensen, P.ChristenP.Perrieri, eds.) Antibiotics and Chemotherapy, 35. Basel, p. 90.

4. Cleary, P.P., L.W.Wannamaker, M.Fischer, N.Laible. 1977. J.Exp.Med. 578. 5. Wagner, M., B.Wagner. 1978. Zbl.Bakt.Hyg., I.Abt.Orig.A 240, 302. 6. Wagner, M., B.Wagner, M.Rye.1978. J.Gen.Microbiol. 108, 283. 7. Rye, M., M.Wagner, B.Wagner. 1979. In: Pathogenic Streptococci (M.T.Parker, edit.) Reedbooks Ltd., Chertsey, England, p. 4 6 . 8. Rye, M., B.Wagner, M.Wagner, R.Straka. 1982. Current Microbiology 7, 187. 9. Rye, M., M.Wagner, B.Wagner, J.HavliXek. 1982.Microbios 34. 7. 10.Beaehey, E.H., J.M.Seyer, A.H.Kang. 1978. Proc.Natl.Acad.Sei. U.S. 75, 3163. 11.Schleifer, K.H., R.M.Krause. 1971. J.Biol.Chem. 2 4 6 . 986. 12.Seidl, P.H., J.R.Golecki, N.Pranken, K.H.Schleifer. 1985. Arch.Microbiol. 142. 121. 13.Gould, G.W., D.L.Georgala, A.D.Hitchins. 1963. Nature (Lond.) 200. 385.

IMMUNOELECTRON MICROSCOPIC STUDIES ON PEPTIDOGLYCAN FROM GRAM POSITIVE BACTERIA: SPECIFIC REACTIONS WITH THE GLYCAN MOIETY, THE PENTAPEPTIDE SUBUNIT AND THE INTERPEPTIDE BRIDGE

Norbert Franken Boehringer Mannheim GmbH, Forschungszentrum Tutzing, 8132 Tutzing, FRG Jochen R. Golecki Institut für Biologie II, Mikrobiologie, Albert-Ludwigs-Universität, 7800 Freiburg, FRG Peter H. Seidl, Peter Zwerenz, Karl H. Schleifer Lehrstuhl für Mikrobiologie, Technische Universität München, Arcisstraße 21, D-8000 München 2, FRG

Introduction The peptidoglycan molecule reveals at least five independent antigenic epitopes, i.e. a) the glycan moiety, b) the pentapeptide subunit, c) N-terminal and d) C-terminal sequences of the interpeptide bridge and e) the tetrapeptide subunit (1; Seidl and Schleifer, this book). This report deals with immunoelectron microscopic studies of the glycan moiety, the pentapeptide subunit and N-terminal interpeptide bridge epitopes in the cell wall of several gram positive bacteria.

Materials and Methods Specific antibodies to peptidoglycan epitopes. The isolation of antibodies to the glycan moiety by affinity chromatography techniques was recently reported (2). Antisera to the C-terminal Dalanyl-D-alanine moiety of H-L-Ala-D-Glu(L-Lys-D-Ala-D-Ala-OH)-NH2 were elicited in rabbits by immunization with synthetic immunoaen albumin-(CH 2 CO-Gly-L-Ala-D-Ala 2 -OH) (3). Antibodies to N-terminal sequences of the pentaglycine interpeptide bridge or of the tri-L-alanine interpeptide bridge were produced as previously described by immunization with tGly^-t-Ahx) »„-albumin or (L-Ala-.-&b J Ahx) 22 -albumin (4). Radioactive hapten binding assays and enzyme immunoassays. Antibodies to the tetrapeptide or to the pentapeptide subunit were determined by radioactive hapten binding assays (5,6) and by an en-

Biological Properties of Peptidoglycan © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

136

zyme-linked immunosorbent assay (ELISA) (7). Glycan strand specific antibodies were measured by a radioactive hapten binding assay (2) or by an appropriate ELISA (Zauner et al., this book). Binding inhibition studies for proving antibodies' specificity were carried out as previously described for the radioactive hapten binding assays (5-7) or for the ELISAs (7; Zauner et al., this book). Bacterial strains used. The origin of Streptococcus pyogenes Avariant A486, Staphylococcus aureus 52A5 and of Bacillus subtilis W23 is given in a recent paper (8). Indirect immunoferritin technique and electron microscopy. Labelling of cells in the indirect immunoferritin technique and electron microscopy were carried out as recently described in detail (8).

Analytical methods. Gas chromatography, quantitative determination of phosphate and protein, amino acid analyses, determination of C-terminal amino acids and of the L-alanine/D-alanine ratio in peptidoglycans were carried out as described (8).

Results and Discussion Specificity of Antibodies to the Glycan Moiety, Pentapeptide Subunit and Interpeptide Bridge Epitopes Antibodies with specificity to the glycan moiety of peptidoglvcan were isolated from antisera to whole cells of Micrococcus luteus CCM 169, known to contain high levels of glycan strand specific antibodies (9). Removal of minor antibody levels in those sera with specificity to the pentapeptide subunit (1,9) was achieved by affinity chromatography on Sepharose-(Gly2-L-Ala-D-Ala-D-Ala-OH)n (10). Glycan strand specific antibodies in the eluent were then prepared by affinity chromatography (2). Peptidoglvcan of Bacillus subtilis NCIB 8060 was used as an immunoadsorbent (2) for the isolation of glycan strand specific antibodies since it lacks as antigenic epitopes interpeptide bridges, pentapeptide subunits or non-crosslinked tetrapeptide subunits (1, 6-9,11). Specificity of the isolated antibody fraction for the glycan moiety was verified by immunoassays for measuring glycan strand specific antibodies (1,2, 9; Zauner et al., this book), and by excluding binding specificity to the tetrapeptide or to the pentapeptide subunit (1,2,5-7). Specific antibodies to the C-terminal D-alanyl-D-alanine moiety of non-crosslinked pentapeptide subunit H-L-Ala-D-Glu(L-Lvs-DAla-D-Ala-OH)-NH0 (10) were elicited in rabbits using as an immuno-

137

gen albumin-(CH2CO-Gly-L-Ala2-D-Ala-D-Ala-OH)3g (3). Exclusive specificity of these antibodies for non-crosslinked peptide subunit pentapeptides was demonstrated in detail by appropriate precipitin and precipitin inhibition studies (3) and by Farr type hapten binding or binding inhibition studies (5,6,8). Specific antibodies to the N-terminal sequences of the pentaglycine interpeptide bridge or to the tri-L-alanine interpeptide bridge were obtained by immunization with synthetic immunogens (Gly^-f-Ahx)2Q~albumin or with (L-Ala^-fc-Ahx)22-albumin, respectively (4). Selective specificity of the particular antiserum for the pentaglycine interpeptide bridge of staphylococci or against the oligo-L-alanine interpeptide bridge typical for many micrococci or pyogenic streptococci was demonstrated in detail by appropriate precipitin and precipitin inhibition studies (4,12) and by latex agglutination (13). Immunoelectron Microscopic Detection of the Glycan Moiety, the Pentapeptide Subunit and of the Interpeptide Bridge Epitope in Gram Positive Bacteria The results obtained from the immunoelectron microscopic studies employing specific antibodies to the particular peptidoglycan epitopes are schematically summarized in Table 1, p. 138. Whole trypsinized cells of Streptococcus pyogenes, incubated with specific antibodies to the glycan moiety followed by incubation with ferritin-labelled anti-rabbit IgG were specifically labelled with ferritin particles as was evident from these sections comparing untreated controls (not depicted) with antibody coated cells (Fig. 1, p. 138). Heavy specific labelling with ferritin particles (data not depicted) was also obtained with trypsinized cells of Staphylococcus aureus and of Bacillus subtilis, compare Table 1 and (2) . Employing in the indirect ferritin technique specific antisera to the pentapeptide subunit (3) , Bacillus subtilis remained completely unlabelled however (Fig. 2a, p. 139 and Table 1), due to missing pentapeptide subunits in its peptidoglycan (3,8). On the contrary, heavy specific labelling was obtained with cells of Streptococcus pyogenes (Fig. 2b, p. 139) or of Staphylococcus aureus (Fig. 2c, p. 139), compare Table 1. These results fully

138

Table 1. Labelling of trypsinized cells of Streptococcus pyogenes, Staphylococcus aureus and of Bacillus subtilis with specific antibodies to the glycan moiety, to the pentapeptide subunit and to the interpeptide bridge epitope (indirect immunoferritin technique). +++, heavy labelling; -, no labelling.

STR. PYOGENES A-VARIANT A486

S. AUREUS 52A5

B. SUBTILIS W23

SPECIFIC ANTIBODIES TO THE GLYCAN MOIETY

+++

+++

+++

SPECIFIC ANTIBODIES TO THE R-D-ALA2 MOIETY OF THE PENTAPEPTIDE SUBUNIT

+++

+++

-

-

+++

-

+++

-

-

SPECIFIC ANTIBODIES TO THE G L Y 5 INTERPEPTIDE BRIDGE

SPECIFIC ANTIBODIES TO THE L - A L A 3 INTERPEPTIDE BRIDGE

Fig. 1. Labelling of trypsinized cells of Streptococcus pyogenes with specific antibodies to the glycan moiety (2) of peptidoglycan (indirect ferritin technique). Bar represents 200 nm.

139

Fig. 2. Labelling of trvpsinized cells of a) Bacillus subtilis W23, b) Staphylococcus aureus 52A5, c) Streptococcus pyogenes A-variant A486 with specific antibodies to the pentapeptide subunit (3) of peptidoglycan (indirect ferritin technique). Bar represents in all Figs. 200 nm.

140

correspond with our previous precipitin studies (3), and with the peptidoglycan structures of bacteria investigated. Due to missing D,D-carboxy-peptidases (14), pentapeptide subunits with C-terminal D-alanyl-D-alanine occur frequently among staphylococci or streptococci (6,7) and were chemically demonstrated in the peptidoglycans of Str. pyogenes A486 or S. aureus 52A5, used in this study (8) .

Employing in the indirect ferritin technique specific antisera to the interpeptide bridge (4), trypsinized cells of Staphylococcus aureus were specifically labelled with antisera to the pentaglycine interpeptide bridge of staphylococci (Fig. 3a) but not with antisera to the tri-L-alanine interpeptide bridge (Fig. 3b). On the other hand, cells of Streptococcus pyogenes were heavily labelled with antiserum to the tri-L-alanine interpeptide bridge but not when using antisera to the pentaglycine interpeptide bridge (not depicted, compare Table 1). Neither interpeptide bridge specific antiserum resulted in labelling of Bacillus sub-

Fig. 3. Labelling of trypsinized cells of Staphylococcus aureus 52A5 with specific antibodies to the interpeptide bridge (4) of peptidoglycan (indirect immunoferritin technique). Bar represents 200 nm. a) ultrathin sections of S. aureus incubated with antiserum to (Gly^-fcrAhx)-Q-albumin; b) ultrathin sections of £3. aureus incubated with antiserum to (L-Ala^-f.Ahx) 00 -albumin.

141

tilis. This selective binding was in excellent agreement with our previous data (4,13) and corresponded to the peptidoglycan structures of strains investigated (11). Immunoelectron Microscopic Controls and Exclusion of Non-Specific Binding Cells not incubated with specific antibodies missed any labelling (8), and incubation of cells with ferritin conjugate alone did not result in any labelling either (8). Non-specific binding of rabbit IgG to possible Fc-binding factor of Str. pyogenes A-variant A486 (15) or traces of protein A of S. aureus 52A5 was ruled out study125 ing direct binding of iodine labelled rabbit IgG to cells of Str. pyogenes A486 or aureus 52A5 (8), and could in addition be excluded from selective binding when using interpeptide bridge specific antibodies (compare Table 1). Application of Specifically Detecting Particular Peptidoglycan Epitopes in the Electron Microscope We have now a tool for specifically detecting particular peptidoglycan epitopes in the electron microscope. For example, pentapeptide subunits represent a precursor sequence from peptidoglycan biosynthesis and accumulate in the wall under sublethal concentrations of penicillin (16) or decrease under cycloserine (17). Recent immunoelectron microscopic studies to localize the points of attack of penicillin revealed for Streptococcus pyogenes the accumulation of pentapeptide subunits close to the septum (18). On the contrary, the rod-shaped Lactobacillus gasseri DSM 20243 (strain AM63) accumulates non-crosslinked pentapeptide subunits at the poles under sublethal concentrations of penicillin (unpublished) . Studies on growing cells of Streptococcus pyogenes revealed secretion or incorporation (?) of pentapeptide subunits near the wall band (unpublished) and studies with reverting spheroblasts are under current research.

142

References 1. Heymer, B., P.H. Seidl and K.H. Schleifer. 1985. Immunochemistry and biological activity of peptidoglycan. In: Immunology of the bacterial cell envelope (Stewart-Tull DES, ed). John Wiley, Chichester, U.K. 2. Seidl, P.H., P. Zwerenz, J.R. Golecki, N. Franken and K.H. Schleifer. 1986. Isolation of specific antibodies to the glycan moiety of peptidoglycan and their application in the indirect immunoferritin technique. FEMS Microbiol. Letters, in press. 3. Schleifer, K.H. and P.H. Seidl. 1974. The immunochemistry of peptidoglycan. Antibodies against a synthetic immunogen crossreacting with peptidoglycan. Eur. J. Biochem. 509-519. 4. Seidl, P.H. and K.H. Schleifer. 1978. Specific antibodies to the N-termini of the interpeptide bridges of peptidoglycan. Arch. Microbiol. 118, 185-192. 5. Heymer, B., D. Bernstein, K.H. Schleifer and R.M. Krause. 1975. A radioactive hapten-binding assay for measuring antibodies to the pentapeptide determinant of peptidoglycan. J. Immunol. 114, 1191-1196. 6. Seidl, P.H. and K.H. Schleifer. 1985. Secretion of fragments from bacterial cell wall peptidoglycan. In: Environmental regulation of microbial metabolism (Kualev IS, Severin AT, Dawes EA, eds). Academic Press, London, 443-450. 7. Franken, N., P.H. Seidl, E. Zauner, H.J. Kolb, K.H. Schleifer, L. Weiss. 1985. Quantitative determination of human IgG antibodies to the peptide subunit determinant of peptidoglycan by an enzyme-linked immunosorbent assay. Mol. Immunol. 22.< 57 3579. 8. Seidl, P.H., J.R. Golecki, N. Franken and K.H. Schleifer. 1985. Immunoelectron microscopic studies on the localization of peptidoglycan peptide subunit pentapeptides in bacterial cell walls. Arch. Microbiol. 142, 121-127. 9. Schleifer, K.H. and P.H. Seidl. 1977. Structure and immunological aspects of peptidoglycans. In: Microbiology (D. Schlesinger, ed). American Society for Microbiology, Washington, U.S.A. 10. Schleifer, K.H. and R.M. Krause. 1971. The immunochemistry of peptidoglycan. The immunodominant site of the peptide subunit and the contribution of each of the amino acids to the binding properties of the peptides. J. Biol. Chem. 246, 986-993. 11. Schleifer, K.H. and 0. Kandier. 1972. Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol. Rev. 36, 407-477.

143

12. Seidl, P.H. and K.H. Schleifer. 1979 . The immunochemistry of peptidoglycan. Serological detection of a difference in a single N-terminal amino acid. Mol. Immunol. J_6, 385-388. 13. Seidl, P.H. and K.H. Schleifer. 1978. Rapid test for the serological separation of staphylococci from micrococci. Appl. Environ. Microbiol. 35.' 479-482. 14. Rogers, H.J., H.R. Perkins and J.B. Ward. 1980. Microbial cell walls and membranes. Chapman and Hall, London, U.K. 15. Wagner, B., M. Wagner, M. Rye. 1983. Morphological evidence for different types of IgG-Fc receptors in group A streptococci. Zbl. Bakt. Hyg. I. Abt. Orig. A 256, 61-71. 16. Munoz, E., J.M. Ghuysen, M. Leyh-Bouille, J.F. Petit, H. Heymann, E. Bricas, P. Lefrancier. 1966. The peptide subunit N0*-(L-alanyl-D-isoglutaminyl)-L-lysyl-D-alanine in cell wall peptidoglycans of Staphylococcus aureus, strain Copenhagen, Micrococcus roseus R2 7, and Streptococcus pyogenes group A type 14. Biochemistry 5, 3748-3763. 17. Spahn, K.F. 1977. Die Wirkung von D-Cycloserin auf die Biosynthese des Mureins. Thesis. University of Munich. 18. Seidl, P.H., P. Zwerenz, J.R. Golecki and K.H. Schleifer. 1985. Streptococcus pyogenes grown under sublethal concentrations of penicillin G accumulates close to the septum pentapeptide subunits of peptidoglycan. FEMS Microbiol. Lett. 30, 325-329.

SOLUBLE PEPTIDOGLYCANS: F O U N D IN M A N

A l l e n R.

LYMPHOCYTE-ACTIVATING

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cell

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bacteria

and

Biological Properties of P e p t i d o g l y c a n © 1 9 8 6 W a l t e r d e G r u y t e r & Co., Berlin • N e w York - Printed in G e r m a n y

its amino

1 shows

the

minimal

cell

t h a t had

been

penicillin

14C-a1 a n i n e .

and SPG

immobility,

amino

sensitivity.

the

glucose,

PG-synthetic

growth medium from Staphylococcus label

the

differentiate

incubated

is g e n e r a l l y

analysis

elution wall

of

for

of

SPG

minimal

cofactors

markedly

S P G c a n be d i s t i n g u i s h e d

In v i t ro, S P G antibiotic,

Seidl

review, the soluble

degradation

its p h y s i o l o g i c a l ,

D e t e c t i o n of

Dr.

as the h i g h m o l e c u l a r w e i g h t

PG-like

University,

knowledge

measure

reported

outset

by s o m e b a c t e r i a w h e n will

One

and

Our

progressed ago.

been

the

peptidog 1 yean SPG

Schleifer

Workshop.

has

years

Jefferson

SPG

the t o p i c of t h i s not

important

Dr.

this

polymers

over

is that

CSPG),

SPG.

to

in o r g a n i z i n g

Workshop then

a n d d e f i n i t i o n of

all

effort

PRODUCTS

Zeiger

Department of Biochemistry, Thomas Philadelphia, Pennsylvania 19107

We

BACTERIAL

G

(2).

146

3

in I O x

2

QQ 1

0 0

20

40

60

80

100

120

140

F R A C T I O N NUMBER F i g u r e 1. Fractionation through a Sephadex m i n i m a l cell w a l l m e d i u m (4 M C i o f L - ( 1 4 C ) a C2x10 CFU/ml) incubated with penicillin G 60 m i n . The first peak coincides with Elution was with distilled water. 4

G - 1 0 0 c o l u m n of spent 1 a n i n e ) f r o m S^. a u r e u s (50 u g / m l ) at 3 7 ° C f o r the void v o l u m e , VQ.

3

«

I o x

2

Q. Q 1

0 0

20

40

60

80

100

1 20

140

F R A C T I O N NUMBER Figure 2. Fractionation through a Sephadex G-100 column of 14C-alanine-1abe1ed SPG (peak 1 in Fig. 1) w h i c h had been digested overnight with lysozyme. Elution was with 0.15 M phosphate buffered saline (pH 7.2).

147 Figure gel

2

is an

following

migrated intact pletely

as

low

to SPG

profile

Figure of

S^.

weight

residues,

immobilized

of

digestion.

molecular

D-Ala-D-Ala

antibodies. labeled

elution 1ysozyme

the

same

SPG

through

At

least

85%

of

components.

SPG

has

vancomycin

3 shows aureus

been

or

an e l u t i o n through

to

By shown

the

virtue to

same now

of

bind

D-Ala-D-Ala

p r o f i l e of t h e a

the label

its com-

specific

14C-a1 a n i n e

vancomyc i n-Sepharose

4B

co1umn. 4

\ i o

Q. o

— O O O O OO0OOO0—I— 5 10 15

20

_°oU 25

FRACTION NUMBER Figure. 3. Fractionation through a vancomycin-Sepharose c o l u m n of 1 4 C - a 1 an i ne-1 abe 1 ed SPG f r o m j>. a u r e u s • The r e p r e s e n t s a c h a n g e f r o m 0.15 M p h o s p h a t e - b u f f e r e d s a l i n e 7 . 2 ) to N H ^ O H (pH 1 0 . 2 ) .

'tB arrow (pH

148 These

specificities

an e n z y m e - 1 i n k e d illustrated with

in

of

purified Ala-D-Ala)

SPG, rabbit 5

'

1

),

first

antibody

avidin

plus

allowed

Figure

vancomycin,

amount

have

immunosorbent the

Plastic fluid

antibody

species

biotlnylated

as

4 U

horseradish

biotin

SPG _i_ri v i v o whose

coated

)

n

steps an

case,

unknown affinity-

'—(A1 a - y - D - G 1 u - L y s - D -

Immunoglobulin Is

covalently

peroxidase

and

of

substrate.

SPG

Rabbit Anti- S P G

Biotinylated A n t i - R a b b i t IgG

E-Jlf-E

'ABC"

• O

Enzyme Substrate

#

•• o

Figure.

4.

Sequential

steps

in the ELISA

to detect

the

bound,

Vancomycin

O

by are

successively

containing

Cin this

against

which

of

(3)

are

urine

( G l u A l a

antibody to

(ELISA)

D-Ala-D-Ala

to

second

detection

wells

such

to

antibody a

the

assay

SPG.

149 Physiological SPG

is

properties

secreted

exposed

to

1uteus

CO,

(6-7).,

S.

by

aureus (9), SPG

gram-negative PG-turnover

(2), and

has

for

(12).

organism

has

been

incubation medium

by

penicillin

trigger

SPG

G,

amounts.

mecilllnam,

by

yet,

from

the

SPG.

St r.

no d e t e c t a b l e G,

SPG

reported in t h e

PG

this (6-7).

to h a v e

secreted

grown

of

no

in w h i c h

penicillin

is

Bac i 1 1 us

any

secreting

SPG

that

by

is

1 uteus

S e c r e t i o n of

cillin occurred

into

PG the

presence

of

pg/ml)

S^. a u r e u s

to

very

from

(2).

be

not (2)

cells

synthesis

on

the

the

penicillin-class amplcillin

mainly that

of

trigger

One

beta-

secretion

on

was

gram-negative

most the

beta-lactam secretion

of

cells.

of on

and

SPG

Is d e p e n d e n t

1 hr.

dependent

dependent

can

cefoxitin.

cause

act

did

In a p p r o x i m a t e l y

antibiotics

approximately

methlclllin and

to

to

different

Maximal

shown not

unable

reported

in

semisynthetic

( 4 ) or S^. a u r e u s

S P G by

cells

oxacillin,

Vancomycin

5 (2).

beta-lactam

cephalothin was

a mechanism (14).

of

S^. a u r e u s

include

like

which

antibiotics

(10-200

of

has been

no

As

is o n e w a y

to h a v e

of

hand, or

variety

like n a f c l l l i n ,

antibiotic

been

secreted

bacteria when

from

These

cephalosporins

although

a

secretion

antibiotics

also

(10).

source

reported

(8),

(10).

Besides

Figure

be

capable

little these

to

presence

on the other Yet,

a n a e robi s

faec i um

Streptococcus

differentiatab1e

the

reported

(10),

when

M i c rococcus

Streptococcus

1 i chen i form i s

bacterial

has been in

bacteria

include

(5),

pyogenes

are c l e a r l y

Yet,

(13).

pen icillin

in

bacteria

reported The

instance,

Escheri ch i a co1i,

S P G by M .

These Bac i 1 1 us

been

products

faec i um,

bacteria

gram-positive

Peptococcus

bacteria.

turnover

lactam

G.

of

Streptococcus

(11)

detectable

and

variety

penicillin

p n e u m o n i ae

same

a

SPG

Brev i b a c t e r i um d i var icatum

s u b t i1i s

turnover

of

SPG

in

Maximal the

on time 50

yg/ml

SPG

density

penicillin

G

as

shown

of

peni-

secretion of

has

bacteria

concentration

150 A. 12 I " 4.5 X 10®DPM 10 £ = 2.7 X 105DPM

x 5

£ = 3.8 X 10SDPM £ = 1.1 X 105DPM

I

V 20

J 20

40

J

L i L 40 20 40 FRACTION NUMBER •

I

20

L

40

F i g u r e . 5. F r a c t i o n a t i o n t h r o u g h a S e p h a d e x G - 1 0 0 c o l u m n of equal portions of s p e n t minimal c e l l , wall medium Ct vCI of L - ( 1 4 C ) - a l a n I n e ) f r o m S^ a u r e u s (2x10 CFU/ml) incubated with p e n i c i l l i n G ( 5 0 y g / m l ) at 3 7 ° C f o r d i f f e r e n t t i m e s . Only the Initial p e a k ( p e a k 1) f r o m e a c h i n c u b a t i o n is s h o w n . P a n e l A , 15 m i n ; Panel B, 30 m i n ; panel C, 60 m i n ; panel D, 120 m i n . T h e VQ is i n d i c a t e d in p a n e l A . T h e t o t a l r a d i o a c t i v i t y r e c o v e r e d in e a c h p e a k is s h o w n in e a c h p a n e l . Elution was with distilled w a t e r.

Studies (11)

have

cillin at

with

G

taining was of

shown

that

treatment.

least

filtered

S^. a u r e u s

four and

evidence

that PG.

s u b t ills

is one

generations

incorporated

derived

SPG In

Incubated

3H-labeled

synthesis

(2),

alanine

into

similar SPG

in

is

SPG to

not

St r.

synthesized

jde n o v o

S^. a u r e u s

in and

over a

and

experiment, the

minimal

that

(9)

presence cell

wall

penicillin a

120

minute

In F i g u r e

5.

degradation

following was

of Only

period This

peni-

grown

for

L-l'tC-a 1 a n I n e ,

growth

G.

p n e u m o n I ae

medium

3H-label

with

kinetics

provides

product

con-

the

of

cell

further wall-

151 Structure

of

Figure

6

depicts

ported

as

40,000

d

for

(5), (63

that

SPG

from

1 uteus

M. and

a

Str.

typical

d for St r. (4),

which

relatively

peptide

however,

results

in t h e

secreted

- NAG

of

lowering

to 5 pg/ml SPG

high

- NAM

SPG

SPG

from

to

modification to

of

be

Shockman of t h e

faec i um

f a e c i urn

lysozyme-

the

glycan

(>_10pg/ml),

highly, has

concentration

hydrolysis

re-

50,000

Str. be

concentrations

appears

been

B_r. d i v a r i c a t u m

shown

if

not

evidence,

from

50

D-Ala-D-Ala

vig/ml

sequence

(7).

NAG - NAM Ca)

- NAG - NAM - NAG | Ala I - y - DA(b,c) etc. I D-Al a I D-Al a

I Ala I D-Glu I (e)

has

(4) and

subt i 1 i s (9),

(4,7,9).

Str.

SPG

1uteus

been

little

the

in s o m e

-

have

of

M.

The B.

the p e n i c i l l i n

by

size

from

penicillin

uncross-1inked

that

The

(2),

(11)

indicates

portion

completely,

aureus

p n e u m o n i ae

sensitive, the

SPG. product

f a e c i urn ( 7 ) . S^

chain.

At

the

- NAM | Ala I etc.

(d)

NAG = N-acety1-D-g1ucosamine NAM = N-acety1-D-muramic acid DA = D i am i no acid Figure. There 6),

are

many

cell

Most

although

contain

The general

structural

of w h i c h

wall.

chain, (5)

some

6.

reflect SPG

the

amide the

SPG

SPG

from

not have

although (4).

appear

acid.

The to

However, been

respectively

in t h e

d i a m i n o p i m e 1 ic

tion

glycine

among

differences

have

a

at

acid. the from M.

SPG

reported

to

at

the

from have

"peptide

SPG

D-glutamic M.

1 uteus (4)

bridge"

S^. a u r e u s

(a-e

in t h e 3 of

and have

in

the

peptide

B_r. d i v a r i c a t u m either

a

acid

residue

is

peptidated

attached (2)

and and

Fig.

bacterial

at

a n d _B. s u b t i1 i s

pentaglycine bridge".

SPG. SPG

PG

position

Most

1uteus

"peptide

the

in t h e i r

lysine

of

f r o m j3. s u b t i 1 i s ( 9 )

a 1pha-carboxy 1 or 2,

differences

contain SPG

structure

to Str.

the

free posi-

with

a

(9)

do

diamino

faec i um

(7)

D-isoasparagine

152 The

muramic

fully to

SPG

only

from

knowledge, (15-16) been

shown

possible

from

substituted lysozyme, was

able

weight an

showed body in

eluant

seen all

all

peptide the

of and

peptide

further

does

been

been

Since

for

for

M.

PG

be

contrast acids

(4,9).

peptide

respective

in

to

muramic

To

described

PG

with

appear

is

reported

the

to

not

95%

of

about

Figure SPG

in

my St r.

M.

1uteus

1 uteus (18),

has

It

in t e r m s o f

Is

their

in

the

glycan

low

The

Into

of

among was

(20).

and

PG

a

chromatothat

SPG

bound The

by

studies an

first of

there

strands. antichange

tripeptide

in e l u a n t w a s

peptide.

This

from

The M.

to

with

arrangement

1 uteus

a

indicates

least m o n o s u b s t i t u t e d

multi-substituted. SPG

the

T4

(19),

molecular

chromatographic

change

the at

as

suggest

concentration

second

randomly

activity small

glycan

1ut e u s

sequences

either

for

This would

strands were the

of

M.

concentration

that most were

be

ha 1 f - u n s u b s t I t u t e d .

peptide

affinity

relatively

the g l y c a n strands

35%

from

D-Ala-D-Ala

higher

the

(19). 7,

to

substitution

unsubstItuted the

a

appear

substituted,

product

in

of

for

study.

This whose

peptide

not

have

peptide

Lys-D-Ala-D-Ala.

hundred-fold that

leaving

fully

was

containing

about

immobile

specific

have

their

half-fully requires

products,

as

with

substituted

1 uteus

nor

that

(7).

faec i um

s u b t i1i s.

(17).

poorly

to d i g e s t

However,

chains

enzymes

SPG m i m i c

M.

almost

St r.

substitution.

which

graphically was

be

from

and

amidases

these

the

peptide

SPG

peptide

s u b t ¡lis

to

that

SPG

substituted

L-alanine B.

of

1ut e u s

half

whereas and

overall

with M.

about

faec i um,

The

moieties

substituted

the

are

acid

must

of

await

153

12

16

20

TUBE NUMBER

15 _

B

10

&

5

12

16

20

TUBE NUMBER F i g u r e 7. Affinity chromatography on purified rabbit antibodies to s y n t h e t i c PG-precursor pentapeptide bound to S e p h a r o s e 4B. The top panel is a n e l u t i o n profile of 580, 000 c p m of l'+Ca 1 an i ne-1 abe 1 ed SPG f r o m M. 1 u t e u s a n d the b o t t o m panel is a n elution profile of 62, 000 c p m 1'tC-g 1 y e a n - 1 a b e 1 e d SPG from M. 1uteus. The f i r s t a r r o w r e p r e s e n t s a c h a n g e to 0.02 m M t - b u t y l o x y c a r b o n y 1 - L y s - D - A l a - D - A l a. The two pronged arrow represents a c h a n g e to 2 m M of t h i s t r i p e p t i d e .

Digestion left

of

10-15%

the

when

produced

and

14C-labeled

covalently not

the

containlng

that

Even

portions

that

no

their

sodium

the

non-SPG

was

of

properties

In

belong

glycerol

sulfate

from

an and

to

recently

the

the

the

affinity Shockman SPG

be

portion

from SPG

rhamnose-

biological

must

SPG

could

obtained

cova1ent1y-attached

to

aureus,

molecule

Barrett

precautions

lysozyme

acid-like dodecyl

investigating

SPG,

S^.

3H-labeled

attached

has

white

from

molecule

Although

laboratory

(22).

egg

SPG

teichoic

1.5% of

hen

The

containing

a

St r . f a e c i u m w i t h

properties the

had

with

CO.

vancomycin.

saccharide

immunological

1uteus

medium

(21).

two

(7), from

M.

material

alanine,

reported

faec i um

preparations

ensure

from

a minimal

containing

originally S t r.

in

attached

separate

column

SPG

undigested

taken of

and to the

154 molecule.

The

saccharide

complexes

this

precaution

a s to h o w SPG

review

the

of by

(23).

the Dr.

inflammatory Schwab

It r e m a i n s

structure

of

properties

should

reinforce

an o p e n

S P G will

field

influence

of the

for

PG-polyneed

for

investigation

their

properties.

in m a n

The

ELISA

man

(24).

mentioned

penicillin within pletely

6

used

to d e t e c t

volunteers

V,

seven

positive

hours

of t h e 1.

had

(test

abolished

Table

was

13 h e a l t h y

Lys-D-Ala-D-Ala none

above

Of

column

with

In

titers Table

a synthetic

(control

SPG

V

of

250 mg d o s e s

of

for

in

1).

Before

in U r i n e

Penicillin

urine

ingested

SPG

in t h e

SPG The

trlpeptide,

column).

pat i ents had d e t e c t a b l e Study of

that

urines

was

com-

t-buty1oxycarbony1 -

penicillin

levels of

ingestion,

in t h e i r

SPG

Samples S i x

Treatment

their

binding

urine.

Hours after

•

A b s o r b a n c e at

490

nm

Subject Sex

and Age

(yr)

M 41

Pre-- t r e a t m e n t

Post- treatment

Test

C o n t ro1

Test

.010

.009

. 043a

.011

a

.015

F 36

.014

.013

. 16 7

F 13

.010

.010

,110a

.009

.010

.013

M 40

C o n t ro1

.0 12 .012 a

.013

F 37

.010

. 007

. 157

F 50

.015

.017

. 042a

.011

M 36

.012

.012

.009

.011

M 27

.010

.013

.012

. 016

F 47

.011

. 014

. 2 3 33

. 021

M

34

.013

.012

.013

.012

M 36

.015

. 014

.013

.015

F 58

.010

.011

. 028a

.0 10

M

26

.007

.009

.008

.009

a

Considered

positive.

155 The

diversity

flects

what

bodies

in

observed

has

been

humans

(25-27).

the t w o o b s e r v a t i o n s SPG Dr.

has

many

Seidl

Figure

of

has

D-Ala-D-Ala

among

found

the

are

several

It

is

related.

criteria

already

portion

individuals

in

discussed

8, t i t e r s of a n t i b o d i e s

sera from patients with

SPG

the

a

anti-

speculate

that

is e v i d e n c e

that

natural

In a d d i t i o n ,

from

patients

who

Furthermore,

there

bodies

correlated

with

that

the

antibody caused

onset titers

of to

was

were

an

endocarditis

treated

increase

with

infection.

of

antibiotic Figure

D-Ala-D-Ala

with

in t h e

of

the

patients

in t h e in the (27).

these rather

time

with

antithan

course S^.

in

bacter-

but not

treatment,

9 shows

among

and

vancomycin

titers

the

as s e e n

to D - A l a - D - A l a a r e e l e v a t e d

S-. a u r e u s - c a u s e d

emia who were treated with beta-lactam antibiotics sera

immunogen.

immunodominance

in SPG.

re-

anti-PG

to

there

for

detection

of

interesting Indeed,

expected

so p r e v a l e n t

in

reports

of

aureus-

bacteremia.

NORMAL BLOOD DONORS (31)

VANCOMYCIN TREATED PATIENTS (14)

p- LACTAM ANTIBIOTIC TREATED PATIENTS (33)

F i g u r e 8. P e r c e n t a g e of b i n d i n g of P G - p r e c u r s o r a n t i g e n by h u m a n sera. T h e h o r i z o n t a l bar r e p r e s e n t s the a v e r a g e p e r c e n t a g e of binding. T h e v e r t i c a l line s h o w s the s t a n d a r d d e v i a t i o n .

156

60 - A. Rb NAFC

40 • •

20

••

•

0

I

I

I

1

1

60 - B. Rw

NAFC

40

•

•

•

•

• •

•

i o 20 z

S 5

o tr

> 0

60 -

1 1 C. Lu

1 1

1 1

|

|

1

40

1

•

20 t

m

• 1 1

0 60 "

OX AC, DAY 0 KEFLIN, DAY 12 KEFLEX, DAY 41

1 1

1 1

i 20

i 30

1 1

•

D. Wa

•

| 1

•

PEN

40

•

20 i 10

i 40

i 50

60

T I M E (days) F i g u r e 9. P e r c e n t a g e o f b i n d i n g of P G - p r e c u r s o r a n t i g e n by s e r a from four staphylococcal bacteremia patients at v a r i o u s times after the initiation of a n t i b i o t i c therapy. N A F C is n a f c i l l i n ; 0XAC is oxacillin; Keflin is cephalothin and Keflex is cephalexin.

At

least

SPG

three

found

genous

in

the

bacteria

appreciable (28). have

possibilities

On

the

SPG

D

other

There

can

colonization

group

secrete

urine. that

hand,

A

account may

be

secrete

by

streptococci

(28).

can

virtually

variation

of

a

SPG.

aureus

which

for

in the

have this

the

diversity

diversity Not

all

their

the

have tract

population

reported

possibility

is

able that

the

indi-

humans

intestinal

entire been

of

of

does to not

157 all

bacterial

amount

of

vitro The of

l o c a t i o n of the

seven were

played

a

across

the

individuals

is

in

host

active

to

The

exclude

host

or

include

genetic

the

the

first

the

random

and

(30). mice the of

Table

antibody

polymer.

for No

may

be

SPG

of

the

for

SPG

concentra-

more

vary

or

less

in

the

exposure

to

SPG-like

Peptidog 1 yeans,

with

synthetic

host

Dr.

poly

polymer

but

random

shows the

not

polymer the

poly

in

humoral (Glu^"

against In

the

H2-q H2-b

random to

in

were

the

observed

of

mice

Low

the

to

titers The

correlated

immunogenic In

the

inbred

obtained.

polymer

the

haplotype

response

polymer.

to

conCGlu^"

in m i c e

the

the

to

poly

in a n y

peptide

was

of

immunogenic

Ala^")

response

titers

of

the

begun

bound

and

and

noted

control

immunogenic

mice

is n o t

PG-precursor humoral

is

PG,

immunogens

Ala^)

to

above,

immunogens.

I have

covalently

(Glu^"

to

Krause

PG-like

pentapeptide

response

mentioned

(29).

Increase

of

possibilities

antibody

reasons

genetic

the

have

in m a n

possible

2

The

These

peptides

on

their

lysozyme may

response

random

the

as

the

titers obtained

found

b1eeds.

to

passage

PG-precursor

haplotypes,

(30).

in may

structure.

the host. such

Six

SPG-secretIng

early

carriers,

latter

the

its

macrophage

SPG

of

on

question

PG-precursor

higher that

d

The

the

immune

from

former

SPG

in

(2).

important. of

of

secretion.

diversity

PG-precursor

The

of

S^. a u r e u s

bacteria

but on

enzymes

the

with

investigating

sequence

Tyr*").

tract,

enzyme

the

the

to

location

same

SPG

further.

Workshop

of

this

taining

H2-b

of

response

examine

for

resulting

importance immune

or

levels

the

secrete

be

vaginal

interplay

diversity

control

tolerance

the

perhaps

cross-reactive

possibilities

PG

that

is d e p e n d e n t

the

secrete to

isolates of

positive

SPG-degrading

can

ability

bacteria may

intestinal

investigated

Antibodies

with

barrier

in o p s o n i z a t i o n

to be

clinical

suggesting

the

potential

organism

in t h e

Alternatively,

also cannot

need

given

SPG-secret1ng

the mucosal

tion of from

a

diversity

female,

role.

bacteria

One

of

Such

has been observed with

urine

At

strains

SPG.

with

random

secondary

158 Table

Dose

2.

I m m u n i z a t i o n of C 5 7 B L / 6 M i c e

CH-2b) with S 1 (A1a-y-D-Glu-Lys-D-Ala-D-Ala) .

(yg)

Bleed

Anti-pentapeptide

(%)

(Glu60

Ala't0)L n

Anti-carrier

C%)

100

primary

22.6 + 3.6

56.9 +

4.7

1

primary

14.6 + 8.0

24.5 +

14.0

100

secondary

19.2 + 5.5

60.8 +

8.2

1

secondary

7.5 + 6.3

The

results

with

the

same

immunogen against

3.

observed

in t h e s e H 2 - d m i c e at

mice.

The

Immunogen

humoral was

immunogen

at

primary

response similar

to

response

again

alone.

especially the

higher

titers

in T a b l e

Secondary

the

1 yg

dose

bleedings.

The

the

to w h a t

PG-precursor has

random polypeptide

Table

3.

been

In H 2 - d the

100

that

responses

to

mice

portion

reported

polymer

portion

of

random

the

of

for

be

higher

did

not

the the

shown

than

mount

were

in H 2 - b of

the

polymer

to the P G - p r e c u r s o r

appeared H2-q

is

peptide

1 yg d o s e s t h a n

random

to

Inbred m i c e

PG-precursor

yg a n d

to the

equivalent

33.8

portion those

an

immunogen.

This

immunogen1city

of

immune of

was the

alone.

I m m u n i z a t i o n of

BALB/c Mice

(H2-d) with CGlu 5 1 .

Ala

n

CA1a-y-D-Glu-Lys-D-Ala-D-Ala) Dose

(yg)

Bleed

Ant 1-pentapeptide

(%)

Anti-carrier

100

primary

36.5 + 10.9

69.5 +

4.5

1

primary

26.4 + 12.3

63.6 +

3.0

100

secondary

47.8 +

14.3

74.9 +

3.4

1

secondary

53.2 +

11.6

69.9 +

4.4

(%)

159 With

a

100

sera

yg

of

H2-b

appears

hapten

and

H2-d

for

the

that

to

of

the

the

one

the g l y c a n

and/or

As

previously

aureus

Infections,

of

PG w o u l d

above

diseases

indigenous important patient disease.

This

associated this

the

ant I-PG

has

done

not

been will

inspire

of

such

as i>.

rheumatoid serology

Some of

the

either

by

these

antibiotics.

Since

be

quite

capable

antibiotics, history

most

previous

with

In the

Often,

with

a

titers

the

titers

with

governed SPG.

infection,

therapeutic

anti-PG-tI ters

review

properties

observation of

should

to

from

murine ability inbred

as

of S^.

a

of

Is

of

the

particular studies

pathogenic

renewal

of

It

our

that

bacteria. efforts

in

can

of

be

its

properties

testimony

PG

are

aureus

SPG

SPG

SPG

study

lymphocytes of

of

that

the

biological stand

properties SPG

specify

anti-PG

the

field.

dimension trum

and

be

Importance.

to

had

classic

with

Thus,

beta-lactam

correlating

elevated

this

Biological The

know

to

organisms.

shown

to

the

with

and adult

beta-lactam

been

exposed

a

diseases,

to b a c t e r i a l

with

have

when

to

linked

as

relationship

juvenile

gram-negative

poly

In

what

analogy

of a

spondylarthritis.

treated

when

Perhaps,

been

or

SPG

reports

to v a r i o u s

to

acting By

associated

to be o f s o m e m e d i c a l

bacteria

secreting

Is

indicated

fever,

to

mice.

immunogenIcIty

are

sera

linked

similar

carrier.

have

rheumatic

have

are

also

in t h e s e

peptide

the

there

ankylosing

gram-positive

was

non-PG material

In h u m a n

appear

Infections

expect

any

reports

antl-SPG and

This

peptide

titers were observed

polymer

polypeptide

mentioned, These

of

arthritis,

mice.

would

titers

antibody

PG-precursor

SPG m o l e c u l e ,

sera.

PG-precursor

random

random

by

human

the

no d e t e c t a b l e

been observed It

dose

Tyr1"),

(Glu^"

has

of

the

related been

In

S^.

aureus

mice.

humans

cell

to

wall-derived

to

effects

Interactions

shown

capable act

gives

properties.

potential

_i_n v i t ro a n d J_n v i v o

from

and c o n g e n l c

to

found

biological

as

of

C31). a

of

with

PG

an

added

The

spec-

and

SPG.

lymphocytes.

Interacting Table

mitogen

MDP Many

4 In

with

shows

the

outbred,

160 Table

4.

Mitogenicity

of

SPG

splenocyte Add i -

Conen

t i on

BALB/c

Cyg/mO

cpm

None

f r o m S^

aureus

in m u r i n e

cultures. Swiss Webster

SI

a

cpm

4, 569

BIO .M

SI

cpm

SI

4, 888

6, 142

1

183,152

40.. 1

Not

LPS

30

111,301

24..4

134,037

2 1,.8

19,103

3.. 9

SPG

100

127,518

27..9

131,978

2 1..5

45,419

9.. 3

SPG

33

177,823

38,.9

119,439

19.. 5

25,010

5., 2

SPG

11

32,488

7. 1

19,254

3. 1

24,003

4,. 9

24,447

5 ,4 .

Con

A

SPG a

3.8

SI,

Stimulation

Table

5 shows

days

1 or

remove activity LPS

Treatment

(Table

indicating cell

that

5.

the

with

did

6).

not

The

murine

mitogens

Table

the m i t o g e n i c

lymphocytes

(another

B

Add i t i on

B cell

of

in e u 1 tu re

effect

Not

is g r e a t e r

anti-theta result

mitogen)

of

(Table

and

any

loss

SPG

gave

done

done

from

day

3

than

complement of

S^. a u r e u s

synergistic stimulated

to

mitogenic with

responses,

by t h e

two

7).

the M i t o g e n i c by

at

serum

in

subpopulations

are different Kinetics

Stimulation

SPG from

1

of

BALB/c

S^. a u r e u s .

2

3

Conen (yg/ml)

None Con

done

combination

cell

Splenocytes Day

Not

Not

index.

that

2.

T

done

cpm

SI

cpm

5,851

1. 9

5 8 , 109

5.. 5

150,371

9.. 6

36,239

3..4

126,886

8.. 1

22,049

2, . 1

SI

cpm

SI

3, 169 A

1

LPS

30

8,549

2 .7

SPG

100

3,532

1 .1

SPG

50

SPG

25

Not done 2,319

0 .7

Not

done

24,097

2. 3

85,782

5. 5

101,125

6.. 5

33,635

2. 2

B

161 Table

6.

Mitogenicity Depleted

of

S P G f r o m JS. a u r e u s

Swiss Webster Complement

Add!-

Conen

tion

on 1 y

Cyg/ml)

+

ant i -6 SI

cpm

1,478

C o n .A

T-cell

Complement

a

cpm

None

in

Cultures.

SI

2,069

1

98,000

66.3

5,522

2.7

LPS

34

12,500

8.5

75,963

36.7

SPG

25

14,674

9.9

24,108

11.7

3

k

C o m p 1 e m e n t o n 1 y resu1 t e d

in a 10%

C o m p l e m e n t + a n t i-6 C a n t i- t h e t a of

loss of

c e l l u l a r v i ab i1 1ty.

serum) resulted

i n a 45%

loss

c e l l u l a r v i a b i1 Ity.

Table Thereof

7.

Effects

on the

Add i t i on

3H-TdR

None

M itogen

cpm

None

of

B Lymphocyte Incorporation C10

SPG

Mitogens by

Combinations

Swiss Webster

ug/ml) add i t i ve

cpm

and

LPS cpm

Splenocytes. C10

ug/ml)

a d d i t i ve

5,333

SPG

11,030

LPS

27,124

46, 0 9 6

CT-G-A-G)

20,049

26,452

Dext ran

17,212

21,077

36,218

23,598

a

46,096

32,821

25,746

57,304

23,800

22,909

61,696

39,003

41,915

72,510

58,009

32,821

sulfate

a

Poly

I.

Poly

C

Underlined

v a l u e s s i g n i fy

ag r e a t e r

than add i t i ve response.

162 SPG

from

Table

S^

aureus

8 shows

secreting blood

the

cells.

cells

immunoglobulin shows

that also

globulin the

SPG has

Antibody

hand

will

a

increases

are

column

are

again,

Effect

of

SPG

effect

to t h e

LPS

Conen

11 o n

(yg/ml)

1

2

None

0 30

5,250

000

SPG

33

7,750

5,250

PG

ND

400

a

ND,

k

The c o n c e n t r a t i o n

The

Not

to p l a s m a c e l l s 100 were

the

pg o f

PBA.

SPG

number

cells

from

of

immuno-

a n d no p r o t e i n A rather

to

In

SRBC.

coupling

significant

cells.

PFC R e s p o n s e

of

Cultures. eel 1 s IgM

IgG

(expt

1) C e x p t

1) C e x p t

2) ( e x p t

52

1,603 NDa

ND

S^.

as w e l l .

immune

secreting

a

column

1,081

ND

2) 17

ND

ND

49,725

4, 492

38,076b

50,808

7,814

73,747

6,263b 17,956

determined.

proliferation

9 shows

as

hand

IgG

0

LPS

giving

nearby left

IgM

Expt

Expt

red

SRBC

a u r e u s on the

Splenocyte

sheep

secreting

the

PFC/106 Addi-

A-coupled

lymphocytes

produce

immunoglobulin

(PBA).

Immunoglobulin-

splenocytes

with

SRBC

activator

of

in t h e

on

immunized

from

BALB/c

the

equivalent

SPG and

of

of

results

directly

Once

protein

The data

both

among

number

Those

lysis

stimulatory

In t h e n u m b e r

8.

a

B cell

the

utilizes

field.

cells

bind

is n e c e s s a r y .

Table

LPS

secreting

right

SRBC

and

polyclonal

SPG on

complement.

cause

the

a

of

assay

and

will

plaque on

also

The

(SRBC)

clear aureus

is

effect

of

of m u r i n e

by

SPG

results of

SPG

or

examined.

mitogens

and

compared

to t h e

SPG for

LPS. SPG

B cell

this

experiment

splenocytes

f r o m S^. a u r e u s iv

inoculations

After

and

three

LPS w e r e

activators,

control.

or

and their

75

pg/ml.

differentiation

a l s o o c c u r s _i_n v i v o . of

Swiss Webster

four

equivalent giving

was

about

days,

the

In t h e i r fivefold

mice

Table with

splenocytes abilities

as

stimulation

163 Table

9.

J_n V i v o

Prol i f e r a t i o n

Lymphocytes with Treat-

Animal

Cells/spleen

ment 1 C o n t ro1

SPG

LPS

st i m u 1 a t e s. factor

SPG as

Further uate

the

term

or

of M u r i n e

PFC/2

cells)

1, 147
-4) -ff-acety lmuramyl-Lalanyl-D-isoglutamine, or GMDP, has been chemically synthesized (1,2). It was found to possess a higher immunoadjuvant activity in comparison to that of MDP (Ai-acetylmuramyl-L-alanyl-D-isoglutamine)

(3). Recently GMDP has been obtained

by an enzymatic degradation of the peptidoglycan of Actinomaduva

R 39 (4). The

structure of the peptidoglycan was demonstrated in (5) and is given in Fig. 1. The peptidoglycan was successively hydrolyzed in jT] by the lysozyme, in [T| by the DD-carboxypeptidase G from Streptomyces albus G and in [3] by the Y - D - g l u ~ tamyl-meso-diaminopimelate endopeptidase I from Bacillus sphaerieus 9602. The enzymes were used in their soluble form.

V

Mur NAc

/

0

Glc NAc X

^/rNAc

ms Azpm

Glc NAc

MurNAc MurNAc

m

Q ] Glc NAc

GlcNAc I ^ Q ] GlcNAc L-Ala-D-GluNHj

Qlpr Glc NAc L-Ala-D-GluNHj H ?

m / Glc NAc

«

I3J "

L-Ala-D-GluNH, I3JD-Ala

j_

ms A2pm

ms Azpm

H]

Fig. 1 : Peptidoglycan of Ae.tinomadura R 39 (from M. Guinand et al. 4).

Biological Properties of P e p t i d o g l y c a n © 1986 Walter d e G r u y t e r & Co., Berlin N e w York - Printed in G e r m a n y

390 We managed to improve the method by immobilization of two of the enzymes, DDcarboxypeptidase G and endopeptidase I, and we produced substantial amounts of GMDP and of related glycopeptides whose chemical synthesis is difficult if not impossible (submitted for publication). A survey of the method is now reported as well as a preliminary study of the biological properties of the glycopeptides.

Immobilization of DD-carboxypeptidase G and properties of the bioreactor. The DD-carboxypeptidase G (EC 3.4.17.8) was purified as described in (6) and 12 units of enzyme were obtained from 25 liters of Streptomyces

albus G. They were

immobilized on Ultrogel AcA 22 treated with glutaraldehyde as recommended in (7); two reactors were prepared : the first one was saturated in enzyme with 0.9 unit of DD-carboxypeptidase G linked to 1 ml of gel and it was used to study the properties of the immobilized enzyme. The second reactor was made with 11.1 units of DD-carboxypeptidase G immobilized on 60 ml of gel ; it was packed inacolumn (2.6 x 12 cm) and it was used for the digestion of the peptidoglycan. The properties of the saturated gel were studied with Ac2 _ L-Lys-D-Ala-D-Ala as substrate. The heat stability of immobilized DD-carboxypeptidase G was examined in comparison with that of the free enzyme. At the inactivation temperature, where the remaining activity is half of the original activity,the immobilized enzyme is more stable than the free enzyme by 10°C. It was also found that the immobilized enzyme retained roughly its initial activity even after one year. The kinetic constant, K' > of the immobilized enzyme was studied at 37°C in a small packed bed reactor of 0.3 ml. Various S q concentrations of Ac^-L-Lys-D-AlaD-Ala we're pulsed through the column at various flow rates and samples were taken out for enzymatic assays of released D-Ala (8) until the maximum substrate conversion rate was obtained. K ' m has been determined according to the MichaelisMenten equation : P . S n

o

- K1

m

. Log(1-P) = V / V (9). S is the initial con6 m s o

centration of substrate and P the maximum substrate conversion rate. The different P . S q datas were plotted versus - Log(1-P). Straight and parallel lines were obtained at the different space velocities and a K' m of 2 mM was calculated from the slopes. It is only 6 times greater than that for the free enzyme (10). Thus the properties of the immobilized DD-carboxypeptidase G (stability towards heat and time as well as kinetic constants) are all suitable to its permanent use as a bioreactor, at any flow rates experimented.

391 Utilization of the immobilized DD-carboxypeptidase G Over a culture of 70 1 of Actinomadura R 39, 7 g of cell walls were obtained as described in (4). They were solubilized by the lysozyme by portion of 1 g and the solutions were separately pulsed and recycled through the reactor thermostated at 37°C at a flow rate of 6 cm.h

Samples have been analysed for amino ter-

minal groups (11) until the completion of the reaction. The products were then eluted and separated through Sephadex G50-G25 columns according to a classical procedure. A disaccharide-peptide monomer (K av 0.60) and a disaccharide-peptide dimer (K^O.42) were subsequently desalted on Trisacryl GF 05. From the 7 g of walls the yield was 750 mg of monomer and 250 mg of dimer. The monomer or GMTP : N-acetyl-B_D-glucosaminyl-( 1->-4)-N-acetylmuramyl-L-alanyl-D-isoglutaminyl-(L) meeo-diaminop ime lyl(L)-(D-alanine) was shown to be a mixture of disaccharidetetrapeptide (87%) and disaccharide-tripeptide (13%) ; the dimer, di-GMTP, possess an osidic linkage (MurNAc-KJlcNac) between two moles of GMTP.

Immobilization of endopeptidase I and properties of the

immobilized enzyme

The endopeptidase I was obtained from 100 liters of Bacillus sphaerieus. The enzyme was extracted from the sporulation medium and from the spores and was purified as described in (12). Five units of enzyme were obtained. We previously demonstrated that endopeptidase I was firmly bound to phenyl-Sepharose by hydrophobic interactions(12) ; the purified enzyme solution was then simply loaded at pH 8 on a phenyl-Sepharose column (2.6 x 12 cm) and the properties of the gel were studied with GMTP as substrate. The conversion of GMTP into GMDP was analysed by quantitative reversed phase HPLC in an ammonium phosphate buffer at pH 5.5. The inactivation temperature which is already very high, 80°C, for the free enzyme is maintained but not enhanced by immobilization. The time stability is constant over several months. A K' of 2.5 mM was determined as described m above in a small packed bed reactor of 0.3 ml. It is smaller than that (4.34 mM) for the free enzyme. This oan be due to the fact that endopeptidase I is naturally particulate and acts better in its immobilized form than in its soluble form. So, the immobilization of endopeptidase I is particularly compatible for its permanent use.

Production of glyco-dipeptides with immobilized endopeptidase I The compounds GMTP (256 mg) or di-GMTP (151 mg) were pulsed and recycled through

392 the reactor of endopeptidase I at a flow rate of 6 cm.h ^. Aliquots w e r e

remo-

v e d and analysed by HPLC until the completion of the reaction. The purification of the resulting compound GMDP or di-GMDP was

achieved by chromatography in w a -

ter o n a Dowex-50 (H + ) column as described previously (4). The yield was 136 m g of GMDP and 95 m g of di-GMDP.

Biological

activities

In the present paper two assays are only reported : the phagocytic activity of macrophages (Table 1)and the antibody

Table 1

production in mice'

Effect of Glycopeptides o n Carbon Clearance by System of Mice*.

Material tested

Amount m g / kg

Phagocytic index(.10 - 4)

(Table 2).

Reticuloendothelial

T95 SEM (.10-4)

Statistical significance

Control MDP MDP

200 2.000

272 378 579

38 115 109

NS S

Control MDP MDP

200 2.000

293 410 715

117 77 88

NS S

Control GMDP GMDP

200 2.000

272 405 459

38 59 92

S S

Control GMDP GMDP

200 2.000

293 443 505

117 67 100

200 2.000

297 213 436

51 74 1 10

Control GMTP GMTP

200 2.000

360 291 436

80 90 120

NS NS

Control di-GMDP di-GMDP

200 2.000

293 531 357

117 90 99

NS

Control di-GMDP di-GMDP

200 2.000

297 297 353

51 51 74

NS NS

Coritrol di-GMTP di-GMTP

200 2.000

360 216 279

80 40 40

S NS

Control GMDP GMDP

- The amount of injected carbon is 80 mg/kg - E a c h assay was performed o n 10 male mice (24 to 26 g).

S

s NS

s

s

393 The phagocytic activity of macrophages was assayed by measuring the rate of clearance of colloidal carbon by the reticuloendothelial system according to the method of Biozzi et at. (13). The phagocytic index K was calculated and K was statistically analysed using Student's test. The compound GMDP seemed to cause a significant increase in the phagocytic activity of macrophages as compared with the injection of MDP. The antibody

production was studied with the plaque-forming-cell (PFC) antibo-

dy response of mice and

the results were statistically analysed using Student's

test. They are presented in Table 2. Only the compound di-GMDP seemed to be an activator of the antibody Table 2

production.

Plaque-Torming-Gell Antibody Response of Mice to the Glycopeptides

Compound

Amount pg/kg

Number of plaques/spleen (mean)

795 SEM

Statistical significance

Control MDP MDP

500 5..000

71 .404 . 120.,301 61 ,545 .

17..762 21..697 13.,639

S NS

Control MDP MDP MDP

50 500 5..000

89.,036 95.,129 93..563 99..439

36.. 139 28..218 36.,860 38..068

NS NS NS

Control GMDP GMDP GMDP

50 500 5..000

73.,131 125.,089 89..684 101..877

22..759 32..689 37..213 29..581

S NS NS

Control GMDP GMDP

500 5..000

71..404 120..745 74.,890

17..762 19..657 14..111

S NS

Control GMTP GMTP GMTP

50 500 5..000

64..611 101..853 94..451 80..473

16..205 26..960 35..514 38..208

S NS NS

Control di-GMDP di-GMDP di-GMDP

50 500 5..000

66..562 123.,878 127..228 151..911

24,.406 41 .838 , 37,.310 34,.950

S S S

Control di-GMTP di-GMTP di-GMTP

50 500 5 .000

64..611 70.478 107,.678 122.,592

16,.205 32,.737 33,.158 45..113

NS S NS

Each assay was performed on 10 female mice (20 to 22 g)

*

394 In conclusion, we managed to perfect a system of bioreactors. It can be used for the production of large amounts of various glycopeptides from bacterial cell-walls. Preliminary assays have shown the interest of those glycopeptides which possess various biological activities.

Acknowledgement Support for research grants from CNRS (UA 528) is gratefully acknowledged.

References 1.

Kusumoto, S., Yamamoto, K. , Shiba, T. (1978) Tetrahedron Lett.45,4407-4410.

2.

Durette, P.L. , Meitzner, E.P., Shen, T.Y. (1979) Carbohydr.Res.77, C1-C4.

3.

Tsujimoto, M. Kinoshita, F. Okunnaga, T., Kotani, S., Kusumoto, S., Yamamoto, K., Shiba, J. (1979) Microbiol. Immunol. 23, 933-936.

4.

Guinand, M., Françon, A.,Vacheron, M.J., Michel G., (1984), Eur. J.Biochem., 143, 359-362.

5.

Ghuysen, J.M., Leyh-Bouille, M., Campbell, J.M., Moreno, R., Frère, J.M., Duez, C., Nieto, M. , Perkins, H.R. (1973) Biochemistry, 2_2, 1243-1251.

6.

Duez, C., Frère, J.M., Geurts, F., Ghuysen, J.M., Dierickx, L., Delcambe, L. , (1978), Biochem. J., 175, 793-800.

7.

Weston, P.D., Avrameas, S., (1971), Biochem., Biophys. Res. Commun. 45, 1574-1580.

8.

De Cohen, J., Lamotte-Brasseur, J., Ghuysen, J.M., Frère, J.M., Perkins, H.R., (1981), Eur. J. Biochem., 121, 221-232.

9.

Tosa,T., Mori ,T., Chibata, I., (1971), J. Ferment, Technol. ,

522-528.

10.

Leyh-Bouille, M., Ghuysen, J.M., Bonaly, R., Nieto, M. , Perkins, H.R., Schleifer, K.H. , Kandler, 0., (1970), Biochemistry,: 9, 2961-2970.

11.

Ghuysen, J.M., Tipper, D.J., Strominger, J.L., (1966). In Methods in Enzymology, (Neufeld, E.T. and Ginsburg V. ed.) Vol. 8, pp.685-699, Academic Press, New-York.

12.

Garnier, M., Vacheron, M.J., Guinand, M., Michel, G., (1985), Eur. J. Biochem., J«S, 539-543.

13.

Biozzi, G., Bennacerraf, 34, 441-457.

B., Halpern, B.N. (1953) Brit. J. Exp. Pathol.,

ADJUVANT ACTIVE PEPTIDOGLYCANS

I N D U C E T H E S E C R E T I O N O F A C Y T O T O X I C F A C T O R BY

MACROPHAGES

F . V a c h e r o n , M. G u e n o u n o u ,

C.

Laboratoire de Microbiologie, Garches, France

Nauciel F a c u l t é de M é d e c i n e de P a r i s - O u e s t ,

92380

Introduction.

Previous studies have shown that peptidoglycans on immune responses growth

in r a t (2) a n d m o u s e

relationship

b e t w e e n the s t r u c t u r e o f P G a n d t h e i r

the P G - i n d u c e d r e s i s t a n c e

to

effect

tumor

(3). It h a s a l s o b e e n o b s e r v e d t h a t t h e r e is a

(3, 4 ) . T h e a i m o f the p r e s e n t s t u d y w a s

Materials and

(PG) e x e r t a n a d j u v a n t

(1) a n d c a n s t i m u l a t e n o n s p e c i f i c r e s i s t a n c e

to t u m o r

immunomodulating

properties

to a n a l y s e the r o l e o f m a c r o p h a g e s

in

growth.

Methods

Activating agents. PG from Bacillus megaterium, Staphylococcus aureus, M i c r o c o c c u s lysodeikticus a n d Corynebact.erium p o i n s e t t i a e w e r e p u r i f i e d as p r e v i o u s l y d e s c r i b e d (4). P e p t i d e s u b u n i t s t r u c t u r e s a r e r e p o r t e d in T a b l e 1. L i p o p o l y s a c c h a r i d e (LPS) from S a l m o n e l l a typhimurium w a s purchased from D i f c o Laboratories. Macrophage cytotoxic activity. P e r i t o n e a l e x s u d a t e c e l l s (PEC) w e r e h a r v e s t e d f r o m 7 to 10 w e e k s o l d D B A / 2 m i c e , 3 d a y s a f t e r the i n t r a - p e r i t o n e a l i n j e c t i o n o f 2 m l o f t h i o g l y c o l l a t e b r g t h . P E C w e r e w a s h e d w i t h H a n k s b a l a n c e d s o l u t i o n ( H B S S ) a d j u s t e d at 1 . 2 5 x 10 /ml in t i s s u e cult.ure m e d i u m (TCM) c o n s i s t i n g o f R P M I 1 6 4 0 s u p p l e m e n t e d w i t h 2 m M glut.amine, a n t i b i o t i c s a n d 10 % f o e t a l c a l f s e r u m (FCS). A v o l u m e o f 0 . 2 ml o f c e l l s u s p e n s i o n w a s d i s t r i b u t e d in 9 6 - w e l l f l a t - b o t t o m e d m i c r o t i t e r p l a t e s . A f t e r a 2 h i n c u b a t i o n at 3 7 ° C , n o n a d h e r e n t c e l l s w e r e r e m o v e d by 3 w a s h e s w i t h H B S S . P 8 1 5 m a s t o c y t o m a c e l l s , m a i n t a i n e d by s e r i a l p a s s a g e s in the p e r i t o n e a l c a v i t y of D B A / 2 m i c e , w e r e w a s h e d in H B S S a n d a d j u s t e d at 1 . 2 5 x 10 /'ml in T C M . T h e n 0 . 2 ml o f this c e l l s u s p e n s i o n w a s a d d e d to the m a c r o p h a g e m o n o l a y e r s . C u l t u r e s ^ e r e i n c u b a t e d a t 3 7 ° C in a 5 % C O ^ a t m o s p h e r e for 24 h . T r i t i a t e d t h y m i d i n e ( H - t h y m i d i n e , 0 . 2 jaCi p e r w e l l ) w a s a d d e d f o r the f i n a l 4 h of i n c u b a t i o n a n d P 8 1 5 c e l l s w e r e c o l l e c t e d w i t h an a u t o m a t i c h a r v e s t e r . I n c o r p o r a t e d r a d i o a c t i v i t y w a s m e a s u r e d by l i q u i d s c i n t i l l a t i o n s p e c t r o p h o t o m e t r y . E a c h d e t e r m i n a t i o n w a s c a r r i e d o u t in t r i p l i c a t e .

Biological Properties of Peptidoglycan © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

396 Table 1. Peptide Subunit Structure of PG

Bacterium

Peptide sequence

B. megaterium

L-Ala-D-Glu-m-A2pm-D-Ala

S. aureus

L-Ala-D-Glu(NH 2 )-L-Lys-D-Ala

M.

lysodeikticus

C. poinsettiae

L-Ala-D-Glu(Gly)-L-Lys-D-Ala Gly-D-Glu-L-Hsr-D-Ala

Cytotoxic factor on. PEC adjusted at 0^5 x 10 /ml in TCM were plated in 16-mm wells (0.5 ml per well) or in 25 cm flasks (5 ml per flask) and incubated for 2 h at 37°C. Nonadherent. cells were removed by washing. Adherent cells were cultured in serumfree RPMI 1640 medium containing 25 mM HEPES. Various concentrations of activating agents were added and culture supernatants were harvested 24 h later. The cytotoxic activity of supernatants was assayed on L - 9 2 9 cells. Target cells in TCM were seeded in microtiter plates (40,000 cells/well) in the presence of actinomycin D (1 yg/ml) and various dilutions of supernatants in a final volume of 0.2 ml. In control wells target cells were incubated w i t h TCM instead of macrophage supernatant. After 18 h of incubation at 37°C, the nonadherent (dead) cells were removed by washing and the remaining adherent cells were stained with crystal violet (5). Absorbance was read at 545 nm . Each determination was carried out in triplicate. Cytotoxic factor characterization. A sample of 100 ml of supernatants from macrophage cultures stimulated with 100 jig/ml of PG from B. megaterium was concentrated by ammonium sulfate precipitation (at a saturation of 80 %) dialysed against 0.1 M Tris-HCl buffer (pH 7.3) applied to a column of Ultrogel AcA 54 (Pharmacia) (2.6 x 90 cm) and eluted w i t h the same buffer. Fractions of 7.5 ml were collected, sterilized by filtration, diluted 1:50 in TCM and tested for cytotoxic activity on L-929 cells. Another sample of concentrated supernatants was dialysed against 0.025 M imidazole-HCl buffer (pH 7.4) and fractionated by chromatofocusing on a PBE 94 column (Pharmacia) (1 x 35 cm) with 300 ml of polybuffer 74 pH 7.4. Fractions of 7.5 ml were collected, dialysed against RPMI 1640, sterilized by filtration, diluted 1:4 in TCM and assayed for cytotoxic activity.

Results

Activation of macrophages for cytotoxicity by PG. Thioglycollate induced macrophages were not cytotoxic for P815 cells. In the presence of PG they inhibit the growth of the target cells. PG from B. megaterium and S. aureus were more active than PG from M. lysodeikt.icus and C. poinsettiae (Table 2).

397 Table 2 Growth Inhibition of P815 Mastocytoma Cells by PG-Stimulated Macrophages

Macrophages

PG

/ i fJg/ml

H-thymidine incorporation

,„. (% growth

-

-

-

19,088 + 2,724

+

-

-

19,606 + 3,505 18,339 + 1,115

\ inhibition)

-

B. megaterium

100

+

B. megaterium

10

5,433 + 735

(71)

+

B. megaterium

100

2,654 + 345

(86)

(4)

+

S. aureus

10

3,485 + 472

(82)

+

S. aureus

100

461 + 135

(97)

+

M. lysodeikticus

10

+

M. lysodeikticus

100

12,550 + 1,577 (34)

C. poinsettiae

10

12,707 + 2,650 (33)

C. poinsettiae

100

7,121 + 1,628 (62)

+

19,539 + 2,023

(0)

Release of a cytotoxic factor by macrophages stimulated by PG. Macrophages incubated with 1 to 10 |ig/ml of PG from B. megaterium or S. aureus released cytotoxic activity in supernatants. PG from M. lysodeikticus

and

C• poinsettiae were active only at higher concentration (100 jig/ml) and the level of activity released w a s low (Table 3).

Properties of the cytotoxic factor. The factor was not inactivated by heating at 56°C for 30 min, but destroyed at 80°C. Cytotoxic activity w a s not inhibited by serum or trypsin inhibitors such as soja bean trypsin inhibitor. The cytotoxic factor was however not stable in the presence of serum. After gel filtration a major peak of cytotoxic activity w a s observed in the range of 60-70,000 daltons (Fig. 1). After chromatofocusing a single peak of activity was present at pH 4.8.

Discussion.

The data presented here show that PG activate the cytotoxic activity of

398

Table 3. Cytotoxic Activity on L-929 Cells

of Supernatants from Macrophage

Cultures Stimulated by PG or LPS.

Stimulant -

B. megaterium PG

S. aureus PG

M. lysodeikticus PG

C. poinsettiae PG

LPS

jjg/ml -

% cytotoxicity

*

0

1

74

10

92

100

89

1

0

10

84

100

93

1

0

10

2

100

25

1

0

10

2

100

32

0.1

77

1

88

* Supernatants were assayed at 1:10 dilution

macrophages and induce the release of a cytotoxic factor. PG from B. megaterium and S• aureus were very active. Previous studies have shown that these preparations exerted immunoenhancing activities, including the ability to inhibit tumor growth (3, 4). Adjuvant inactive PG from M. lysodeikticus or C. poinsettiae have only a slight effect on macrophage cytotoxicity. Several studies have shown that macrophages can release cytotoxic factors after stimulation by various inducers such as LPS. Different factors have been described. Some of them are low molecular weight components : oxygen metabolites (6), thymidine (7) or C3a (8). Arginase is of a molecular weight higher than the factor described in the present study (9). Factors with protease activity have also been reported (10). Unlike these factors the cytotoxic factor we have found is not inhibited by serum or trypsin inhibitors. It shares several properties with a factor released by macrophages after induction by calcium ionophore (11) or BCG and LPS (12). Its relationship with tumor necrosis factor has been suggested (12).

399 Figure 1. Ultrogel AcA 54 Chromatography of Cytotoxic Factor

BSA I

OVA I

FRACTION

CHYM i

NUMBER

Molecular weight markers were bovine serum albumin (BSA), ovalbumin (OVA) and chymotrypsinogen (CHYM)

The ability of PG from some bacterial species to induce the secretion of a cytotoxic factor by macrophages may explain, at least in part, their activity against experimental tumors. Furthermore our results confirm that macrophages are an important target of PG (13).

References

1. Nauciel, C., J. Fleck, J.P. Martin, M. Mock, H. Nguyen-Huy. 1974. Eur.J. Immunol. 4, 352. 2. Nauciel, C., A.F. Goguel. 1977. J.Natl.Cancer Inst. 59, 1723. 3. Goguel, A.F., G. Lespinats, C. Nauciel. 1982. J.Natl.Cancer Inst. 68, 657.

400 4. Guenounou, M., A.F. Goguel, C. Nauciel. 1982. Ann.Immunol.(Paris).

133D, 3.

5. Fish, H . t G.E. Gifford. 1980. J.Immunol.Methods. 57, 311. 6. Nathan, C.F., S.C. Silverstein, L.H. BrUhner, Z.A. Cohn. 1979. J.Exp.Med. 149, 100. 7. Opitz, H.G., D. Niethammer, R.C. Jackson, H. Lemke, R. Huguet, H.D. Flad. 1975. Cell.Immunol. 18, 70. 8. Ferluga, J., H.U. Schorlemmer, L.C. Baptista, A.C. Allison. 1978. Clin.Exp. Immunol. 31, 512. 9. Currie, G.A. 1978. Nature. 273, 758. 10. Reidarson, T.H., G.A. Granger, J. Klostergaard. 1982. J.Natl.Cancer 69, 889.

Inst.

11. Drysdale, B., C.M. Zacharchuck, H.S. Shin. 1983. J.Immunol. 131, 2362. 12. Männel, D.N., R.N. Moore, S.E. Mergenhagen. 1980. Infect.Immun. 30, 523. 13. Vacheron, F., M. Guenounou, C. Nauciel, 1983. Infect. Immun. 42, 1049.

STIMULATION AND

OF N O N S P E C I F I C

BACTERIAL

TREHALOSE

RESISTANCE

BY M U R A M Y L

AGAINST

AEROGENIC

DIPEPTIDE

VIRAL

COMBINED

WITH

DIMYCOLATE

K.N. Masihi, Robert

INFECTIONS

Koch

W. B r e h m e r , Institute,

W.

Lange

Federal

Health

Office,

1000 Berlin 65, F.R.G.

Introduction There

are a m u l t i t u d e

animals.

Many

rubella,

rabies,

ted by ces,

of i n f e c t i o n s

infectious mumps,

and m o r t a l i t y . or a c c e p t a b l e for many

still

It is d i f f i c u l t

infections

like

factory.

For

instance,

the

inherent

problems.

face

antigens

of the

of the

vaccine

possessing

WHO

report

influenza

tion light host

completely

(No. 6 9 8 , epidemics

achieved...

of d e v e l o p i n g of this

defence

Biological

emphasis

vaccines

different mechanisms

avenue

response

against for

modifiers

u s e d as i m m u n o s t i m u l a n t s

of

this, presur-

annual

influenza when A

yet

on the

specificity

viru-

recent

control

h a s not

of

or v a r i a n t s " .

of e n h a n c i n g

infections

satis-

of the an

appear.

be p l a c e d

A viruses

the c o n c e p t

future serotypes

viruses

drift

"effective

infections

should

vaccines

to

situation

antigens

that

100

necessitates

The e f f i c a c y

advan-

morbidity

near

over

influenza

with a wide

influenza

background,

and an a t t r a c t i v e

stated

individual

of

not c o m p l e t e l y

in a s h i f t

different

1983) or

Particular

against

limited

preven-

specific

in the

antigenic

viruses

composition.

is p a r t i c u l a r l y

ses

frequent

influenza

source

that

domestic fever,

major

In a d d i t i o n

are

of the

and

yellow

B can be

with

23 t y p e s .

vaccines

man

despite

an i m p o r t a n t

nature

The

but

rhinoviruses

viral

sent

vaccines

and h e p a t i t i s

be d e v e l o p e d

B viruses with

of the c u r r e n t

affect

smallpox,

to e n v i s a g e

will

the

which

like

vaccines,

constitute

chemotherapy

or the c o x s a c k i e certain

bility

measles,

the use of a p p r o p r i a t e

infections

review

diseases

of been

feasiprotecIn

offers

an

alternative

origin

are

widely

of h o s t

immune

research. of b a c t e r i a l

to e n h a n c e

a variety

the

nonspecific

Biological Properties of Peptidoglycan © 1986 Walter de Gruyter & Co., Berlin • New Y o r k - Printed in Germany

402 responses. teria, tion

Detailed

of t h e i r

cell

moieties.

The

vanticity

is t h e

u/alls are

structure

structure

glycolipids

the

to

nonspecific

virus

NMRI

and

mice

pretreated

treatment

and

employed,

no

was

observed

nor

TDM,

were long

infection.

1).

restrict

the

slightly

increased

the

in were

adjuthis

on

of t r e h a l o s e .

We

response

modifiers

aerogenic

influenza

and

obtained

combination

with

A greater

to

of

TDM w i t h

desmethyl-MDP.

of

of

mouse-viru-

between MDP

virus

the

MDP

of the

aerosol against

or

on of

analogs

protection

tested, the

bacteria.

two

were

infections influenza of

virusM.tuin

of c o l o n i e s

analogs

preparations

lipophilic

analog infection

multiplication number

pre-

that were

multiplication both

the

organisms.

of the

a mixture

6-0- acyl

degree

or

of M. t u b e r c u l o s i s ;

reduced

when

TDM,

aerosol

analogs

protection

the

only

an

MDP

or

resistance by

natural

influenza

the

1 % squalane-in-water

of M D P . of

to

present

of 3 - 4 w e e k s

against

Significant

obtained

were

TDM,

ineffective

mortality

by

to

growth

of M D P ,

the

analog

bacterial

M.tuberculosis

of the

they

indicated

vative

the

In a d d i t i o n

Regardless

None

to be

TDM

acid-arabinoga-

components

MDP,

interval

(Table

shown).

results

mycobacidentifica-

infections.

infected

pathogenic

b e r c u l o s i s , as with

synthetic

resistance

not

lung,

MDP.

against

tuberculosis

or

aerosol

suspensions

induced

for

biological

detectable

found

(data

these

the

immunopotentiating

6,6-diesters

could

contrary, also

virus

u/as a r e l a t i v e l y

Aqueous

monomer

resistance

with

of M D P + T D M

influenza

There

to

as a m y c o l i c contains

essential

like

of

led

Discussion

combination lent

ability

and M y c o b a c t e r i u m

Results

have

immunopotentiating

investigated stimulate

which

peptidoglycan

other

on a d j u v a n t - a c t i v e

nocardia

complex

minimum

glycopeptide,

and

wall

lactan-mucopeptide

cell

investigations

corynebacteria,

were

(Table

and

combined 1).

Similar

ubiquinone

was

derivatives

induced like

deri-

by

the

seryl

403 Table 1.

Effect of MDP and Analogs Alone and in Combination with TDM Against Influenza Virus and fl^ tuberculosis Infections in Mice.

MDP analog

TDM

% surviving influenza infection

MDP

0 60

0.001

7.43 5.74

0.05

+

0 55

0.001

7.20 5.36

0.01

+

0 80

0.001

7.17 5.19

0.01

+

0 58

0.001

7.30 6.50

0.05

MDP-Abu

Groups

15 0

+

vehicle control (1 squalane)

of 1 0 - 2 0

mice

virus

or

after

intravenous

tions

containing

enza,

150

were

6.86 6.61

were

given

tuberculosis, MDP

or

analog

with

300+150

H 3 7Rv , respectively

(300

^.g), T D M

(75 ng

or M D P + T D M

influenza

3 or 4

squalane-in-water

p.g for

respective

of A / P R / 8 / 3 4

with

tuberculosis)

influenza,

compared

an a e r o s o l

strain

pretreatment

^.g for

75 p.g for

P

+

MDP-seryl MDP-des-seryl

Viable median count of tuberculosis per lung (log 10)

P

for

influ-

combination

(150+

t u b e r c u l o s i s ) . MDP

MDP+TDM

group

for

the

weeks

prepara-

groups

statistical

analysis.

The

efficacy

measurement by

the

hemadsorption

cultures. the

of M D P + T D M of

Peak

MDP+TDM

of v i r u s cured

the

earlier

MDP+TDM

group. ted

challenge strain

was

guinea-pig

this

pretreated

showed of

group mice

(Figure but

only

that

for

later,

even

on

all

day

the

7 in the

rechallenge.

virus on

oc-

day

5 in

control

of the

against

if a h e t e r o l o g o u s

3 but

levels

hemagglutina-

already

survivors

resistance

the

tissue

on day

infectious

detected

by

lungs

on B H K - 2 1

decreased

1). A l s o ,

be

checked

infected

attained

markedly

detectable

a long-lasting

6 months

were

could

be m e n t i o n e d

showed used

mice

further in the

erythrocytes

concentrations

antibodies

It s h o u l d

groups

was

concentration

clearance

in

t i o n - ! nh i bi t ing the

with

virus

pretreated

and

combination

virus

pretrea-

lethal

influenza

reA

404

DAYS AFTER INFLUENZA CHALLENGE

Figure 1. Comparison of lung virus titers and serum hemagglutination-inhibiting antibodies of mice pretreated with 1 % squalane-in-water preparation alone (•) or containing HDP+TDM combination (o). The

mechanisms

killer

cell

nificantly the

MDP+TDM

activity

the

role

of the

combination, the

killer for

the

2-4 weeks

macrophage

in

the

immunostimulants

were

given

hours

activity

MDP+TDM Natural

the

animals

time

of

macrophages pretreatment.

resistance

pretreated

later,

silica,

an a e r o s o l

of

not

sig-

to

be

activity

of

To

induced

21 d a y s

dextran

was

infection. appear

immunopotentiating

were

24

at

after

that

and

of

experiments.

pretreated

cells,

animals

carrageenan,

further

from c o n t r o l s

cells

combination

protective in

of 3 - 4 w e e k

to n a t u r a l

target

in t h e

investigated

different

In c o n t r a s t among

involved

were

combination

elucidate by

MDP+TDM

earlier

sulfate, influenza

with and virus.

405 The

results

influenza gated

by

inhibit

presented

virus

that

treatment or

impair

in T a b l e was

with

2 show

induced all

the

that

the

by M D P + T D M three

the m a c r o p h a g e

agents

function

resistance

combination known

in

to

to

was

abro-

selectively

vivo.

Effects of Macrophage Inhibitory Agents on the In Vivo Resistance

Table 2.

of MDP+TDM Combination Against Influenza Virus Infection Pretreatment

Compound

Dose

(150+75 p.g)

% survival

P

(mg)

MDP+TDM

-

-

90

MDP+TDM

silica

3

10

0.01

MDP+TDM

carrageenan

1

10

0.01

MDP+TDM

dextran sulfate

1

20

0.01

Groups

of 10 m i c e

preparations influenza

3 weeks

pretreated

before

with

aerosol

1 %

infection

compounds

were

administered

experiments

were

functional

alterations

in the m a c r o p h a g e s

ted

animals.

the

phagocytic shunt

A/PR/8/34

intravenously

Phagocytosis cells

stimulates

which

the from

oxidative

in i n c r e a s e d

aim

of

h

of a c t i v a t e d

superoxide

anion,

hyaroxyl

radical

with

the

of

light

form

dent

chemiluminescence.

with

the

combination

miluminescence aureus

zymosan.

of M D P + T D M

activity (Figure

Spleen

can

be

cells

exhibited

in r e s p o n s e

2) a n d

detected

similar

to

by

animals markedly

stimulation

results

were

of

monophos-

oxygen

hydrogen

of p h o t o n s .

from

pretrea-

hexose

generation emission

detecting

MDP+TDM

the

coccus

24

metabolism

oxygen,

in t h e

and

results

with

singlet

of e n e r g y

activity

performed

like

bolites and

with

infection.

Additional

phate

squalane-in-water

virus.

Anti-macronhage before

were

The

meta-

peroxide, release

luminol-depenpretreated enhanced by

che-

Staphylo-

obtained

with

406

1

2

3 t

4

5

6 7 TIME Imial

8

9

10

11

12

S.aureus

F i g u r e 2. C h e m i l u m i n e s c e n c e r e s p o n s e o f s p l e e n c e l l s from mice p r e t r e a t e d w i t h 1 % s q u a l a n e - i n - w a t e r p r e p a r a t i o n a l o n e o r c o n t a i n i n g MDP+TDM c o m b i n a t i o n .

In and

summary,

the

nonspecific

intracellular

biological

bacterial

response

host

defence

infections

modifiers

and

mechanisms

can

warrants

be

against

enhanced

further

by

viral potent

investigation.

INCREASED ADJUVANT ACTIVITY OF MDP BY DIRECT COUPLING OF MDP TO THE IMMUNOGEN

M. Jolivet, F. Audibert, L. Chedid Immunothérapie Expérimentale, Institut Pasteur, Paris, France E.R. Clough International Minerals and Chemical Corporation, Northbrook, Illinois 60062

Introduction It is generally assumed that synthetic antigens and vaccines require the addition of an adjuvant. Fifteen years ago several investigators identified the active cell wall structure that can substitute the Mycobacteria in Freund's complete adjuvant (1,2). This molecule is N-acetylmuramyl-L-Ala-D-isoGln, designated muramyl dipeptide or MDP (3). This glycopeptide was effective in stimulating the immune response in vivo even when administered in saline (4) . However, after intravenous injection of labelled MDP solution about 65 % of the injected dose was recovered in the urine within 30 min (5). In order to delay its elimination, MDP can be conjugated to the immunogen (6). In our study, we coupled MDP derivatives to different immunogens including a natural vaccine (diphtheria toxoid) and two synthetic vaccines (carboxy terminal peptide of HCG beta subunit and a synthetic malarial peptide copying a part of the circumsporozoite protein coupled to tetanus protein).

Results 1. Diphtheria toxoid Toxin was treated with formalin or with glutaraldehyde in presence of L-lysine or a derivative of MDP, the MDP-L-lysine (MDP-Lys) which has a reactive amino group and the same activities as MDP.

Biological Properties of Peptidoglycan © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

408 The conjugates contained 8 to 11 molecules of MDP per molecule of diphtheria toxoid and presented a single band by SDS PAGE (data not shown). Table 1. Antibody Response to Toxoids Conjugated to MDP-L-Lysine. Immunogen Coupling agent Primary response Secondary response formalin 2.5 1,357 A B formalin 4.5 2,833 C D E F

formalin glutaraldehyde glutaraldehyde glutaraldehyde

15 6.5 12 17

7,071 2,218 3,500 8,722

Toxoid was formed according to the method of Relyveld (7) with the addition of MDP-Lys to the reaction mixture. (A) Toxin treated by formalin (3Lf = 7 . 5 yg protein), (B) Toxoid (3Lf) mixed with 0.5 yg of MDP, (C)Toxoid (3Lf) linked to 0.5 yg of MDP, (D) Toxin treated by glutaraldehyde (3Lf) , (E)Toxoid (3Lf) mixed with 0.8 yg of MDP, (F)Toxoid (3Lf) linked to 0.8 yg of MDP. Mice (eight per group) received subcutaneously 3 Lf toxoids in PBS. On day 30, animals were boosted with the same amount of toxoid without adjuvant. Mice were bled at days 21 and 36. Sera were titrated by passive hemagglutination and the results meaned. Protein content in each conjugate was measured by Folin reaction and MDP content by a colorimetric method (8). As can be seen on Table 1, in secondary response 0.5 yg of MDP-Lys derivative mixed with the toxoid enhanced the antibody titer, but a more marked increase was observed in the case of MDP-Lys conjugated to the protein with formalin or glutaraldehyde. In this case, MDP was not immunogenic because anti-MDP antibody was not detected using ELISA. 2. Synthetic vaccines Synthetic vaccines are usually constructed with a synthetic antigen coupled to a protein carrier and administered with adjuvants. In laboratory models, tetanus toxoid is often used as a carrier and Freund's complete adjuvant as an adjuvant. However, FCA is unacceptable for human use because of its unwanted side effects. Thus, we have constructed two hapten-carriers with or without MDPLys coupled to the conjugate. The first example is constructed from synthetic carboxy terminal peptide of HCG beta subunit (residues 109-145). This peptide is lacking in the human luteinizing hormone (hLH) and other pituitary derived glycoprotein hormone (9).

409 Table 2. Antibody Response of Rabbits Injected with Synthetic Beta HCG subunit peptide-tetanus toxoid. „ response 2 Primary response 2 Secondary Rabbit Immunogen Adjuvant anti-ßhCG anti-TT anti-ßhCG anti-TT 1

pep-TT

2

pep-TT-MDP

3

pep-TT

4

pep-TT

5

pep-TT-MDP

MDP FCA FIA + MDP

37

1

6

4

82

4

50

32

53

52

100

256

25

8

32

128

94

26

97

64

The 109-145 synthetic peptide was conjugated to tetanus toxoid (TT) with glutaraldehyde used as the coupling agent, according to a previously described method (10). Fourteen pg of MDP are present in 200 pg of conjugate. Five rabbits received 200 yg of conjugate (multiple intradermal sites) in PBS without or with adjuvants (MDP 100 yg, FCA or FIA). On day 75, animals were boosted with the same amount of conjugates in PBS. Rabbits were bled on days 40 and 97 and sera tested. Anti-BhCG antibodies were measured by a radioimmunoassay (RIA) and results were expressed in percentage of binding. Anti-tetanus antibodies titers are expressed as the inverse of the maximal dilution of serum agglutaning tetanus-sensitized sheep erythrocytes x lO - ^. As can be seen on Table 2, a strong primary response was observed against HCG beta subunit when MDP was coupled to the conjugate. In aqueous medium, the percentage of binding with the B-subunit is of 8 2 % when MDP is coupled compared to 37 % when MDP is only mixed with the conjugate.

The same results were obtained in oil medium

(rabbits 4 and 5). Analysis of the secondary response show that a strong antibody response is obtained against the carrier and all the rabbits. Nevertheless, the carrier antibody reponses of rabbits treated with the MDP conjugates were only slightly elevated as compared to rabbits treated with pep-TT mixed with adjuvant. The second example of a synthetic vaccine is constituted by a synthetic 24 amino acid peptide representing the immunodominant epitope of the sequence of the circumsporozoite protein of P.knowlesi (12). This peptide contains two additional amino acids : tyrosine at the N-terminus and cystein at the C-terminus. Moreover, Gysin (13) has shown that antibodies to the peptide react with the surface membrane of sporozoites and that they neutralize the infectivity of the parasite.

410

In our study, we have made conjugates containing different substitution ratios of the synthetic malarial peptide and MDP-Lys on tetanus toxoid. Figure 1.

15H

10-

V) a.

UJ

< if)

5'

Anti

P

26

Anti

TT

This peptide (P26) represents 24 amino acids present in the natural sequence of the P.knowlesi circumsporozoite protein and two additional amino acids. Pep-TT was made according to a previously described method (10). For conjugates L (low), H (high) varying amounts of the peptide and MDP-Lys were cross-linked to tetanus toxin using glutaraldehyde. Amino equivalents peptide/MDP-Lys ratio are the following : L 1/0.5, H 1/2. Six adult female BALB/c mice received pep-TT (50 yg in PBS) ( ) or in presence of MDP (100 pg) ( ) or conjugate L(L) or conjugate H(H). On day 21, animals were boosted with the same amount of conjugate in PBS. Mice were bled on day 28. Sera from animals in each group were pooled and tested for total anti-peptide and anti-tetanus toxoid antibodies by ELISA as previously described (11). Titers

411

are expressed as inverse of the dilution of serum giving an O.D. in ELISA of two times background x 10~3. The results in Figure 1 show that the mice treated with conjugates containing MDP-Lys had higher antibody responses to both the peptide and the carrier. Moreover, the secondary antibody response of the conjugate containing 0.3 |ig of MDP-Lys is about the same as when the same quantity of antigen is mixed with 100 ng of this adjuvant. Antibodies were tested in circumsporozoite protein assay and were shown to be effective.

Discussion These experiments demonstrate that the covalent linkage of MDP-Lys to an immunogen molecule increases the adjuvant activity of MDP. In addition, it does not seem that MDP-Lys became a hapten since no anti-MDP antibody was detected by ELISA. Pharmacokinetic studies showed that 5 % of MDP was recovered in urine 30 minutes after the injection of MDP linked to the macromolecule in comparison with 65 % of free MDP. If synthetic peptides are to be used for the production of future vaccines, methods of increasing the immunogenicity of conjugates must be found. Our results demonstrated that very low doses of adjuvant can stimulate the production of protective antibodies. Finally, we have constructed a synthetic vaccine with three parts which can provoke protective antibodies : an antigenic (synthetic peptide copying part of protein natural sequence), an immunogenic (protein carrier) and adjuvant (MDP derivative). This type of vaccine excludes the parts of conventional vaccines which provoke side effects. Acknowledgements We thank Dr. Jean Choay and Dr. Pierre Lefrancier for the samples of synthetic compounds. We are grateful to M. Hattab for his technical assistance, and to C. de Champs for typing the manuscript .

412

References 1. Lederer, E. 1971. J.Med.Chem. 23^, 819. 2. Azuma, I., S. Kishimoto, Y. Yamamura, J.F. Petit. 1971. Jap.J. Microbiol. 1_5, 193. 3. Ellouz, F., A. Adam, R. Ciorbaru, E. Lederer. 1974. Biochem. Biophys.Res.Com. 59, 1317. 4. Audibert, F., L. Chedid, P. Lefrancier, J. Choay. 1976. Cell. Immunol. 21_, 243. 5. Parant, M., F. Parant, L. Chedid, A. Yapo, J.F. Petit, E. Lederer. 1 979. Int. J. Immunopharmac. 1_, 35. 6. Arnon, R., M. Sela, M. Parant, L. Chedid. 1980. Proc.Natl.Acad. Sei. USA 77, 6769. 7. Relyveld, E.H. 1973. C.R.Acad.Sc.Paris 277, 613. 8. Reissig, J.L., J.L. Strominger, L.F. Leloir. 1956. J.Biol.Chem. 217, 959. 9. Morgan, F.J., S. Birken, R.E. Canfield. 1975. J.Biol.Chem. 250, 5247 . 10. Audibert, F., M. Jolivet, L. Chedid, R. Arnon, M. Sela. 1982. Proc.Natl.Acad.Sei. USA 7_9, 5042. 11. Jolivet, M., F. Audibert, E.H. Beachey, A. Tartar, H. Gras-Ma Masse, L. Chedid. 1983. Biochem.Biophys.Res.com. 117, 359. 12. Godson, G.N., J. Ellis, P. Svec, D.H. Schlesinger, V. Nussenzweig. 1983. Nature 305, 29. 13. Gysin, J., J. Barnwell, D.H. Schlesinger, V. Nussenzweig, R.S. Nussenzweig. 1984. J.Exp.Med. 160, 935.

CONTRIBUTORS AND PARTICIPANTS

R. Barot-Ciorbaru Université Paris Sud, Institut de Biochimie, Bât 432, 91405 Orsay, France

G. Barratt Université Paris Sud, Institut de Biochimie, Bât 432, 91405 Orsay, France

G. Baschang CIBA-GEIGY Limited, K-122.4.39, Postfach, CH-4002 Basel, Switzerland

H. Bauer Institut für Kristallographie, Takustr. 6, D-1000 Berlin 33 Germany

W.G. Bessler Arbeitsbereich Mikrobiologie und Immunologie Universität Tübingen, Auf der Morgenstelle 28 D-7400 Tübingen Germany

U. Bläsi Institut für Genetik und Mikrobiologie der Universität München, Maria-Wardstr. 1a D-8000 München 19. Germany

H. Brade and L. Brade Forschungsinstitut Borstel, Parkallee 4b D-2061 Borstel, Germany

E. Bräutigam Institut für Biologie II, Mikrobiologie, AlbertLudwigs-Universität, Schänzlestr. 1, Germany

H. Brunner Bayer AG, Pharma-Forschungszentrum, Institut für Chemotherapie , D-5600 Wuppertal, Germany

B. Christensson Department of Infectious Diseases, University Hospital, S-221 85 Lund, Sweden

R. Dziarski Indiana University School of Medicine, Northwest Center for Medical Education, 3400 Broadway, Gary, IN 46408, U.S.A.

J. Endl Soehringer Mannheim GmbH, Bahnhofstr. 9-15, D-0132 Tutzing, German"

H.B. Evans Department of Immunology, Royal Liverpool Hospital, P.O. Box 147, Liverpool, L69 3BX, U.K.

H. Feucht Institut für Immunologie der Universität München, Schillerstr. 42, D-8000 München 2, Germany

414

F. Fiedler Institut für Mikrobiologie und Genetik der Universität München Maria-Ward-Str. 1a D-8000 München 19 Germany

H. Finger Städtische Krankenanstalten Krefeld, Institut für Hygiene und Laboratoriumsmedizin, Lutherplatz 40, D-4150 Krefeld 1 Germany

A. Fleer Department of Clin. Bacteriology, University Hospital, Utrecht, Netherlands

H. Formanek Botanisches Institut der Universität München Menzingerstr. 67 D-8000 München 19, Germany

S. Formanek Botanisches Institut der Universität München Menzingerstr. 67 D-8000 München 19, Germany

A. Fox Department of Microbiology and Immunology, USC School of Medicine, Columbia SC 29208, U.S.A.

N. Franken Boehringer Mannheim GmbH Bahnhofstr. 9-15 D-8132 Tutzing, Germany

K. Gadilhe Lehrstuhl für Mikrobiologie Technische Universität München, Arcisstr. 21, D-8000 München 2, Germany

I. Ginsburg Department of Oral Biology Hebrew University, Hadassah School of Medicine, Ein Kerem, Jerusalem, Israel

J. Gmeiner Institut für Mikrobiologie Technische Hochschule Darmstadt Schnittspahnstr. 9 D-6100 Darmstadt, Germany

P. Goroncy-Bermes Institut für Medizinische Mikrobiologie und Immunologie Ruhr-Universität Universitätsstr. 150 D-4630 Bochum 1, Germany

M. Guinand Université Cl. Bernard Laboratoire de Biochimie microbienne, Lyon I 4 3 Bd du 11 novembre 1918 69622 Villeurbanne, France

F. Hagen Lehrstuhl für Mikrobiologie Technische Universität München, Arcisstr. 21 D-8000 München 2, Germany

R. Hakenbeck Max-Planck-Institut für Molekulare Genetik Ihnestr. 63-73 D-10 0 0 Berlin 33, Germany

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C. Herzog Department für Innere Medizin Universitätskliniken CH-4 0 31 Basel Switzerland

B. Heymer Universität Ulm Abteilung für Pathologie Oberer Eselsberg D-7900 Ulm, Germany

J.-V. Höltje Max-Planck-Institut für Entwicklungsbiologie Abteilung Biochemie Spemannstr. 35 D-7400 Tübingen, Germany

K. Huber Fachbereich Biologie (10) Mikrobiologie, Technische Hochschule Darmstadt Schnittspahnstr. 9 D-6100 Darmstadt, Germany

L. Johannsen Robert-Koch-Institut Nordufer 20 D-10 00 Berlin 65 Germany

M. Jolivet Immunothérapie Expérimentale Institut Pasteur, 28, rue du Dr. Roux, 75724 Paris cedex 15 France

U.J. Jürgens Institut für Biologie II Mikrobiologie, AlbertLudwigs-Universität Schänzlestr. 1 D-7800 Freiburg, Germany

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H.J. Kolb Städtisches Krankenhaus München-Harlaching Klinisch-chemisches Institut, Sanatoriumsplatz 2 D-8000 München 90 Germany

T. Komuro Max-Planck-Institut für Immunbiologie Stübeweg 51 D-7800 Freiburg Germany

416

S. Kotani Department of Microbiology and Oral Microbiology Osaka University Dental School 1-8 Yamadoakoa, Suita Osaka 565, Japan

H.P. Kroll Bayer AG Pharma-ForschungsZentrum Institut für Chemotherapie D-5600 Wuppertal Germany

J.M. Krueger Department of Physiology The Chicago Medical School 3333 Green Bay Road North Chicago, IL 60064 U.S.A.

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E. Kwa Robert-Koch-Institut Nordufer 20 D-1000 Berlin 65 Germany

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417

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419

H.I. Wergeland Department of Microbiology and Immunology The Gade Institute MFH-Bygget 5016 Haukeland Sykehus Norway

C.H. Wirsing von Koenig Städtische Krankenanstalten Krefeld Institut für Hygiene und Laboratoriumsmedizin Lutherplatz 40 D-4150 Krefeld 1, Germany

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AUTHOR INDEX

Adkinson,N.F., 83 Anderle,S.K., 273 Audibert,F., 407 Berger,R., 113 Bessler,W.G., 335 Biemann,K., 49 Bläsi,U., 215 Brade,H., 341 Brade,L.f 341 Brase,A., 267 Brehmer,W., 401 Briese,T., 83 Brown,R.R., 273 Chedid,L., 353, 407 Christensson,B., 21 Clough,E.R., 407 Cox,M., 335 Cromartie,W.J., 273 Duoassieu,P. , 389 Dzlarski,R., 229 Ellerbrok,H., 83 Endresen,C., 99 Evans,H.B., 89 Farkas,J., 379, 383 Feucht,H.E., 267 Finger,H., 371 Fleer,A., 261 Fleming,T.J., 313 Formanek,H., 43 Fox,A., 61 Franken,N., 135 Giesbrecht,P., 191, 197 Gilbart, J. , 61 Ginsburg,I., 167, 197 Golecki,J.R., 135 Guenounou,M., 3 95 Guinand,M., 389 Hagen,F., 221 Hakenbeck,R., 83 Halfmann,G., 215 Hammer,M., 61 Harkness,R.E., 215 Harrison,J., 61 Haslberger,H., 121 Herzog,Ch., 113 Heymer,B., 291, 305

Höltje,J.-V., 209 Hrsak, I. , 203 Huber,K., 187 Huber,M., 121 Ishikawa,Y., 61 Janusz,M.J., 27 3 Jaspers,F.C.A., 261 Jezek,J., 379, 383 Johannsen,L., 37, 197 Johnson,P.M., 89 Jolivet,M., 407 Jürgens,U.J., 55 Jung,G., 335 Just,M., 113 Just,V., 113 Kawahara,K., 341 Kleine,B., 335> Krueger,J.M., 329 Kuchenbauer,T., 105 Kwa,E., 197 Labischinski,H., 37, 191 Ladesic,B., 203 Lahav,M., 197 Lange,W., 401 Ledvina,M., 379, 383 Leitherer,G., 75, 221 Lex,A. , 335 Loo s,M., 319 Ludewitz,H., 209 Lüderitz,Th., 341 Luwitz,W., 215 Mar,P., 121 Marget,W., 121 Martin,H.H., 187 Martin,S.A., 49 Martinez-Alonso,C., 335 Masihi,K.N., 401 v. Mayenburg,J., 291, 305 Michel,G., 389 Montoya,F.R., 313 Morel,P., 389 Morgan,S.L., 61 Nauciel,C., 395 Nogami,W., 313 Nolibe,D., 249 O'Neill,G.J., 267

422 Parant,M., 353 Pararajasegaram,G., 61 Pekarek, J. , 383 Petit,J.-F., 249 Phua,K.K., 89 Reissenweber,S., 75 Riethmüller,G., 267 Rietschel,E.Th., 341 Rosenthal,R.S., 49, 313 Rotta,J., 379, 383 Ryc,M., 129, 379, 383 Schade,U., 341 Schleifer,K.H., 1, 67, 75, 105, 1 35 , 221 , 31 9 Schumann,G., 255 Schwab,J.H., 27 3 Seidl,J., 67 Seidl,P.H., 1, 67, 75, 105, 135, 221, 267, 319 Stimnson,S.A., 273 Straka,R., 383 Suhr,B., 335 Tacken,A., 341

Tenu,J.-P., 249 Tomasic,J., 20 3 Tympner,K.-D., 105 Vacheron,F., 395 Vacheron,M.J., 389 Valinqer,Z., 203 Verhoef, J., 261 Wagner,B., 129 Wagner,M., 129 Wecke,J., 197 Weckesser,J., 55 Wells,A., 61 Wergeland,H.I., 99 Whiton, R. , 61 Wiesmüller,K.-H., 335 Wirsing von Koenig,C.H. Witte,A., 215 Yang ,C.H. , 61 Yavordios,D., 389 Zahringer,U., 341 Zaoral,M., 379, 383 Zauner,E., 7 5 Zeiger,A.R., 95, 145 Zwerenz,P., 135, 221

SUBJECT INDEX The abbreviation of Peptidoglycan is PG.

N-ACETYLATION partial lack of, among bacilli 2, 7, 76, N-ACETYLGLUCOSAMINE 211, G-N-ACETYLGLUCOSAMINIDASE

206,

N-ACETYLMURAMIC ACID 211, antibodies to 5, N-ACETYLMURAMYL-L-ALANINE AMIDASE 205, O-ACETYLATION biological properties of PG, effects on 197, of gonococcal PG 313-317, of staphylococcal PG 191, 1 97 , ACTINOMADURA 389, ACTIVATION of B-cells 213, 298, 299, of T-cells 231 , of complement 264, 284, 298, 299, 319, 323, of macrophages 298, 299, of neutrophils 298, 299, polyclonal, of lymphocytes 232 , polyclonal, by soluble PG 162, polyclonal, of B-cells 299, 339, ACTIVITY adjuvant 231, 236, 371, 381 , antimetastatic, of PG 204, 206, antitumor, of MTP-PE 256, antiviral, of MTP-PE 256-258, biological, of lipopolysaccharide 341 , biological of PG, in man 291, 305,

cancerostatic, of MDP analogs 37 9, immunomodulating 230, 236, 249, 255, 395, inflammatory, of soluble PG 163, mitogenic, of lipoprotein 335, 336, mitogenic, of soluble PG 159, of PG, in man 291, 305, pyrogenic, of MDP analogs 37 9, 381 , 383, 384, somnogenic, of MDP 329, synovial activator activity 230 , ACUTE PHASE PROTEINS 231, ADJUVANT ACTIVITY 231, 236, 371, 381, 407, AGAMMAGLOBULINEMIA TYPE BRUTON 107, AGE DEPENDENCY of antibodies to lipid A 127, of antibodies to peptidoglycan 106, AIDS-HTLV III INFECTION 305, ALLERGIC bronchitis 298, diseases 295, 298, 305, encephalitis, experimental 384, 387, rhinitis 298, vasculitis 298, ALPHATOXIN 25, ALTERNATE PATHWAY MENT activation of, by 319, 323, activation of, by activation of, by acid 319,

OF COMPLEcell walls PG 264, 319, teichoic

424 AMIDASE 3, in human serum 205, 209, ANAPHYLAXIS 373, 1,6-ANHYDROMURAMIC ACID 212, 314, 315, 331, ANIONIC POLYELECTROLYTES 168, ANTIBODIES age dependency of titers 106, 127, IgE antibodies to S. aureus cell walls 67, IgG antibodies to S. aureus PG 24, reacting with soluble PG 157, to A polysaccharide of streptococci 6, to A variant polysaccharide of streptococci 10, to ß-lactam antibiotics 83, diagnostic role of 21 , to di-alanine 10-14, 135, to the glycan moiety 4, 5, 75 to interpeptide bridge 8, 9, to lipase 27, to lipid A 121, to lipopolysaccharide 127, 342, to Lys—D-Ala-D-Ala 95, 148, in man 5, 1 1 , 99, 105, 310, to MDP 5, 6, to Micrococcus luteus 5, 136, to muramic acid 5, 6, 354, to pentapeptide subunit 5, 11, 95, 105, 136, 310, to PG, ferritin conjugated 130, to PG, in animals 4, 99, to PG-polysaccharide polymers 90, to Staphylococcus aureus 113, to Staphylococcus aureus cell walls 67, to Staphylococcus aureus PG 24, to Streptococcus pyogenes A-variant 10, to streptococcal PG 90, to Streptolysin 0 107, to teichoic acid 43, 113, to tetanus toxoid 267, to the tetrapeptide subunit 5, ANTIBODY DEFICIENCY SYNDROME 308, ANTIGENIC EPITOPES OF PG 4, 131, 133, 135,

ANTIGENICITY of lipopolysaccharide 342, of peptidoglycan 4, ANTI—IDIOTYPE 90, ANTI-INFLAMMATORY EFFECT of MDP 281, ANTI-METASTATIC ACTIVITY of peptidoglycan 204, 206, ANTI-TUMOR ACTIVITY of peptidoglycan 204, 249, 255, of liposome-entrapped MTP-PE 256, ANTI—STREPTOLYSIN-0 107, ANTI-VIRAL ACTIVITY of liposome-entrapped MTP-PE 256-258, ARACHIDONIC METABOLITE PRODUCTION induction by MDP 283, 285, induction by PG-polysaccharide polymers 283, ARTHRITIS 173, 174, 176, induction of 277-279, 314, 315, 317, reaction of, induced by MDP 279, ATOPIC DERMATITIS 109, diseases 305, AUTOLYSINS 170, 171, 215, AZTREONAM 188

B BACILLI lack of N-acetylation 7, BACILLUS LICHENIFORMIS secretion of soluble PG 149, BACILLUS SPHAERICUS endopeptidase I 389,

425 BACILLUS SUBTILIS carboxypeptidase of 11, glycopeptides of 6, 75, immunoelectronmicroscopy 136, PG of, as immunoadsorbent 136, secretion of PG 149, 224,

BREVIBACTERIUM DIVARICATUM biological activity 203, metabolic fate of PG monomer 203 , PG monomer 203, secretion of soluble PG by 149,

BACTEREMIA 113, 115,

BRONCHITIS allergic 298, bacterial 297,

BACTERIAL bronchitis 297, hypersensitivity 306, lysis 215, morphogenesis 209, rhinitis 297 , sinusitis 2 97,

C

BACTERICIDAL REACTIONS 167, BACTERIOLYSIS 167, BACTERIOPHAGE PHI-X-17 4

215,

BASOPHILS effect of PG on 283, B-CELLS activation of 213, 298, 299, B-LACTAM ANTIBIOTICS action on Proteus mirabilis 187 antibodies to 83, aztreonam 188, cefotaxime 188, cefoxitin 188, ceftizoxime 188, functional damage of PG 190, growth of bacterial spheroblasts in the presence of 189, imipenem 188, inhibition of PG synthesis 190, latamoxef 188, penicillin 187, secretion of soluble PG 145, BIOLOGICAL ACTIVITY of lipopolysaccharide 341, of PG in man 291, 305, BIOREACTORS 390, B-LYMPHOCYTE MITOGENS 335, BOOSTER REACTION 37 4, BORDETELLA PERTUSSIS 371,

C4B2.9 IMMUNOGENETIC MARKER 268, CANCEROSTATIC ACTIVITY of MDP analogs 37 9, A-CARBOHYDRATE of streptococci, antibodies to 6 , CARBON CLEARANCE by reticuloendothelial system 392 , CARBOXYPEPTIDASE 4, 11, 13, of Bacillus subtilis 11 , of Staphylococcus aureus 11 of Streptomyces albus 389, lack of 4, 140, CARCINOMA 204, CATIONIC POLYELECTROLYTES 169, 171 , CEFOTAXIME 188, CEFOXITIN 188, CEFTIZOXIME 188, CELL CHEMILUMINESCENCE 231, 262, 299, 405, 406, CELLULAR RESPONSE to PG 267, CELL WALL models 129, 133, structure 37, 129, 341,

426

CEPHALOSPORIN 13,

CYANOBACTERIUM 55,

CHEMILUMINESCENCE 231, 262, 299, 405, 406,

CYTOCHROME P-450

CHEMOTACTIC FACTOR 230, CHEMOTAXIS for macrophages 229, 262, COLITIS 125, COLONY STIMULATING FACTOR 230, COLLAGENASE release of 231, COMPLEMENT activation of 262, 284, 298, 299, 319, 323, alternate pathway 264, 319, 323, genotype 267, CONFORMATION 37, CONJUGATES glycopeptide-protein 6, 75, lysozyme-peroxidase 130, MDP-carrier 6, MDP-immunogen 407, peptide-polypeptide 95, 148, peptide-protein 8, 10, 11, 135, 137, CONTACT ECZEMA 2 38. CORE of LPS 343, COUNTERIMMUNOELECTROPHORESIS 113,

207,

CYTOSTATIC ACTIVITY 230, CYTOTOXIC ACTIVITY 230, 249, CYTOTOXIC FACTOR 395,

D DEGRADATION of PG 191, 197, 215, 230, resistance of PG to 313-315, DELAYED HYPERSENSITIVITY 206, 375, 383, 385, DERMATITIS, ATOPIC 109, DIAGNOSTIC ROLE of PG antibodies 21, DI-ALANINE 131, antibodies to 10-14, 135, DIPHTERIATOXOID 407, DISACCHARIDE MDP ANALOGS biological properties 379, 384 , preparation 379, DISACCHARIDE PEPTIDE MONOMERS 314, 315, 317,

CROHN'S DISEASE 125,

E

CROSSLINKAGE of PG 2,

EDEMA 276, 278, 279, 300,

CROSSREACTIVITY between B-lactams and R-D-Ala2 12, 13, between different PGs 95, 101, between L-Lys and meso-A2pm 95, between rheumatoid factor and PG-polysaccharide 89, CULTURE FILTRATES 79, 221,

ELECTRON DIFFRACTION 43, ELECTRON MICROSCOPY 43, 129, 1 71 , 200 , 201 , 293 , 385 , ELISA 11, 75, 113, see also: enzyme immunoassay ENCEPHALITIS, ALLERGIC 384, 387 , ENDOCARDITIS 24, 115,

427

ENDOGENOUS PYROGEN 230,

G

ENDOPEPTIDASE 3, 210, 389,

GAS CHROMATOGRAPHY-MASS SPECTROMETRY levels of muramic acid in tissue 64, levels of rhamnose in tissue 64,

ENDOPEPTIDASE I from Bacillus sphaericus 389, ENDOTOXIN 341, ENTERITIS 125, ENTEROCOCCUS FAECALIS secretion of PG 223, ENTEROCOCCUS FAECIUM PG-polysaccharide polymers of 275, 276, secretion of soluble PG 149, ENZYME IMMUNOASSAY antibodies to glycan moiety of PG 75, antibodies to pentapeptide subunit of PG 11, antibodies to PG-polysaccharide 90, antibodies to teichoic acid 113, levels of PG-polysaccharide complexes in vivo 62, levels of soluble PG 95, 148, EPITOPES of PG 4, 131, 133, 135, ERYTHEMA 300, ESCHERICHIA COLI carboxypeptidase of 11, glycan strands released 209,

F FAST ATOM BOMBARDMENT MASS SPECTROMETRY 49, 20 3, FERRITIN-CONJUGATED PG-ANTIBODIES 130, FERRITIN TECHNIQUE, INDIRECT 135, FEVER induction of 213, FIBROBLAST ACTIVATING FACTOR 2 30, FIBROBLAST PROLIFERATION 231 ,

GENE lysis gene 215, GENETIC CONTROL of immune response 267, GENOTYPE of comnlement 267, GLOMERULONEPHRITIS 267, GLUCOSAMINIDASE 3, GLYCAN MOIETY OF PG antibodies to 4, 5, 75, detection of, by RIA 221, immunoelectron microscopic studies 132, 135, structure of 2, GLYCAN STRANDS length distribution 209, unsubstituted by peptides 78, GLYCOPEPTIDES from Actinomadura 389, from Bacillus subtilis 6, 75, synthetic 379, 383, GONOCOCCAL PG 313-317, GRAM NEGATIVE BACTERIA cell wall structure of 341, PG of 2, GRAM POSITIVE BACTERIA PG of 2, GROUP A STREPTOCOCCI 129, GROUP A POLYSACCHARIDE antibodies to 6, edema induced by 274-276, 278, effect on mast cells 283, intraarticular injection with 278,

428

GROUP A-VARIANT STREPTOCOCCI 129, a n t i b o d i e s to 10,

IMMUNOGENICITY of P G 4, of s o l u b l e P G 159, I M M U N O G L O B U L I N A 17, 373,

H HEMAGGLUTINATION, HEMORRHAGE i n d u c e d by P G

INDIRECT

113,

283,

HERPES SIMPLEX VIRUS

I M M U N O G L O B U L I N E 37 3, to P G 17, 67, t o t a l levels of 297, 310,

256,

HISTAMINE RELEASE i n d u c e d by P G 283, HISTONE

IMMUNOGLOBULINEMIA

HISTOMORPHOLOGY

IMMUNOGLOBULIN M

293,

IMMUNOHISTOLOGY

H L A - R E G I O N 26 7,

HYDROGEN FLUORIDE action on cell walls HYPERSENSITIVITY

209,

319,

306,

IMMUNOSUPPRESIVE of P G 235,

384, EFFECT

IMMOBILIZED ENZYMES

390,

IMMUNOCHEMISTRY Of P G 1,

401-405,

INHIBITION of m a c r o p h a g e m i g r a t i o n 230, of P G s y n t h e s i s 190, of p h a g o c y t o s i s 230,

113,

IMMUNOELECTRON MICROSCOPY 129, 132, 135, 136, 141, of S. a u r e u s 137, of Str. p y o g e n e s 129, 137, IMMUNOFERRITIN TECHNIQUE 1 29, 1 35 , 1 41 , IMMUNOGENETIC MARKER

2 97,

I N F L A M M A T I O N 172, 296, b y s o l u b l e P G 163, INFLUENZA VIRUS

107,

268,

113,

21,

INFECTIOUS DISEASES

188,

230,

401,

INDIRECT HEMAGGLUTINATION

I

IMMUNODIFFUSION

295,

IMMUNOSTIMULATION

INFECTIONS b y S. a u r e u s

IMMUNODEFICIENCY 297, 308,

373,

IMMUNOMODULATING ACTIVITY 236, 249, 255, 395,

305,

H U M A N S E R U M A M I D A S E 205,

IMIPENEM

108,

I M M U N O G L O B U L I N G-j 37 3,

171,

H T L V III V I R U S

IMMUNOGLOBULIN CLASSES of a n t i b o d i e s to L i p i d A 126, of a n t i b o d i e s to P G 11, 16, 75,

INTERLEUKIN

230, 283,

229,

285,

INTERPEPTIDE BRIDGE OF PG s t r u c t u r e 2, a n t i b o d i e s to 5, 8, 137,

429

INTRAARTICULAR INJECTION with MDP 278, with PG-polysaccharide polymers 278,

LIPID A 121, 341, antibodies to 121, synthetic 347,

INTRADERMAL INJECTION of bacterial substances 306,

LIPOPEPTIDES polyclonal B-cell activation 339", synthetic 335,

J JOB'S SYNDROME 68,

K KAWASAKI SYNDROME 109, KILLER CELLS 404, KLEBSIELLA PNEUMONIAE resistance to 375,

L LACTAM see: S-lactam LACTOBACILLUS ACIDOPHILUS secretion of PG 224, LACTOBACILLUS CASEI PG-polysaccharide polymers of 275, 276, LACTOBACILLUS GASSERI immunoelectron microscopic studies 141, point of attack of penicillin 141 , secretion of PG 224, LATAMOXEF 188, LEUCOCYTOSIS 231, 265, LEUCOPENIA 231, 265, LEWIS LUNG CARCINOMA 204, LIPASE antibodies to 27,

LIPOPOLYSACCHARIDE 121, 249, 341 , antigenicity of 342, antibodies to 127, 342, biological activity of 341, chemical structure of 341347, core 343, O-specific chains 343, as pyrogen 204, 344, repeating units 343, LIPOPROTEIN interaction with lymphocytes 335, 336, PG associated 335, 336, mitogenic activity 335, 336, LIPOSOME-ENTRAPPED MTP-PE (MTP-PE/MLV) antitumor effects 256, antiviral effects 256-258, effects in virus models 256, effects in tumor models 256, induction of tumoricidal macrophages 256-258, induction of virucidal monocytes 256-258, LIPOTEICHOIC ACID 169, 172, and autolytic enzymes 171, LIQUOID 172, LISTERIA MONOCYTOGENES 375, LOCALIZATION OF PG immunoelectron microscopy 129, 135, in tissues 316, LUNG CARCINOMA 204, LYMPHOCYTES interaction with lipoprotein 335, 336, interaction with PG 229, 231, 232 ,

430 B-LYMPHOCYTE MITOGENS 335, polyclonal activation of 232, LYSIS, BACTERIAL 215, LYSIS GENE 215, LYSOSOMAL ENZYMES 167, LYSOSOMAL HYDROLASES 167, LYSOSTAPHIN 118, LYSOZYME 48, 167, 191, 206,

effect on PMN's 284, effect on REM sleep 330, effect on slow wave sleep 329, effect on T-cells 285, IL-2 production suppressed by 285, in vitro degradation 61, intraarticular injection with 278, oxygen radical production induced by 284, 357, reactivation of arthritis induced by 279, somnogenicity 32 9,

LYSOZYME-PEROXIDASE CONJUGATE 130,

MDP ANALOGS 353, 379, 383, immunoadjuvant activity of 379, 383, macrophage cytotoxicity of LYTIC ENZYMES 395 , of PG 3, 48, 118, pyrogenic activity of 379, 381, LYTIC MUREIN TRANSGLYCOSYLASE 212, 383, 384, MELANOMA M

B 16 melanoma 204, 257,

MACROPHAGES 173, 197, 404, 405, activation of 249, 284, 298, 299, 355-359, cytotoxicity by MDP analogs 395, inhibition of migration 229, 230, inhibitory agents 404, 405, interaction with PG 229, 231, 232,

MEDIATORS

MAMMARY CARCINOMA 204, MASS SPECTROMETRY 49, 20 3,

of inflammation 294, MEMBRANE perturbation 215, potential 215, protein 215, METABOLIC FATE of MDP 61, of PG monomers 204, METHANOBACTERIUM FORMICICUM 276,

MAST CELLS degranulation 294, effect of A polysaccharide on 283, effect of PG on 283,

MICELLAR STRUCTURE OF PG 45,

MDP 1 73 , 31 5 , 353 , 371 , 383, 401-406, adjuvanticity of, coupled to immunogen 407, antibodies to 5, 6, 354, anti-inflammatory effect of

MICROCOCCUS LUTEUS antisera to 5, 136, partial lack of peptide substitution in PG 3, 5, 7, secretion of PG 149,

280,

281,

arachidonic metabolite production induced by 283, 285, coupled to immunogen 407, effect on anti-PG antibody formation 289,

MICROCOCCI antibodies to interpeptide bridge of 8, 9,

MITOGENS of B-lymphocytes 335,

431

MITOGENIC ACTIVITY of lipoprotein 335, 336, of soluble PG 159, MODELS of cell wall structure 129, 133, of peptidoglycan 37, 44, MOLECULAR WEIGHT ANALYSIS 64, 65, MONOKINES secretion 229, MORPHOGENESIS, BACTERIAL 209, M-PROTEIN SEQUENCE 130, MTP-PE/MLV see: liposome entrapped MTP-PE MURALYTIC DIGESTION of PG-polysaccharide polymers 276, 278, 279, MURAMIC ACID antibodies to 5, 6, levels of, in tissues 64, MURAMINIDASE 3, MURAMYL PEPTIDES see: MDP and MDP analogs MUREIN see: peptidoglycan MUTANOLYSIN 276, 279, 280, MYCOBACTERIUM TUBERCULOSIS 402, 403,

N NATURAL KILLER CELLS 404, NEISSERIA GONORRHOE 313, 314, NEUTROPHILS activation of 298, 299, NMRI-MICE 372, NON-SPECIFIC RESISTANCE 372,

O O-ACETYLATION see: ACETYLATION O-specific chains of LPS 343, OXYGEN METABOLISM stimulation by PG 231, OXYGEN RADICALS 167, 168, 284, 357 , production of, induced by MDP 284, 357,

P PATHOMECHANISMS 305, PATIENT'S SERA 21, 22, 26-28, 99, 105, PBP 83, of Proteus mirabilis 188, PENICILLIN 170, 172, 187, 204, 225, see also: ß-LACTAM ANTIBIOTICS, antibodies to 83, cross-reactivity with R-D-Ala,, 13, growth of S. aureus 225, point of attack 141, PENICILLIN BINDING PROTEINS, see PBP, PENTAPEPTIDE SUBUNIT OF PG antibodies to 5, 11, 95, 105, 136, 310, as inhibitor, 131, detection of, by RIA 221, immunoelectron microscopic localization 135, structure of 2, PEPTIDE SUBSTITUTION OF PG partial lack in Micrococcus luteus 3, 5, 7 , PEPTIDOGLYCAN anti-metastatic activity of 204, 206, anti-tumor activity 204, 249, 255, 256,

432 arthritis induced by 277-279, 314, 317, biological activity of, in man 291, 305, biosynthesis 46, complement activation by 264, 284, 298, 299, 319, 323, crossreactivity between PGs 95, 101, degradation of 191, 197, 209, 215, 230, digestion of 48, disaccharide peptide monomers 314, 315, 317, effect on cells of the immune system 229, effect on human basophils 283, effect on mast cells 283, effect on phagocytic cells 261, effect on PMN function 26 3, glycan strand 2, 4, 5, 75, 78, 132, 135, 209, 221, hemorrhage in gut lympoid tissue induced by 283, histamine release induced by 283, inhibition of synthesis 190, interaction with lymphocytes 229, 231, 232, interleukin 1 production induced by 283, intraarticular injection with 278, immunochemistry of 1, 61, 95, 145, macrophage activation by 284, 298, 299, micellar structure of 45, models of 37, 44, molecular architecture of 37, 44, monomers, metabolic fate of 204, pentapeptide subunit of 2, 5, 11, 95, 105, 131, 136, 310, peptidoglycan types 2, persistence of 316, 317, phlogistic properties of 273-283, platelet aggregation or lysis induced by 284, 285, polar caps of 209, precursor sequence of 3, preparation of 320, 321, removal of teichoic acid from 320, 321, sacculus 209, self association of 46,

of staphylococci 7, 9, 191, 197, soluble PG 7, 79, 95, 145, 190, 221, spatial arrangement of 37, 43, stimulation of oxygen metabolism by 231, structure of 2, 37, 43, 49, 55, 61 , 209, structure-function relationship of 1-20, 37, 43, 145, 273, 285, synthesis 215, synthesis, inhibition of 190, synthetic analogs of 383, T-cell activation by 231, tetrapeptide subunit of 2, 5, 131 , thrombocytopenia, induced by 284, 285, tissue localization of 316, turnover products of 221 , ultrastructural localization of 130, 135, PEPTIDOGLYCAN-POLYSACCHARIDE POLYMERS 167, antibodies to 90, arachidonic acid metabolite production induced by 283, complement activation by 284, edema induced by low mol. wt. fragments of 276, 278, 279, effect on PMN's 284, effect on T-cells 285, from Streptococcus pyogenes (group A) 273-286, from Streptococcus faecium (group D) 275, 276, from Streptococcus agalactiae (group B) 275, 276, from Lactobacillus casei 275, 276, from Peptostreptococcus productus 275, 276, from Propionibacterium acnes 275, 276, intraarticular injection with 278, levels in vivo 62, molecular weight analyses of 64, 65, muralytic digestion of 276, 278, 279, N-acetylation or de-O-acetylation of 276, 278, 279,

phlogistic properties of 273-286, sedimentation field flow fractionation 64, 65, thrombocytopenia induced by 284, PEPTOCOCCUS ANAEROBIS secretion of soluble PG 149, PEPTOSTREPTOCOCCUS PRODUCTUS PG-polysaccharide polymers of 275, 276, PEROXIDASE-LYSOZYME CONJUGATES 1 30, PERSISTENCE of PG 316, 317, PHAGOCYTIC CELLS effects of PG on 261 , PHAGOCYTOSIS 405, inhibition of 229, 230, PHI X-174

215,

PHLOGISTIC PROPERTIES of PG 273-283, of PG-polysaccharide polymers 273-286, PLAQUE FORMING CELL ANTIBODY RESPONSE 393, PLASMIDS 215, PLATELET AGGREGATION induction by PG 284, 285, PLATELET LYSIS induction by PG 284, 285, POLYANETHOLE SULFONATE (LIQUOID) 172, POLAR CAPS of PG 209, POLYARTHRITIS 62, POLYCLONAL ACTIVATION of lymphocytes 232, POLYELECTROLYTE S anionic 168, cationic 169, 171,

POLY-L-ARGININE 171, PORES 215, POSTOPERATIVE WOUNDINFECTION 124, PRECURSOR SEQUENCE Of PG 3, PREDICTIVE VALUE of iS. aureus serology 28, PROPRIONIBACTERIUM ACNES PG-polysaccharide polymers of 275, 276, PROSTAGLANDINS 230, PROTEUS MIRABILIS action of ß-lactam antibiotics on 187, conversion to spheroplasts 1 defective cell walls of spheroblasts 190, growth in spheroplast form 189, penicillin binding proteins

188,

PYELONEPHRITIS 127, PYODERMA 297, PYROGEN 344, endogenous 2 30, PYROGENICITY of MDP derivatives 379, 381, 383, 384,

R RADIOACTIVE HAPTEN BINDING ASSAY 6, 105, 135, detection of secreted PG 221 inhibition of 11, RADIOIMMUNOASSAY see also: SPRIA levels of secreted PG 221 , levels of teichoic acid anti bodies 113,

434 RAPID EVE MOVEMENT 326, RAST 71 , REPEATING UNITS of LPS 343, RESPIRATORY BURST 168, 170, 263, RHAMNOSE gas chromatography-mass spectrometry, levels in tissue 64, RHEUMATOID FACTOR 89, RHINITIS, BACTERIAL 297, RIBITOL TEICHOIC ACID 118, RIFT VALLEY FEVER VIRUS 256,

S SARCOMA T 241 sarcoma 257, SALMONELLA ENTERITIS 125, SCARLET FEVER 109, SECRETION OF PG 145, 149, 189, 221, 223, 224, levels of, by RIA 221, SEDIMENTATION FIELD FLOW FRACTIONATION 64, 65, SELF ASSOCIATION of PG 46, SEPTICEMIA 24, 123, 127, 261, by S^. aureus 21 , SEPTUM 209, SHIGELLA ENTERITIS 125, SINUSITIS, BACTERIAL 297, SKIN REACTIONS induced by PG 292, 299, 308, 386, 387,

SKIN TUMOR UV induced 257, SLEEP see: SLOW WAVE SLEEP SLOW WAVE SLEEP 329, induction of 231, slow wave sleep promoting activity 230, SOLUBLE PG 7, 79, 95, 145, 190, 221, antibodies cross-reactive with 157, definition 145, detection 145, 148, 221, immunogenicity 159, inflammatory properties 163, in man 154, mitogenicity 159, physiological properties 149, polyclonal activation by 162, secretion 149, 221, 224, structure 151, SOMNOGENIC MURAMYLPEPTIDES 329, SPATIAL ARRANGEMENT of PG 37, 43, SPHEROBLASTS 187, 189, SPRIA, SOLID-PHASE-RADIOIMMUNOAS SAY levels of IgE antibodies to S. aureus cell walls 67, levels of IgG antibodies to S. aureus PG 24, levels of antibodies to teichoic acid 113, STAPHYLOCOCCAL PG O-acetylation 7, 191, 197, STAPHYLOCOCCAL SCALED SKIN SYNDROME 109, STAPHYLOCOCCI antibodies to interpeptide bridge of 9, degradation of PG 191, 197, STAPHYLOCOCCUS AUREUS 405, 406, antibodies to 113, bacteremia 113, 115, carboxypeptidase of 11,

435

degradation of PG 191, endocarditis 115, growth under penicillin G 225, immunoglobulin E antibodies to 67, immunoelectronmicroscopy 1 37, infections 21, 149, labelled with 14C-GlcNAC 171, 174, S. aureus 52A5 136, 319, S. aureus Cowan I 305, S. aureus H 114, S. aureus Wood 46 114, secretion of PG 223, septicemia 21 , serology 113, 114-116, serology, predictive value of 28, soluble PG from 145, teichoic acid 113,

STRUCTURE calculation- 45, of cell walls 37, 129, 341, determination 49, of glycan moiety of PG 2, of interpeptide bridge of PG 2, micellar, of PG 45, of PG 2, 37, 43, 55, 61, of soluble PG 151, structure-function relationship 1-20, 37, 61-66, 145-166, 341-352, 353-370, SYNECHOCYSTIS 55, SYNOVIAL ACTIVATOR ACTIVITY 230,

STAPHYLOCOCCUS EPIDERMIDIS 305,

SYNTHESIS of PG 215,

STEM PEPTIDE 2,

of PG, inhibition of 190,

STREPTOCOCCAL A-CARBOHYDRATE antibodies to 6,

SYNTHETIC immunogens 6, 8, 10, 11, 75, 95, 135, 137, 148, 190, lipid A 347 , lipopeptides 335, MDP analogs 37 9, vaccine models 408,

STREPTOCOCCUS AGALACTIAE PG-polysaccharide polymers of 275, 276, STREPTOCOCCUS FAECALIS see: ENTEROCOCCUS FAECALIS STREPTOCOCCUS FAECIUM see: ENTEROCOCCUS FAECIUM STREPTOCOCCUS PNEUMONIAE PBP from 84, secretion of soluble PG 149, STREPTOCOCCUS PYOGENES 129, 131, 136, immunoelectron microscopy 137, M-protein sequence 130, PG-polysaccharide polymers of 273-286, secretion of soluble PG 149, 223,

T T-CELL ACTIVATION by PG 231, T-CELL REPLACING (HELPER) ACTIVITY 231,

STREPTOLYSIN 0 antibodies to 107,

TEICHOIC ACID 113, 168, 180, activation of alternate pathway of complement 319, antibodies to 43, 113, counterImmunoelectrophoresis 113, ELISA 113, immunodiffusion 113, indirect hemagglutination 113, preparation of 114, radioimmunoassay 113, removal from PG 320, 321, ribitol teichoic acid 118,

STREPTOMYCES ALBUS D,D-carboxypeptidase 389,

TETANUS TOXOID antibodies to 267,

STREPTOCOCCUS PYOGENES A-VARIANT antibodies to 10,

436 TETRAPEPTIDE SUBUNIT OF PG 131 , antibodies to 5, structure of 2, THROMBOCYTOPENIA 2 65, induced by PG 284, 285, induced by PG-polysaccharide polymers 284, THYMOCYTE ACTIVATING FACTOR 230, TISSUE LOCALIZATION, OF PG 316, levels of muramic acid 64, levels of rhamnose 64, TRANSGLYCOSYLASE 212, TREHALOSE DIMYCOLATE 401-406, TRIALANINE 11, 131, TUMORICIDAL EFFECT 255, 258, TUMORS Lewis lung carcinoma 204, mammary carcinoma 204, B 16 melanoma 204, 257, T 241 sarcoma 257, skin tumors, UV-induced 257, TURNOVER OF PG 221, ULCEROUS COLITIS 125, ULTRASTRUCTURAL LOCALIZATION of PG 130, 135, URINARY TRACT INFECTION 123, 127, UVEITIS 62 VANCOMYCIN 148, VASCULITIS, ALLERGIC 298, VIRUSES Rift Valley Fever Virus 256, Herpes simplex virus 256, HTLV III 305, WOUND INFECTION 124, 127, X-RAY DIFFRACTION 4 3

The Target of Penicillin The Murein Sacculus of Bacterial Cell Walls Architecture and Growth Proceedings • International FEMS Symposium Berlin (West), Germany, March 13-18,1983 Editors

R. Hakenbeck, J.-V. Holtje, H. Labischinski

1983.17 cm x 24 cm. XXVIII, 663 pages. Numerous illustrations. Hardcover. DM 180,-; approx. US $85.70 ISBN 311009705 2

Immunoassay Technology Volume 1 Editor

S. B. Pal

1985.17 cm x 24 cm. VIII, 192 pages. Numerous illustrations. Softcover. DM 118,-; approx. US $56.20 ISBN 311010062 2 This is the first volume of a series on Immunoassay Technology which includes Review Articles and Methods and deals essentially with immunological methods of biological, commercial and environmental importance, without introducing radioactive isotopes.

Immunoassay Technology Volume 2 Editor

S. B. Pal

1986.17 cm x 24 cm; approx. US $70.50

ISBN 311010948 4

The second volume of this series, Immunoassay Technology, contains several articles which it is hoped readers will find as interesting, useful and thought-provoking as those presented in the previous volume, in particular to recent entrants in this field.

Prices are subject to change without notice

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WALTER DE GRUYTER • BERLIN • NEW YORK

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Verlag Wfeüter de Gruyter & Co., Genthiner Str. 13, D-1000 Berlin 30, Tel.: ( 0 3 0 ) , 2 6 0 0 5 - 0 , Telex 1 8 4 0 2 7 Walter de Gruyter, Inc., 200Saw Mill River Road, Hawthorne, N.Y. 10532, Tel. : (914) 747-0110, Telex 64 6677

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Concise ^yclopedia B i o c h

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s t r y Edited by Thomas Scott and Mary Brewer 2nd printing with corrections. 1983.14 cm x 21,5 cm. VI, 519 pp. Approx. 650 illustrations. Hardcover. DM 78,-; US $34.95 ISBN 3110078600

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The Concise Encyclopedia of Biochemistry, with more than 4,200 entries, is the foremost collection of current information in this rapidly expanding field. The contents are complemented by numerous structural formulas, metabolic pathways, figures and tables. All those interested in or working in the field of Biochemistry and Biology (Life Sciences), will profit from the information contained in this encyclopedia. This truly remarkable book is an essential reference for Biochemists, Clinical Chemists, Clinical Biochemists, Clinicians, Medical Researchers and Experimental Biologists. It will also serve as a very useful source of information for students.

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WALTER DE GRUYTER • BERLIN • NEW YORK Verlag Walter de Gruyter & Co., Genthiner Str. 13, D-1000 Berlin 30, Tel.: (030),26005-0, Telex 184027 Walter de Gruyter, Inc., 200Saw Mill River Road, Hawthorne, N.Y. 10532, Tel. : (914) 747-0110, Telex 64 6677