International Review of Cytology: A Survey of Cell Biology, Volume 118

INTERNATIONAL REVIEW OF CYTOLOGY VOLUME118 SERIES EDITORS GEOFFREY H. BOURNE JAMES F. DANIELLI KWANG W. JEON MARTIN ...

Author: Geoffrey H. Bourne | Kwang W. Jeon

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INTERNATIONAL

REVIEW OF CYTOLOGY VOLUME118

SERIES EDITORS GEOFFREY H. BOURNE JAMES F. DANIELLI KWANG W. JEON MARTIN FRIEDLANDER

1949-1988 1949- I 984

19671984-

ADVISORY EDITORS H. W. BEAMS HOWARD A. BERN DEAN BOK GARY G. BORISY BHARAT B. CHATTOO STANLEY COHEN RENE COUTEAUX MARIE A. DiBERARDINO DONALD K. DOUGALL BERNDT EHRNGER CHARLES J. FLICKINGER NICHOLAS GILLHAM M. NELLY GOLARZ DE BOURNE MARK HOGARTH KEITH E. MOSTOV AUDREY MUGGLETON-HARRIS

ANDREAS OKSCHE MURIEL J. ORD VLADIMIR R. PANTIC M. V. PARTHASARATHY LIONEL 1. REBHUN JEAN-PAUL REVEL L. EVANS ROTH JOZEF ST. SCHELL HIROH SHIBAOKA JOAN SMITH-SONNEBORN WILFRED STEIN RALPH M. STEINMAN HEWSON SWIFT MASATOSHI TAKElCHI M. TAZAWA ALEXANDER L. YUDIN

INTERNATIONAL

Review of Cytology A SURVEY OF CELLBIOLOGY

Editor-in-Chief

G. H. BOURNE (Deceased)

Editors

K. W. JEON

M. FRIEDLANDER

Depurtment of Zoology University of Tennessee Knoxville, Tennessee

Jules Stein Eye Institute UCLA School of Medicine Los Angeles, California

VOLUME118

ACADEMIC PRESS, INC. Harcourt Brace Jovanovich, Publishers

San Diego London

New York Berkeley Boston Sydney Tokyo Toronto

This book is printed on acid-free paper.

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COPYRIGHT 0 1989 BY ACADEMIC PRESS, INC. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher.

ACADEMIC PRESS, INC. San Diego, California 92101 United Kingdom Edition published by ACADEMIC PRESS LIMITED 24-28 Oval Road. London NWI 7DX

LIBRARY OF CONGRESS CATALOG CARD NUMBER: 52-5203

ISBN 0-12-364518-2

(alk. paper)

PRINTED IN THE UNITED STATES OF AMERICA 84

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Contents CONTRIBUTORS ............................................................................................ GEOFFREY H. BOURNE ..................................................................................

vii ix

Differentiation of the Bacterial Cell Division Site WILLIAMR. COOK,PIETA. J.

DE

BOER. AND LAWRENCE I. ROTHFIELD

I. Introduction ....................................... I I . Structure o f t n Apparatus ..................... 111. Biogenesis and Localization of the Division Site ..... ........................... IV. Formation of the Septum ......... ............................................ V. Regulation of the Division Process ....................... VI. Conclusions ................... ......................................... ........................................ ........ References .....................

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27 28

Properties of the Cell Surfaces of Pathogenic Bacteria R. J. DOYLEAND E. M. SONNENFELD 1. Introduction ....................................................................................... ......... 11. Surface Structures of Bacteria ............................................... 111. Biological Reactions to Peptidoglycans ................................................... IV. Bacterial Cell-Surface Amphiphiles ........................................................ V. Surface Adhesins of Bacteria and Pathogenesis ...................................... VI. Turnover of Cell Wall a thogenesis .................................................. VII. Concluding Remarks ... ......................................... References ........................ .........................................

33 34 43 49 58 14 83 84

Cellular Studies on Marine Algae AHARONGIBOR

.......... .............................. ........................ ................................ ......................................... Eoergesenia ........... .............................. .................. Porphyra ...............

1. Introduction

11. Acetabularia

Ill. IV. V. Concluding Remarks References ...................

V

93 94 102

vi

CONTENTS

The Centrifugal Visual System of Vertebrates: A Century-Old Search Reviewed J . REPERANT,D. MICELI,N . P. VESSELKIN.A N D S. MOLOTCHNIKOFF

I. 11. 111. IV. V. VI. Vll. VIII.

Introduction .... ........ ..................................... ...................................... C yclostomes ............... Fish ....... ....... ............ .......... .... ................... . ................................................................. Amphibians ..... ...

................. ................................................................................... ns ....................................................................................... s ...................................................

1 I5 116

i20 127 130 133 151 160 164

Cell Biology and Kinetics of Kupffer Cells in the Liver K. W A K ~K. . D ~ c K ~A. K ,KIKN.D. L. KNOOK,R. S. M C C U S K ~ Y , L. B O U W ~ N SA.N D E. Wissk

I. 11.

Ill. IV. V. VI.

VII. VIII.

1x.

Introduction ... ................................., .................................................. Morphology of Kupffer Cells ........................................................ Population Dynamics of Kupffer Cells s .............. ............ Isolation, Purification, and Culture of Metabolic Responses of Stimulated Rat Kupffer Cells in Vitro Endocytosis ........ ....................................................................... Kupffer Cells and Endotoxin ................................................................. ......................... Kupffer Cells in Infectious Diseases .... Concluding Remarks .......... References .... .....................................................................................

173 176 181 187 191 198

203 210 220 22 1

Cellular and Molecular Biology of Capacitation and Acrosome Reaction in Mammalian Spermatozoa K. S. SIDHIJ A N D S. S. GUKAYA 1.

Introduction .................,

11. Capacitation ..._............., 111. Acrosome Reaction ............ ............ . ............

1V. Conclusion and Prospects References ....................

INDEX.........................................................................................................

281

Contributors

Numbers in parentheses indicate the pages on which the authors' contributions begin.

L. BOUWENS (173), Laboratorium voor Celbiologie en Histologie (VUB), 1090 Brussel-Jette, Belgium

WILLIAM R. COOK (l), Department of Microbiology, University of Connecticut Health Center, Farrnington, Connecticut 06032 PIET A. J. DE BOER ( l ) , Department of Microbiology, University of Connecticut Health Center, Farrnington, Connee ticut 06032

K. DECKER(173), Biochernisches Institut, Albert-LudwigsUniversitat, Federal Republic of Germany R. J. DOYLE (33), Department of Microbiology and Immunology, Health Sciences Center, University of Louisville, Louisville, Kentucky 40292 AHARONGIBOR(93), Department of Biological Sciences, University of California, Santa Barbara, California 93106 S. S. GURAYA (23 l), I.C.M.R. Regional Advanced Research Centre in Reproductive Biology, Department of Zoology, College of Basic Sciences and Humanities, Punjab Agricultural University, Ludhiana, India A. KIRN (173). Laboratoire de Virologie, Faculte' de Mede'cine and INSERM U74, 67000 Strasbourg, France

D. L. KNOOK (173), Instituut voor Experimentele Gerontologie TNO, 2280 HV Rijswijk, The Netherlands vii

...

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CONTRlBUTORS

R. S. MCCUSKEY(173), Department of Anatomy, School of Medicine, University of Arizona, Tucson, Arizona 85724 D.

MKELI ( 1 15), Laboratoire de Neuropsychologie, UniversitP du Que'bec, Trois-Rivieres, Que'bec, Canada

S. MOLOTCHNIKOFF( 1 15), De'partement de Sciences Biologiques, UniversitP de Montre'al, Montre'al, Que'bec, Canada

J. R E P ~ R A N( 1T1 9 , Laboratoire de Neuromorphologie U106, INSERM, H6pital de la Salpetrie're, Paris, France

LAWRENCE I. ROTHFIELD(l), Department of Microbiology, University of Connecticut Health Center, Farmington, Connecticut 06032 K . S. SIDHU(231), I.C.M.R. Regional Advanced Research Centre, in Reproductive Biology, Department of Zoology, College of Basic Sciences and Humanities, Punjab Agricultural University, Ludhiana, India E. M. SONNENFELD (33), Department of Microbiology and Immunology, Health Sciences Center, University of Louisville, Louisville, Kentucky 40292 N . P. VESSELKIN ( 1 15), Institut Sechenov, Leningrad, U.S.S.R.

K . WAKE(173), Department of Anatomy, Tokyo Medical and Dental University, Faculty of Medicine, Yushima, Bunkyoku, Tokyo 113, Japan E. WISSE(173), Laboratorium voor Celbiologie en Histologie (VUB), 1090 Brussel-Jette, Belgium

This Page Intentionally Left Blank

GEOFFREY H. BOURNE (November 17, 1909-July 19,1988)

In Memoriam: Geoffrey H. Bourne On July 19, 1988, the International Review of Cytology-Cell Biology lost its Editor-in-Chief and remaining founder, Professor Geoffrey H. Bourne (co-founder, Professor James F. Danielli, passed away on April 22, 1984). Professor Bourne died rather unexpectedly in a New York hospital while visiting the city to attend a graduation ceremony for the 1988 class of St. George’s University School of Medicine, Grenada, West Indies, of which he was Vice Chancellor and Professor of Nutrition. Among his many prominent achievements, Professor Bourne, along with Professor Danielli, founded the International Review of Cytology in 1950, while he was teaching histology at the University of London. The first volume was published in 1952. Bourne intended to keep the scope of the new international series “as wide as possible to deal with all aspects of cell biology including morphological and chemical studies of both cells and tissues.” Papers presenting new theories of general interest were to be welcomed also. The editors initially published only one volume a year, until 1967,when the number was increased to two. Thereafter, the number of volumes published each year gradually increased to the present rate of five to seven volumes per year, with occasional special volumes introduced during the 1970s. Until 1970, the two founding editors and assistant editor, Kwang W. Jeon, who joined them in 1967, searched the literature to select authors and subjects for review. The task of covering the wide range of rapidly advancing cell biology became too onerous for them alone, so in 1970, they established an advisory board of 22 members drawn from eight different countries. Eight of the original board members still serve. Professor Bourne led a very colorful and successful professional life as a teacher, scientist, administrator, author, editor, and even diplomat. He has many monographs, edited series, and more than 500 scientific articles published in medical and scientificjournals to his credit. Born on November 17, 1909, in Perth, Australia, he received his B.Sc., M.Sc., and D.Sc. degrees in biology from the University of Western Australia. He continued his studies at the University of Oxford, where he received his doctorate in histology in 1943. Between 1943 and 1946, he served in the British

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IN MEMORIAM: GEOFFREY H.BOURNE

Armed Forces as an officer in charge of R&D for special forces and as a nutritional advisor in Southeast Asia. In 1957, he moved from the University of London to the Emory University Medical School in Atlanta to take up chairmanship of the Department of Anatomy. In 1962, he became director of the Yerkes Primate Research Center of Emory University while continuing his active research in histochemistry and ultrastructural studies of various tissues and organs. After retiring from the Yerkes Primate Research Center, Professor Bourne joined the newly established St. George’s University School of Medicine in 1978, as a professor of nutrition. Soon thereafter, he assumed the role of Vice Chancellor. The scientific community at large has greatly benefited from Professor Bourne’s creativity. leadership, vision, and wisdom, and owes him a great debt of gratitude. All who knew and worked with Professor Bourne have suffered a great loss. We shall miss him for his keen insight, charm, and sense of humor. However, we are comforted to know that the fruits of his labor, including the International Review of Cytology-Cell Biology, will remain with us and continue to enrich us for many years. KWANCW. JEON MARTINFRIEDLANDER

INTERNATIONAL REVIEW OF CYTOLOGY, VOL. 118

Differentiation of the Bacterial Cell Division Site WILLIAM

R.

J. DE BOER, AND LAWRENCE I. ROTHFIELD

COOK, PIET A.

Department of Microbiology, University of Connecticut Health Center, Farmington, Connecticut 06032

I. Introduction

A. BACKGROUND In most bacterial species cell division occurs by the ingrowth of a division septum at the midpoint of the cell. This process is under strict topological and temporal control. We will discuss this subject as a problem in subcellular differentiation, in which a complex structure is constructed at a specific location in the cell in a process that must be coordinated with a variety of other cellular events, most notably with chromosome replication and segregation. In recent years a major change in thinking about bacterial division has come from the demonstration that early events in the differentiation process can be detected long before the initiation of septa1 ingrowth. In addition, more recent evidence suggests that the residual division site continues to carry out specific functions even after cell separation is completed. Thus, a major theme of this review will be the idea that the division site itself has a developmental history in which the stages of genesis, maturation, and localization, and ultimate fate have become accessible to study. In this context we will describe evidence obtained from several disciplines: genetics, microscopy, and, to a lesser extent, biochemistry. We will begin by briefly discussing differences between eukaryotic and prokaryotic cell division, and will describe aspects of bacterial cell envelope organization and chromosomal replication that are relevant to the later discussion of the division process itself. The main body of the review will be divided into three parts concerning (1) the formation and localization of the division apparatus, (2) the formation of the division septum, and (3) the coordination of cell division with other cellular events, such as DNA replication and chromosome segregation. The discussion will be largely limited to information derived from studies of gram-negative bac1 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

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WILLIAM K. COOK

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teria, most especially of Escherichiu coli, because the combined application of genetics with other techniques to study cell division in this organism has been particularly fruitful in recent years. It should be pointed out that a substantial body of important work has also been done in other species. Although division in both bacteria and eukaryotic cells is accomplished by the circumferential invagination of the cytoplasmic membrane and cell envelope, the details differ considerably, reflecting the different organization of surface structures in the two groups. For example, contractile elements have not been described in most bacterial species. Therefore, it is not surprising that the contractile ring that appears t o play an important role in cytokinesis in animal cells (Beams and Kessel, 1976) is absent from dividing bacteria. On the other hand, the bacterial cell envelope contains a highly crosslinked peptidoglycan structure, the murein (described more fully later), that is not found in eukaryotic cells but plays a key role in the bacterial division process. The murein is the only known rigid structure in bacterial cells and, therefore, although it lies outside of the cytoplasmic membrane, may fulfill some of the roles ascribed t o the intracellular cytoskeleton of higher cells. The driving force in formation of the bacterial division septum may come from the circumferential inward growth of the rigid murein layer. In the simplest model, inner membrane is passively pushed ahead of the ingrowing murein while outer membrane is pulled behind, thereby leading to the coordinate inward movement of the three layers of the septum. This simplistic model provides a convenient starting point for thinking about the septation process.

B. ORGANIZATION OF THE BACTERIAL CELLENVELOPE As indicated in Fig. 1 , the cell envelopes of E. coli and other gramnegative bacteria contain three morphological layers: cytoplasmic (inner) membrane, murein, and outer membrane (Inouye, 1979). The compartment between the inner and outer membranes is termed the periplasmic compartment or periplasmic space. Formation of the division septum requires the ingrowth of this complex structure at the proper site within the cell. The cytoplasmic membrane acts as the major osmotic barrier of the cell, and contains a large number of specific transport proteins as well as proteins involved in energy transduction and other cellular processes. In contrast, the outer membrane contains a more limited spectrum of proteins, including several “porins” that permit the passage of various low molecular weight solutes into the periplasm (Nikaido, 1979). The murein layer is composed of an extensively crosslinked peptidogly-

DIFFERENTIATION OF BACTERIAL CELL DIVISION SITES

3

FIG. 1. Diagram of the Escherichia coli cell envelope. OM, Outer membrane; OmpA, OmpA protein; Mur, murein layer; PPS, periplasmic space; IM, inner membrane; MLP, bound form of murein-lipoprotein.

can that completely surrounds the cell. This bag-shaped molecule determines the shape of the organism and is responsible for the rigidity and the resistance of the cell to mechanical and osmotic stresses. Cell elongation and septal ingrowth both require that new peptidoglycan units be inserted into the continuous murein structure (Mirelman, 1979). This requires the controlled breaking of covalent bonds within the structure to permit the directed insertion of the new material. There is reason to believe that different biochemical mechanisms are used, at least in part, for insertion of septal murein as opposed to “elongation murein” (i.e., the murein along the length of the cell cylinder) (Mirelman, 1979). When examined by conventional electron microscopy, the murein layer of gram-negative bacteria appears as a thin, dense layer of 2-3 nm thickness that is closely apposed to the outer membrane (Fig. 1). Studies with alternative methods of preparation were interpreted as showing a less densely packed peptidoglycan domain between the dense murein layer and the inner membrane (Hobot et a / . , 1984; Leduc et al., 1985). Biochemical evidence shows that a number of intrinsic outer-membrane proteins (e.g., OmpA and murein-lipoprotein) are involved in attaching the outer membrane to the murein layer (Fig. 1). The outer-membrane proteins that mediate this attachment are sufficiently abundant (-lo5 copies per cell) to provide -400,000 contact points between murein and outer membrane (Park, 1987). The periplasmic compartment contains a number of water-soluble proteins. These proteins include degradative enzymes, proteins that inactivate toxic exogenous substances, and proteins that participate in solute transport and in chemotaxis (Brass, 1986). It is likely that other functional periplasmic proteins remain to be identified, and the possibility must be considered that some of these may play a role in the septation process.

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Proteins can diffuse within the periplasmic space, although the diffusion rate is slower than in free aqueous solution (Brass, 1986; Foley rt ul., 1989). The periplasm can be operationally divided into two regions: the inner periplasm that lies between the inner membrane and the dense murein layer, and the outer periplasm located between murein and outer membrane (Fig. I). Free diffusion within the outer periplasm may be limited by the close apposition of murein and outer membrane and by the very high concentration of fixed proteins (most prominently OmpA and murein-lipoprotein) in this region. Significant movement of proteins within the periplasmic space may therefore be restricted to the inner periplasm.

C. DIFFERENCES BETWEEN CHROMOSOME SEGREGATION IN PROKARYOTIC AND EUKARYOTIC CELLS There are several differences in the process of chromosome segregation between bacteria and eukaryotic cells. Most strikingly, microtubules or analogous structures have not been shown to exist in bacteria, suggesting that the link between the chromosome and the cellular site responsible for directing daughter chromosomes to progeny cells in bacteria is likely to be less complex than in eukaryotes. Consistent with this fact, a mitotic apparatus has not yet been described in bacteria. It is widely believed-though without any direct evidence-that chromosome segregation occurs in bacteria by the attachment of daughter chromosomes to cell envelope sites that direct each of the daughter chromosomes to a different progeny cell. In the model originally proposed by Jacob et ul. (19631, the chromosomal site is located at o r near the origin of replication. In this model, still the most widely accepted one, the processes of chromosome replication and chromosome segregation are coupled and the driving force for chromosome separation is provided by insertion of new cell envelope material between the attachment sites. We will discuss in this review relevant aspects of this problem together with the question of how the cell coordinates the placement and synthesis of the division septum with the processes of DNA replication and chromosome segregation.

XI. Structure of the Division Apparatus A. THE PERISEPTAL ANNULARAPPARATUS Until the past few years, the only visible event in the cell division process was the formation of the division septum. However, earlier stages in the differentiation process have now become accessible to study. This


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was made possible by the discovery of a new organelle, the periseptal annular apparatus, which is present at the future division site before the onset of septal ingrowth (Anba et al., 1984; MacAlister et al., 1983). Studies of the structure and biogenesis of the periseptal annuli have provided a large amount of new information dealing with the process of formation and localization of the division site. The periseptal annuli are two concentric rings that surround the cell at the site of cell division (Fig. 2A). Electron microscopy has revealed that each annulus consists of a narrow zone within the cell envelope, in which the inner membrane, murein, and outer membrane lie in close apposition to each other (Fig. 2B). Three-dimensional reconstructions from serialsection electron micrographs showed that the zones of membrane-murein association are continuous structures that run completely around the cell cylinder. As discussed in more detail later, the annuli appear at the future division site long before the onset of septal invagination, thereby defining the future division site (Cook et al., 1986, 1987). The septum is later formed within the cell envelope domain (the periseptal domain) that lies between the two annuli. In bacterial cells the osmotic pressure resulting from the high intracellular concentration of impermeant solutes pushes the cytoplasmic membrane against the murein layer. Therefore, to visualize structures such as the annular attachments, cells are first plasmolyzed by brief exposure to hypertonic solutions of sucrose or other solutes that can enter the periplasm but do not readily cross the cytoplasmic membrane barrier. This results in a loss of cytoplasmic volume, causing the inner membrane to retract from the rigid murein-outer membrane layer. As shown by Bayer

FIG.2. Diagrammatic representation of periseptal annuli in surface view (A), and cross section (B) at several stages of progression through the cell cycle. PSA, Periseptal annuli; PA, polar annuli: OM, outer membrane; IM, inner membrane; M, murein. From Rothfield er al. (1986).

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WILLIAM R. COOK t r

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et al. in an important series of papers (Bayer, 1979), random electron micrographs of plasmolyzed cells reveal numerous sites where the inner membrane fails to retract from the outer layers of the cell envelope. These attachments were named zones of adhesion by Bayer, and will be referred to as “Bayer bridges” in this review to avoid confusion with the adhesion zones that comprise the periseptal annuli. It is interesting to note that zones of adhesion between inner and outer membranes of isolated chloroplasts and mitochondria can also be visualized following exposure to hypertonic solutions (Cremers et al., 1988; Hackenbrock, 1968). The circumferential membrane-murein attachments that form the periseptal annuli and their biogenetic progenitors resemble the Bayer bridges when viewed in individual thin sections (MacAlister et al., 1983). It is not known whether all of the Bayer bridges that are visible in electron micrographs of random sections are in fact components of continuous structures such as the periseptal annuli, o r whether there are several types of adhesion zones that are structurally and functionally distinct.

B. ROLE OF T H E PERISEFTAL ANNULARAPPARATUS Although there is good evidence that the periseptal annuli are associated with the division process, the physiological role of the annular apparatus has not been established. Two possible roles, which are not mutually exclusive, have been suggested: ( I ) the annuli act as gaskets to segregate the division site from the remainder of the cell envelope, and (2) essential elements of the machinery are components of the annuli themselves. The gasket hypothesis was based on the morphological appearance of the annular attachments. The circumferential membrane-murein adhesion zones that comprise the annuli appear in electron micrographs to separate the periplasmic space into separate compartments, one of which defines the periseptal domain that lies between the paired annuli at midcell. It therefore was hypothesized that the annuli might act as physical barriers to prevent the unrestricted movement of molecules into and out of the periseptal domain. This would permit the cell to accumulate at this site proteins o r other components required for septum formation, ensuring that septation was restricted to the proper location. Studies by Foley et al. (1989) have provided experimental support for the gasket hypothesis. In these studies proteins labeled with fluorescent groups were introduced into the periplasm. The ability of the labeled proteins t o diffuse into different regions of the periplasmic space was followed by measuring the recovery of local fluorescence after the irreversible photobleaching of probe molecules within localized regions. The


7

results indicated that certain regions of the periplasm were not in free communication with the remainder of the periplasmic space. These sequestered regions were preferentially located at potential division sites and at cell poles. This supports the view that the periseptal and polar annuli act as functional barriers to the passage of molecules into and out of the periseptal and polar domains that previously had been identified morphologically. It therefore is reasonable to suggest that one role of the annuli is to segregate the periseptal domain from the remainder of the cell envelope. It will be of interest to extend these studies to the inner and outer membranes to determine whether the barrier function is limited to the periplasm or whether membrane components are also sequestered at division sites by the annular gaskets. It is also possible that essential division components may be part of the annular attachments themselves. This question has not been accessible to experimental study because the annuli have not yet been isolated.

c. MOLECULARORGANIZATION OF PERISEPTAL ADHESIONZONES The molecular organization of Bayer bridges and of the periseptal adhesion zones is unknown. It appears likely t o us that the adhesion zones represent sites where inner membrane is attached to murein. This is based on the observation that the inner membrane at these locations resists the strong inward pull resulting from the plasmolysis procedure. This requires that inner membrane be attached to a rigid structure that can itself resist the inward pull. Since the murein is the only rigid structure that could provide an anchor for the inner membrane, we think it likely that protein(s) at these sites interact simultaneously with inner membrane and with the peptidoglycan that comprises the murein sacculus. The following general models are compatible with this formulation (Fig. 3): 1 . The adhesion zones are sites at which proteins directly attach inner membrane to murein without any involvement of outer membrane (Fig. 3A). This would explain the fact that inner membrane remains associated with the murein-outer membrane layer at these sites despite the inward pull of the plasmolysis procedure. 2. The adhesion zones contain protein that attach both the inner and outer membranes to the murein layer (Fig. 3B). This would explain the fact that inner membrane remains associated with the murein-outer membrane layer at these sites despite the inward pull of the plasmolysis procedure and would also be compatible with the evidence that the annular adhesion zones act as diffusional barriers within the periplasm. It should be noted that an outer membrane-murein attachment

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WILLIAM R. COOK

ET AL

FIG.3. Hypothetical models of zones of adhesion. For details see text and Fig. 1.

would not be needed to explain the barrier function if lateral diffusion of proteins did not occur at a significant rate in the outer periplasm (i.e., the region between murein and outer membrane), as discussed in Section I,B. Though less likely, in our opinion, than the models just described, it is also possible that the adhesion sites represent regions of fusion between the lipid matrices of inner and outer membranes. Two such models are illustrated in Fig. 3C and D. In Fig. 3C the adhesion zones represent sites where the fusion of inner- and outer-membrane bilayers results in a continuous bilayer that connects the two membranes. This model is untenable in its simplest form, since an aqueous pore would be formed between cytoplasm and the external medium. This problem could be avoided if the sites contained protein(s) that plugged the potential pore. In Fig. 3D the adhesion zones represent sites where the inner- and outer-membrane bilayers have fused to form a new mixed bilayer. This structure would pre-


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C

D FIG.3. (con?.)

sumably be relatively unstable because of the suboptimal packing of phospholipid molecules at the junctions. This problem might be avoided if the sites contained proteins that served both to stabilize the common bilayer phase and to anchor the membrane to murein at these sites.

D. MEMBRANE-PEPTIDOGLYCAN ATTACHMENT AT THE LEADING EDGE OF THE SEPTUM A second differentiated structure based on the attachment of murein to inner and outer membranes has also been identified in high-resolution electron micrographs of the nascent septum (Fig. 4; MacAlister et al., 1987). The structure, called the septal attachment site (SAS) or membrane attachment at the leading edge (MALE), is located at the leading edge of the ingrowing septum throughout the septation process, forming a pursestringlike zone at the leading edge of the ingrowing septum. It consists of a sharply localized zone in which inner membrane, murein, and outer membrane are tightly apposed. The attachment zone at the septal edge

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WILLIAM R. COOK

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FIG 4. Membrane attachment at the leading edge (MALE) of the nascent septum. ( A ) Electron micrograph of septa1 region of a dividing cell from a culture of Esclii~richirrc d i . The cells were plasmolyzed in 20% sucrose prior to fixation. X 88,320. (B) Enlargement of (A) showing the characteristic bulbous enlargement of the murein (M)-outer membrane (OM) layers that lie in close apposition to the inner membrane (IM) at the leading edge. x 320,000. (C) Schematic representation of (B) indicating the relationships of the various cell envelope layers. PPS, Periplasmic space. From MacAlister et al. (1987).

has a characteristic ultrastructural appearance that distinguishes it from the adhesion zones that form the periseptal annuli. After septal closure and cell separation, the attachment site appears to remain at the pole of the newborn cell, where it can be detected as a bacterial birth scar. It has been suggested that MALE may represent the site at which new murein units are inserted into the murein layer of the nascent septum (MacAlister et ul., 1987). If correct, this would explain the vectorial movement of the septum toward the interior of the cell, with the inner membrane being pushed inward by the ingrowing murein. This idea has gained support from the demonstration that incorporation of the murein precursor [3H]diaminopimelic acid ([3H] DAP) occurs predominantly at the leading septal edge, as determined by autoradiography of septating cells that were pulse-labeled with [3H]DAP (Wientjes and Nanninga, 1989). 111. Biogenesis and Localization of the Division Site

A. DIVISIONSITESARELOCALIZED LONGBEFORE THE ONSETOF SEFTATION The association of periseptal annuli with the division septum was originally established by serial-section electron microscopy (MacAlister et uf., 1983). The same experiments showed that structures resembling perisep-

DlFFERENTIATION OF BACTERIAL CELL DIVISION SITES

11

tal annuli were also located at other sites along the length of the cell, where septation was not occurring. Because these structures did not extend completely around the cell cylinder, it was suggested that they might be precursors of the circumferential periseptal annuli that flanked the division septum (Cook et al., 1987; MacAlister et al., 1983). As noted before, the annular attachments form the limiting borders of localized regions of plasmolysis (plasmolysis bays). Thus, although visualization of the attachments themselves requires electron microscopy, the plasmolysis bays that mark their locations can easily be visualized by light microscopy (Cook et al., 1986). The positions of the plasmolysis bays thereby have been used to identify the locations of the annular structures along the length of the cell. This made it possible to study large numbers of cells to establish the pattern of genesis and localization of the annular apparatus during the course of the division cycle. These studies led to the unexpected observation that the future division site was placed at its proper position along the length of the cell during the cell cycle that precedes the cycle in which it participates in septum formation (Cook et al., 1987). In these studies the fact that plasmolysis bays were already localized at midcell in newborn cells originally suggested that new annuli were formed very early in the life of the cell. Evidence that formation and localization of the periseptal annuli in fact occurred during the preceding cell cycle came from the demonstration that predivision cells contained annular structures at one-quarter and three-quarters cell lengths, in addition to the periseptal annuli at midcell. The annuli at the cell quarters were retained during division to become the midcell annuli of the newborn cells. Since the annuli mark the site at which the division septum is formed, the question of how the cell determines the correct location of the division site therefore becomes a question of how the cell localizes the annular apparatus at one-quarter and three-quarters cell lengths.

B. AREDIVISIONSITES GENERATED FROM PREEXISTING SITES BY A REPLICATION-DISPLACEMENT MECHANISM? Studies of annulus distribution at intermediate stages of the division cycle showed that new pairs of annuli were first detected immediately adjacent to the midcell annuli that are present in the youngest cells in the population (Cook et al., 1987). As the cells elongated, the paracentral annuli that were located on both sides of the periseptal annuli appeared to move progressively away from midcell toward the two poles until, in the longest cells, the structures were tightly clustered at one-quarter and three-quarters cell lengths (Fig. 5). These results led to the proposal that new division sites are generated

WILLIAM R. COOK

12

ET A L

I

I

. .

*. 1

.....

i.:. . ,

.

D

FIG 5. Formation and displacement of nascent periseptal annuli during the division cycle. From Cook el ul. (1987).

and localized in a three-step process by a repetitive cycle of (1) replication of the preexisting sites at midcell, generating nascent division sites on each side of the central annuli; (2) displacement of the nascent annuli toward the two poles; and (3) arrest of the lateral displacement when the annuli have arrived at their final positions at one-quarter and three-quarters cell lengths. It is likely that the nascent annuli continue to mature during the displacement stage, since the annuli at intermediate positions appear in electron micrographs not to extend completely around the cell. Although this is difficult to prove, the micrographs suggest that the annuli continue to grow circumferentially around the cylinder during the displacement period with final closure of the ring occurring sometime after their final arrival at one-quarter and three-quarters cell lengths. If, as seems likely, septum formation cannot begin until the periseptal annuli extend completely around the cylinder to seal off the future division site, the coupling of the localization process with the process of annulus maturation would help to ensure that septation is not initiated at ectopic sites. Because the two new sets of annuli are generated on opposite sides of the preexisting central annuli, each of the nascent annular pairs is com-


13

mitted to one of the two daughter cells from the moment of its generation at midcell. It has been pointed out that the nascent annuli are attractive candidates to serve as the structures that anchor the daughter chromosomes to the cell envelope during the process of DNA replication and chromosomal segregation (Cook et al., 1987). This would ensure that each daughter cell received one copy of the genome. Although we find this idea attractive, it is at present a hypothesis without supporting evidence. The recent demonstration that the E. coli origin of DNA replication binds specifically to a cell envelope fraction in vitro may facilitate identification of the cell envelope proteins responsible for chromosome binding, and this in turn may make it possible to test the hypothesis that the DNA-binding sites are located within the annuli (Ogden et al., 1988). c . RESIDUAL DIVISIONSITES REMAINAT THE CELL POLES AFTER CELL DIVISIONIs COMPLETED When cells divide, each daughter cell inherits one of the two periseptal annuli that had flanked the septum prior to cell separation. The residual annulus remains at the cell pole, defining a polar domain that lies between the polar annulus and the end of the cell. Because the polar domain is derived from the periseptal domain that delimited the septation site during the preceding division cycle, it is not unreasonable to expect that elements of the division machinery might be retained in this domain after cell separation is completed. Evidence that the residual division site at the new pole has the potential to support another cycle of septum formation has come from studies of minicell mutants of E. coli. These mutants are characterized by the frequent aberrant placement of the division site at the pole of the cell, leading to formation of large numbers of very small spherical cells (“minicells”) that lack chromosomal DNA (Frazer and Curtiss, 1975). It was originally suggested by Teather et al. (1974) that the minicell mutation results in loss of an activity that is needed to inactivate residual polar division sites. Later studies showed that deletion of the minicell genetic locus (minB) results in appearance of the minicell phenotype (de Boer et al. 1989). These studies have identified two gene products of the minB locus, the products of the minC and minD genes, that act together to form an inhibitor of septation that is required to prevent minicell formation. A third gene product, the minE protein, appears to give the minCD division inhibitor specificity for polar sites as opposed to normal sites at midcell. The demonstration that a septation inhibitor is required to prevent formation of polar septa is consistent with the view that the residual sites at

the cell poles retain the capacity to undergo additional cycles of septum formation. The phenotype of minicell-forming strains also includes cells that span a broad range of cell lengths, ranging from normal-sized cells to mediumlength filaments. Analysis of the length distribution of minB cells led Teather et af. ( 1974) t o propose that in minicell-forming strains the number of septation events per unit increase in cell mass remains normal. Thus, only one septum would be formed with each cell doubling, with an equal probability that septation will occur at the “normal” site at midcell or at one of the two residual division sites at the cell poles. If correct, this implies that at least one essential division component is present in limiting amounts, reaching a sufficient level at some point to support one septation event per division cycle. The hypothetical “division potential” would be used up after triggering septation, thereby preventing extra septation events. Alternatively, one could imagine that the initiation of one septation event could provoke a cellular change that excluded subsequent events until cell growth diluted the hypothetical inhibitor. A possible candidate for the hypothetical “division potential” molecule might be the products of the ftsZ gene, since overexpression of ftsZ appears to increase the number of septation events, resulting in formation of minicells and of chromosome-containing cells that are shorter than normal (Lutkenhaus, 1988; Ward and Lutkenhaus, 1985). It has been shown that some septation events in minicell strains result in the formation of rod-shaped cells lacking chromosomes (Jaffe et al., 19881, and that nucleoid distribution patterns are disturbed in the minB filaments (E. Mulder and C. L. Woldringh, personal communication). This suggests that the products of the minB gene may also be involved in maintaining the normal DNA segregation pattern.

IV. Formation of the Septum A. COORDINATION OF INGROWTH OF OUTER MEMBRANE-MUREIN-INNER MEMBRANE Septum formation requires the invagination of the inner-membrane, murein, and outer-membrane layers of the cell envelope. In E. coli the three layers appear to invaginate coordinately, based on thin-section and freeze-etch electron micrographs of septating cells. In a contrary view, Murray and collaborators have suggested that the invagination of inner membrane and murein precedes the invagination of outer membrane during normal septation in E. cofi, based on the use of alternative fixation


15

procedures to the usual glutaraldehyde fixation protocol (Burdette and Murray, 1974a,b; Gilleland and Murray, 1975). For a more complete discussion of this question the reader is referred to Rothfield et al. (1986). However, it is clear that the ingrowth of the three layers is not obligatorily coupled, since mutants strains of E. coli and Salmonella typhimurium have been identified in which the ingrowth of the inner membrane and murein is uncoupled from the ingrowth of the outer membrane (Donachie et al., 1984; Donachie and Robinson, 1987; Fung et al., 1978; Weigand et al., 1976). The affected genes are cha and IkyD, respectively. In the uncoupled mutants the inner membrane and murein appear to invaginate normally, resulting in formation of filaments that contain multiple inner membrane-murein crosswalls that divide the cytoplasm into cell-length units. The units are held together by bridges of outer membrane that failed to participate in septal ingrowth. Thus invagination of outer membrane requires one or more proteins that are not required for invagination of inner membrane and murein. These proteins could, for example be involved in attaching outer membrane to the leading edge of the invaginating septum.

B. BIOCHEMISTRY OF SEPTUMFORMATION 1. Role of the ftsl Protein (PBP3) The only division-related protein for which a biochemical function has been identified is the product of theftsl gene (Spratt, 1983). Theftsl gene encodes a 60-kDa inner-membrane protein that has been identified as penicillin-binding protein 3 (PBP3). The specific interaction of the f ts l gene product with p-lactams implies that the protein plays a role in murein biosynthesis or metabolism. It has also been reported that purified preparations of PBP3 show two enzymatic activities presumably involved in murein biosynthesis, a transglycosylase and a transpeptidase, the transpeptidase being the most sensitive to inhibition by p-lactam antibiotics (Ishino and Matsuhashi, 1981). Evidence that PBP3 is required for septum formation has come from studies of thermosensitiveftsl mutants, in which growth at elevated temperature results in formation of nonseptate filaments (Nishimura et al., 1977; Spratt, 1977; Suzuki et al., 1978). Additional support for this view comes from the observation that filament formation also is induced by treatment of normal bacteria with antibiotics, such as cephalexin, that have a high affinity for PBP3 (Botta and Park, 1981; Spratt, 1975). Taken together, the aforementioned evidence strongly suggests that PBP3 plays a direct role in synthesis of septal murein. This implies that PBP3 either

I6

WILLIAM R. COOK

ET Al.

is preferentially located at division sites or is more randomly distributed and can be functionally activated at division sites at the appropriate time in the cell cycle. These possibilities should be accessible to study by immunoelectron microscopy using antibody directed against PBP3 or by autoradiography of cells labeled with isotopically labeled PBP3-specific compounds. 2 . Other Proteins Implicated in Cell Division A number of other gene products have been implicated in the division process based on the isolation of conditional mutants that form nonseptate filaments when grown under nonpermissive conditions. It is not known whether any of the gene products participate directly in the differentiation process. They have been reviewed elsewhere (Donachie and Robinson, 1987; Lutkenhaus. 1988). V. Regulation of the Division Process A. THEE. coli CELLCYCLE 1. Description of the Division Cycle

In E. coli the cell division cycle is a repeating series of events, punctuated by chromosome replication and septum formation (Helmstetter, 1987). For E. coli B/rA grown at a generation time of 60 minutes, chromosome replication (defined as the C phase) requires -40 minutes. Septa1 ingrowth begins 8-10 minutes after completion of replication. Approximately 10-12 minutes are then required for completion of septation and physical separation of daughter cells, although the time required for septum ingrowth increases when doubling times are >60-70 minutes. An additional interdivision period is present before the initiation of the next round of chromosome replication (Helmstetter and Pierucci, 1976; Kubitschek et al., 1967; Skarstad et a / . , 1983, 1985). This may be equivalent to the G , phase of the eukaryotic cell cycle (Cooper, 1984). Similar values have been obtained in both B/r and K-12 strains (Helmstetter, 1987). The components required for formation of the division septum are generated prior to the termination of chromosome replication. This is shown by the observation that in cells that have just completed chromosome replication, septation is not prevented by inhibitors of DNA, RNA, and protein synthesis(BrehmerandChuang, 198lb;Clark, 1968; Dix and Helmstetter, 1973; Helmstetter and Pierucci, 1968; Jones and Donachie, 1973; Kubitshek, 1974; Pierucci and Helmstetter, 1969; Woldringh et al., 1977). The durations of the C phase and septation periods are generally invariant


17

when generation times fall between 20 and 60 minutes, but they gradually slow down when generation times are prolonged beyond 60 minutes.

2. Initiation of Chromosomal Replication Slowly growing cells (generation time >50-60 minutes) contain approximately two completely replicated chromosomes per cell at the time of septum formation. On the other hand, the number of partially or completely replicated chromosomes per cell increases by a factor of 2-4 in rapidly growing cells (Helmstetter, 1987). This reflects the fact that initiation of new replication forks at the chromosomal start site (oriC in E. cofi K-12) can begin prior to completion of ongoing rounds of replication. As a consequence, rapidly growing cells contain multiple replication forks at the time of septation. Therefore, replication events can be initiated in one division cycle and completed in the next. This demonstrates that division cycle events can be overlapped. The number of replication forks (and therefore initiation events) per cell (Cooper and Helmstetter, 1968; Helmstetter and Cooper, 1968) and the average mass per cell are both dependent on the growth rate. Donachie (1968) deduced that the ratio of initiation events to cell mass was invariant over a broad range of growth rates, and concluded that initiation began when the ratio of chromosomal origins to cell mass fell below a critical value. This led to suggestions that a positive-acting substance required for initiation of chromosome replication is synthesized at a rate proportional to the overall increase in cell mass (Donachie, 1968; Jacob et al., 1963; Lark, 1979; Margalit et al., 1984; Sompayrac and Maaloe, 1973). Following the initiation event, levels of the initiator would be depleted, thereby preventing further initiations until the critical concentration was reestablished. In the alternative model, initiation of replication would be prevented by an inhibitor molecule that is synthesized once during each replication cycle, after replication of the appropriate gene locus (Pritchard et al., 1969; Pritchard, 1984). The intracellular concentration of the inhibitor would be diluted with increasing cell mass, until the level was too low to prevent initiation. At this point a new round of initiation would take place, followed by a new burst of inhibitor synthesis. 3. Termination of Chromosome Replication It has been suggested that formation of the division septum may be coupled to the termination of chromosome replication. This suggestion was originally based on studies of cells in which DNA replication was permitted to resume after 2 hours of thymine starvation. Both RNA and

18

WILLIAM R. COOK ET

AL

protein synthesis were required to restore septum-forming ability to these cells. By adding rifampicin o r chloramphenicol at various times after thymine supplementation, Jones and Donachie (1973) showed that septation was dependent on a 5- to I0-minute period of RNA and protein synthesis occurring 35-40 minutes after resumption of DNA synthesis. Septation occurred shortly thereafter. The initial round of chromosome replication also terminated at this time, leading to the proposal that “termination protein(s1” formed shortly after termination of chromosome replication are required for the initiation of septation (Jones and Donachie, 1973). The possibility also exists that the period of required protein synthesis reflected the time required for recovery from the SOS response (see later) and that the apparent relationship to termination was coincidental. A correlation between termination of chromosome replication and septum formation was also observed by Grossman et al. (1989). In these experiments, the presence of methionine during amino acid starvation was used to slow chromosome replication, resulting in a delay in the time of termination. There was a corresponding delay in the first burst of division that was seen when protein synthesis was allowed to resume, consistent with the hypothesis that termination is required for septum formation. It is also possible that the delay in division was not causally related to the delay in termination but rather reflected other secondary effects of the rnethionine treatment. It should be noted that septation can occur in cells blocked in DNA elongation and termination (see later), demonstrating that termination of chromosome replication is not absolutely required for septum formation. B.

COUPLING OF SEPTATION TO CHROMOSOME

REPLICATION AND

SEGREGATION Under normal conditions the formation of anucleate cells is extremely rare. This implies that septation is both temporally and topologically coupled to the processes of DNA replication and segregation. Temporal coupling requires that chromosome replication and the decatenation and separation of the daughter chromosomes must be completed prior to completion of the septal crosswall. Topological coupling requires that the new septum must be located between the daughter chromosomes. If temporal and topological coupling did not occur, one can imagine two possible results: ( 1) a mixture of anucleate cells and cells containing more than one chromosome would be produced, and/or (2) septal closure would have a guillotine effect on the unreplicated o r unseparated DNA. Under normal circumstances neither of these effects occurs with significant fre-


19

quency, implying that an effective cellular mechanism exists to coordinate septation with DNA replication and/or segregation. Septa1 ingrowth can begin prior to the completion of DNA replication and segregation (Fig. 6; Woldringh et al., 1977). Thus, if direct coupling does exist between DNA segregation and the timing of septum formation it appears unlikely to involve a control on the initiation of septal invagination. On the other hand, a mechanism to prevent closure of the crosswall until DNA replication and separation are completed remains possible. 1. Prevention of Formation of Cells Lacking Chromosomes When DNA

Synthesis Is Blocked

a. Negative Regulation of Septation by the SOS System. The best understood of the pathways that prevent formation of anucleate cells is a part of the SOS response, a complex cascade of reactions in which the presence of damaged DNA or the inhibition of DNA replication results in the induction of a number of genes required for DNA repair and other processes (D’Ari, 1985; Holland, 1987; Little and Mount, 1982; Walker, 1984; Witkin, 1976). One of the consequences of SOS induction is that septation is reversibly inhibited, preventing formation of daughter cells until the defective DNA has been repaired. The mechanism by which septation is halted after induction of the SOS response begins with the activation of the product of the recA gene to an active protease in response to damaged DNA (Little and Mount, 1982;

FIG.6. Thin-section electron micrograph of Escherichiu coli B/rK showing initiation of septal constriction prior to complete separation of nucleoids. X 66,000. From Woldringh et al. (1977).

20

WILLIAM R. COOK Er A L

Walker, 1984). The RecA protease cleaves LexA, a repressor of the SOS regulon (Little and Mount, 1982; Walker, 1984). One of the derepressed genes, the sfiA gene (also known as sufA),codes for an inhibitor of septation (Huisman and D’Ari, 1981; Huisman et a f . , 1984; Mizusawa et a f . , 1983). After removal of the inducing stimulus, LexA is no longer cleaved by RecA protease, and normal division is restored (Maguin et a f . , 1986a; Mizusawa et al., 1983; Mizusawa and Gottesman, 1983; Shoemaker et al., 1984). For further information on the SOS response the reader is referred to Walker (1987). Genetic evidence indicates that the target of SfiA action is the essential cell division gene, f t s Z , or its gene product (Gayda et a f . , 1976; George et a f . , 1975; Huisman et a f . , 1984; Jones and Holland, 1984, 1985; Lutkenhaus, 1983). It has been suggested that inhibition of septation involves a direct interaction between the SfiA and FtsZ proteins. This is based on the observation that the rate of degradation of the SfiA protein by Lon protease is much slower in cells containing wild-type FtsZ protein than in certainftsZ mutants [i.e., sfiB (also known as sufB)mutants] in which the SfiA-mediated division inhibition is lost (Jones and Holland, 1984; Mizusawa and Gottesman, 1983; Mizusawa et al., 1983; Shoemaker et ul., 1984). Consistent with this interpretation is the observation that overproduction of FtsZ can suppress the division-inhibitory effect of SfiA (Lutkenhaus et a f . , 1986; Ward and Lutkenhaus, 1985). Alternative explanations are possible, and a final judgment on the idea that SfiA acts directly on the FtsZ protein must await more direct biochemical experiments. The SOS response appears to be a damage control system rather than a mechanism responsible for coupling DNA replication and septation under normal conditions, since sfiA(sufA)and sfiB(sufB)mutations that inactivate the SOS division inhibition response have no apparent effect on the normal division pattern. A second RecA-dependent coupling between chromosome replication and septation is mediated by the sfiC gene, which is present in some but not all strains (D’Ari and Huisman, 1983). As with SfiA, SfiC is derepressed after RecA activation. However, sfiC derepression is not dependent on LexA cleavage, indicating that an alternative repressor, also subject to RecA cleavage must exist. sfiC has been identified as a component of excisible element €14 (Maguin et a f . , 1986b). Genetic evidence indicates that the target of SfiC action, like SfiA, isftsZ (D’Ari and Huisman, 1983). The action of SfiC is irreversible, and cells do not recover after removing the original inducer (Maguin et a f . , 1986a), making the physiological significance of SfiC action questionable.


21

b. Regulation of Septation by SOS-Zndependent Mechanisms. Filamentation also occurs when DNA replication is blocked in strains containing mutations that result in an inoperative SOS pathway. This indicates the presence of a non-SOS mechanism that couples chromosome replication and septum formation (Burton and Holland, 1983, Huisman et al., 1980, 1983; Jaffe et al., 1986; Jaffe and D’Ari, 1985). Such a mechanism could play a role in coordinating chromosome replication and septation in unperturbed cultures (Huisman et al., 1983; Jaffe et al., 1986; Jones and Donachie, 1973), but this will remain conjectural until the responsible gene or genes have been identified and it has been shown that inactivation of these genes perturbs the normal division pattern. Although nonseptate filaments are formed when DNA synthesis is blocked in strains that lack the SOS system, a number of chromosomefree cells are also produced. This indicates that septation inhibition in the non-SOS system is not as effective as that provided by the action of sfiA in the SOS system (Jaffe et al., 1986; Jaffe and D’Ari, 1985). It is possible to envision two types of mechanism by which the SOSindependent coupling of cell division and DNA replication might be mediated. In the first mechanism, coupling could be mediated by a positive control element similar to the “termination protein” proposed by Jones and Donachie (1973), as discussed earlier. In this view the effector would be required for initiation of septation during each division cycle. It has been suggested that the product of thefrsA gene may be the coupling effector (Tormo et al., 1980, 1985a,b, 1986; Tormo and Vicente, 1984). Conversely, the non-SOS coupling could be mediated by a negativecontrol element. In this case the partial inhibition of septum formation that occurs when DNA synthesis is inhibited in SOS-defective cells could reflect the induction of a division inhibitor analogous to the SfiA septation inhibitor of the SOS system.

2. Coordination of Chromosome Replication and Cell Division As noted before, the evidence for direct coupling between chromosome replication and cell division-in the sense that a signal is passed from the replicating or postreplication chromosome to the septation machinery to trigger septation-is not compelling. In an alternative model, DNA replication and septum formation are not directly coupled but both processes are independently triggered by factors related to progression through the cell cycle. This would result in the temporal coupling of the two processes without requiring direct signaling between the chromosome replication process and the septation machinery. This is consistent with the observation that septation occurs in the absence of chromosome replication pro-

22

WILLIAM R. COOK

ET A1

vided that the SOS-induced division inhibition mechanism is not present (see later). The idea that chromosome replication and septation are separate, parallel pathways is also consistent with the fact that the timing of initiation of chromosome replication is much more precisely regulated than the initiation of septation (Brehmer and Chuang, 1981a; Koppes et al., 1978; Kubitschek, 1%2; Newman and Kubitschek, 1978; Schaechter et al., 1962; Skarstad et al., 1986).

c. RELATIONOF CHROMOSOME SEGREGATION TO LOCATIONOF THE DIVISIONSITE 1. Mutations that Affect Nucleoid Segregation

A number of genes have been identified that affect the fidelity of nucleoid segregation into daughter cells. These fall into three general groups: genes coding for products that are known to affect DNA synthesis, genes that affect DNA supercoiling (DNA gyrase mutants), and genes the biochemical function of which is still unknown (par mutants). a. DNA Synthesis Mutants. Inhibition of DNA synthesis induced by temperature shift in thermosensitive mutants blocked in the initiation or elongation steps of DNA replication, or by thymine starvation, results in formation of filaments. Nucleoids are restricted primarily to the center of the filament, presumably representing the original unreplicated chromosome. This was shown most clearly by the studies of Jaffe and collaborators, who examined the effects on the division pattern of blocking DNA synthesis in a number of different ways in strains that d o not express the SOS response (Jaffe et al., 1986; Jaffe and D’Ari, 1985). A two-phase response was observed. During the first few hours, filaments accumulated in the culture, ascribed to the non-SOS-mediated coupling of DNA replication and septum formation. Later, however, substantial numbers of anucleate cells appeared (620% after 4 hours), reflecting the resumption of septation after the initial period of blocked division. Thus the septation block that is mediated by the non-SOS-mediated DNA-division coupling system was transient (Jaffe et al., 1986). The formation of anucleate cells was abolished by cya or crp mutations and therefore is dependent on the presence of of cAMP and the CAMP-binding protein. The mechanism that underlines the cAMP dependence of this septation activity has not yet been defined (D’Ari et ul., 1988; Jaffe et ul., 1986).


23

b. DNA Gyrase Mutants. i. gyrA(parD). Hussain et al. (1987a,b) have described a strain containing a mutation (parD) at 88.4 units on the E.coli genetic map, which resulted in the apparent failure to segregate nucleoids, leading to formation of filaments and to the production of anucleate cells. Subsequent experiments showed that the abnormal phenotype was due to an amber mutation in gyrA, coding for the a subunit of DNA gyrase (Hussain et al., 1987b); a second uncharacterized mutation in the same strain also may play a role in the abnormal phenotype. No effects on chromosome replication were noted. The aberrant pattern of septation in this strain has been described in detail, and the results show the presence of chromosome-free cells that span a broad range of cell sizes. The mechanism(s) that couple initiation of septation to cell mass appeared not to be altered, as shown by the fact that the total number of septa formed per mass doubling were similar to the numbers of septa formed in a wild-type strain. ii. gyrB. Mutations in the gene coding for the p subunit of DNA gyrase (gyrB) (Fairweather et al., 1980) fail to decatenate newly replicated chromosomes (Steck and Drlica, 1984). Presumably as a result of this, the cultures contain filaments with centrally localized nucleoids. The cultures also contain small anucleate cells (Orr et a / . , 1979), reflecting the uncoupling of septum formation from chromosome segregation. c. par Mutants. Two other mutations [parA, at 95 units (Hirota et al., 1968a; Norris et al., 1986) and pa&, at 65 units (Kato et a / . , 1988)]affect chromosome partition without any apparent effect on DNA synthesis. In both cases filaments are formed that contain a large centrally located nucleoid. DNA-less cells are also present in the cultures. The fact that chromosome segregation was affected in these mutants whereas DNA synthesis appeared to be unperturbed suggests that the primary defect may be in the process of chromosome segregation. It should be noted that a third par mutant (parB) was originally thought also to fill these criteria (aberrant chromosome segregation in the presence of normal DNA synthesis). The parB mutation was later found to be located in the dnaG gene and was shown to affect the initiation of DNA replication (Filutowicz and Jonczyk, 1983; Norris et al., 1986).

2. Does Nucleoid Position Determine Septa1 Position? a. Nucleoid-Occlusion Model. All of the mutationsjust described result in the formation of filaments with centrally positioned nucleoids, as

24

WlLLlAM R. COOK

ET AL.

well as substantial numbers of smaller, nonfilamentous cells that lack nucleoids. Thus, the cells are capable of septation but appear not to form septa at the normal midcell position. Studies of the pattern of division in these strains have led to the suggestion that the nucleoid(s) play an essential role in establishing the location of the division septum by exerting a local inhibitory effect on septum formation (Donachie et d . , 1984; Woldringh et d . , 1985; Taschner et al., 1987). We will call this the nuclear-occlusion model. We will now examine the implications of this interesting model and the evidence on which it is based. The model states that the location of the nucleoids is the sole determinant of septal positioning (Woldringh et al., 1985; Hussain et al., 1987a,b). In this view the entire cell envelope is competent to initiate septal ingrowth. Following chromosome replication in wild-type cells, the two daughter nucleoids are positioned at regular intervals along the length of the cell by an active nucleoid segregation mechanism, or perhaps by the passive diffusional redistribution of daughter chromosomes. The nucleoids then would act as local inhibitors of septum formation, thereby restricting formation of the septum to a region near the middle of the cell. Three possible mechanisms can be considered for a nucleoid-occlusion effect, if it exists. 1. The nucleoid provides a simple physical block to the inward move-

ment of the septum. 2. The proximity of the nucleoid perturbs the organization of the overlying cell envelope by local physicochemical effects. 3. The nucleoid elaborates a diffusible septation inhibitor that prevents the initiation of septal ingrowth within a limited distance from the nucleoid itself.

b. Evidence for the Model. The idea that nucleoid placement is the sole determinant of septal placement was first suggested by Woldringh et al. (1983, based on studies of dnaZTs cells in which division was permitted to resume after a period of inhibition of DNA synthesis. This resulted in the production of anucleate cells and minicells. It is of interest that rninicell production has not been described when DNA synthesis has been blocked by inactivation of other dna gene products. The authors noted that the coefficient of variation in cell length was much larger for the temperature-shifted culture because both minicells and long chromosome-free cells were formed. On the basis of the increased coefficient of variation, it was suggested that there were no predetermined sites for the execution of constriction in the downshifted cells. E. Mulder and C. L.


25

Woldringh (personal communication) have also noted similarly broad coefficients of variation of placement of constrictions in filaments of other DNA mutants in which anucleate cells were produced. The use of coefficients of variation as a indication of the randomness of placement of septa is based on the assumption that the distance from septal constriction to cell pole represents a single unimodal length distribution population. It should be noted that this assumption need not be correct. For example, if preexisting sites were distributed at regular intervals between cell pole and nucleoid, septation at any one of these sites would be possible. Measurement of the distance from constriction to cell pole in a large number of cells would then yield a mixture of discrete size classes, each class representing a multiple of the unit cell length. In this hypothetical case, the coefficient of variation of segment length for the entire data set would be high despite the fact that septal placement was nonrandom. It will be of interest to see whether it is possible to resolve the length distribution pattern into more than one Gaussian population before making a decision on the randomness of septal placement in these experiments. Studies of mutants with DNA gyrase defects have also provided evidence that has been interpreted to support a nucleoid-occlusion model. Growth of gyrA(parD) cells under nonpermissive conditions (see earlier) led to formation of filaments with large centrally located nucleoids, leaving long regions of the filaments devoid of visible nucleoid material. This presumably was due to failure to decatenate daughter chromosomes after completion of replication. Septation within the anucleate regions of the filaments subsequently led to formation of small chromosome-free cells (Hussain et al., 1987a). The chromosome-free cells were said to consist of short rods of all lengths between minicells and normal-length cells, although this is difficult to evaluate based on the resolution of the published results. It was pointed out that these observations are compatible with the idea that the absence of nucleoids near the cell poles led to the random placement of septa in these nucleoid-free regions (Hussain et al., 1987a). It should be noted that inactivation of DNA gyrase changes the expression of a number of chromosomal genes in which the state of chromosomal supercoiling affects promoter activity (Schmid, 1988; Wang, 1985). This raises the possibility that any septal localization defect could be secondary to altered expression of genes that affect septal placement by a mechanism other than nucleoid occlusion. c. Evaluation o f t h e Model. The idea that the entire cell envelope is competent to initiate septation during the latter portion of the division cycle raises certain problems. It is not compatible, for example, with the

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WILLIAM R. COOK

ET A L .

observation that in slowly growing cells periseptal annuli are localized at the future division site during the previous cell cycle, long before the onset of the C phase of the division cycle in which septation occurs at this site (see Section 11). It is therefore unlikely, in our view, that the potential to initiate septum formation is randomly distributed along the entire length of the cell at the time of formation of the new septum. The most direct way t o test the nucleoid-occlusion model is to determine the size distribution of newborn cells in which nucleoids are absent from the greater part of the cell body secondary to inhibition of DNA replication. This avoids the possible complications of other secondary effects of gyrase defects or of par mutations. If future division sites were localized prior t o chromosome segregation, septa would be expected to form at regular intervals along the filaments, with the exception of the occluded site at midcell. On the other hand, if nucleoid positioning were the sole determinant of septa1 positioning as stated by the nuclear-occlusion model, then septa would be randomly distributed and the anucleate cells that are formed would show a continuum of cell lengths, down to the size of minicells. In the most extensive published study of this type, Jaffe et al. (1986) have studied the division patterns of cultures in which DNA synthesis was blocked in the absence of the SOS system. The inhibition of DNA synthesis was accomplished in a variety of ways, including thymine starvation and temperature shift in dnaA, dnaB, and dnaC mutants. This led to the formation of filaments that contained only one or two nucleoids, primarily located near the midpoint of the filament, plus significant numbers of anucleate cells. The chromosome-free cells were of normal length and were relatively uniform in size, suggesting that the positioning of septation sites was not affected by the absence of nucleoid in the vicinity of the septation event. Similar results were reported by Hirota et a / . (1968b) in studies of a thermosensitive dnaA mutant. In our view, these studies provide the most cogent argument against the proposition that division sites are randomly located along the length of the cylinder and that selection of the site depends only on the absence of nucleoid. One can imagine a more permissive variant of the model in which the nucleoid would affect the placement of the septum by vetoing septation at nearby division sites, while permitting septation to occur at other potential division sites that are located at regular intervals along the length of the cylinder. If this were correct, newly born chromosome-free cells would fall into discrete size classes equivalent to multiples of the presumed unit cell length. The possibility can also be considered that aberrant nucleoid placement affects an earlier stage in differentiation of the


27

division site, such as the formation or localization of periseptal annuli, rather than affecting the septation event itself. Further studies will be needed to determine whether nucleoid position indeed plays a role in the selection of the septation site. VI. Conclusions

In recent years, significant advances have been made in defining the E. coli cell division process. At the morphological level, the discovery of periseptal annuli has permitted the developmental history of the division site to be followed throughout the cell cycle. At the same time, genetic studies have identified a number of proteins that are implicated in formation and localization of the division apparatus, and in the process of septa1 morphogenesis. Much useful information remains to be obtained in these areas, most notably an understanding of the coordination of division site development and localization with other events of the division cycle. In this regard, the use of immunoelectron microscopy to study the cellular localization of division-related gene products has hardly been exploited. This promises to provide new information that will be essential to any detailed understanding of the mechanism of the division process. Nevertheless, if this rich body of information is to be extended to the molecular level, it will be necessary to develop methods for isolation of the division site itself. Until this is achieved, the picture that emerges will necessarily be incomplete. A second major area of interest concerns the relation between cell division and chromosome replication. It is clear that the two processes are temporally coupled under normal conditions. A considerable body of evidence has permitted the formulation of specific hypotheses regarding the basis of the coupling. However, it is still not known whether or not there is direct coupling between the two processes or, alternatively, whether both respond to a common division clock. Does the initiation or termination of chromosome replication generate a signal that normally regulates the timing of septum formation? Does the differentiating division site send a signal that affects chromosome replication? Do both processes respond to signals generated by other events of the division cycle? It has proved difficult to obtain unequivocal answers to these significant questions, and this general area remains an important field for future study. A third key question is, how does E. coli determine where to place the

28

WILLIAM R. COOK

ET A L

division septum? There was little experimental information dealing with this question until the past few years, when early stages in the localization process have been described at a morphological level. These studies have suggested that the localization of the division site takes place long before initiation of septum formation. At the same time, studies of cells in which chromosome partition is defective have led to the proposal that the position of the nucleoid plays a role in determining the placement of the division septum. The identification of gene products of the minicell locus that affect the site selection process suggests that considerable additional information about the localization process remains to be obtained from genetic approaches. Exploitation of these relatively recent advances is likely to lead to a more complete understanding of this important but poorly understood aspect of the division process.

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INTERNATIONAL REVIEW OF CYTOLOGY. VOL. 118

Properties of the Cell Surfaces of Pathogenic Bacteria R. J. DOYLEAND E. M. SONNENFELD Department of Microbiology and Immunology, Health Sciences Center, University of Louisville, Louisville, Kentucky 40292

I. Introduction The bacterial cell surface possesses many properties that are important in pathogenesis and immunity. The surface may contain components that stimulate or depress the immune response, depending on concentrations of the components. The surface serves as a site of antigenic recognition. Many bacterial vaccines are composed of surface structure materials. Vaccines against the pneumococcus or Haemophilus influenzae, for example, utilize surface-associated poly saccharides. In many cases, surface structures tend to inhibit phagocytosis of an invading bacterium. Almost any encapsulated bacterium is more resistant to phagocytosis and subsequent intracellular digestion than a nonencapsulated mutant of the same species. The surface also may serve as an anchoring point for a bacterium. It now seems clear that some adherent bacteria have growth advantages over their nonadherent siblings. In both gram-positive and gramnegative bacteria, there are specialized surface structures that enable the bacteria to be tethered to complementary sites on animal cells. In most cases, adhesion of the bacteria to a surface is the initial step in the infectious process. For some bacteria, surface components have direct toxic effects on host cells. Lipopolysaccharides (LPS), surface constituents of many gram-negative pathogens, exhibit several manifestations of toxicity in infected hosts.' Lipoteichoic acids (LTA) from gram-positive bacteria also seem to possess toxic and immunomodulating activities. Finally, the 'Abbreviations used in the text are as follows: Ab, antibodies: Ag, antigens: DAP, diaminopimelic acid: DIC, disseminated intravascular coagulation; EA-IJI, extractable antigens I and 11; FN, fibronectin; Fuc, fucose: GalNAc, N-acetyl-D-galactosamine:GlcNAc, Nacetyl-D-glucosamine: HLA, human lymphocyte antigen (histocompatibility); Ig, immunoglobulins; IL-I , interleukin 1 ; LPS. lipopolysaccharides: LTA, lipoteichoic acids: Man, mannose: MDP, muramyl dipeptide: MS, mannose-sensitive: PG, peptidoglycan. 33 Copyright 0 1989 by Academic Press. Inc. All rights of reproduction in any form reserved.

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R.J.DOYLE A N D E.M. SONNENFELD

surface may serve as a target for antibiotic action. Penicillin and penicillinlike antibiotics and drugs prevent the synthesis of peptidoglycan (PG), resulting in cell death. The purpose of this article is to review a few aspects of bacterial cell surfaces, with an emphasis on the role of surface structures in pathogenesis and in biological responses.

11. Surface Structures of Bacteria

Macromolecular syntheses and nucleic acid replication occur in the bacterial cytoplasm. Most pathogenic bacteria, except for members of the genus Mycoplusma, possess reasonably high internal concentrations of solutes. This requires that the bacteria have extracellular supporting structures to circumvent lysis. Gram-positive bacteria are thought to possess approximately a molar equivalent of internal solute. Such an internal concentration of intracellular material would give rise to a force of -22 atm or about the equivalent of the pressure in an inflated bicycle tire of 70 psi (Koch, 1983). The gram-negative cell is thought to possess an internal solute concentration of -0.2-0.4 M. Solutes are derived from transported materials, precursors, ATP salts, and macromolecules. The extracellular supporting material for bacterial pathogens is PG (also called murein). A simplified structure for a PG is shown in Fig. 1. The PG is composed of p-I ,Clinked repeating units of N-acetylmuramic acid and N-acetyl-D-glucosamine (GlcNAc). Most bacteria do not fully acetylate the amino sugars, whereas others 0-acetylate the C-6 of muramic acid. Resistance to lysozyme is frequently associated with O-acetylation or with free amino groups on the amino sugars. The glycan chains may also be substituted on the lactyl group of the muramic acid by amino acids. Generally, the amino acid directly bonded to the muramic acid is L-alanine. The sequence L-Ala-D-Glu-L-Lys-D-Ala from the muramic acid is common for many bacteria, although diaminopimelic acid (DAP) may replace the L-LYSresidue. The L-LYSor DAP amino acids represent sites for cross-linkages because they possess free amino groups that may form peptide bonds with terminal D-Ala residues of nearby chains. The cross-linkage may be direct, as in an L-Lys-D-Ala bond, or it may involve other amino acids to connect L-LYS(or DAP) to a D-Ala. Staphylococcus izureils, for example, possesses a pentaglycine as a crosslinking unit. The extent of crosslinking varies from bacterium to bacterium, but may be as low as 12% for Bacillirs anthrucis (Zipperle et al., 1984), to as high as 8090% for Legionelh pneurnophila (Amano and Williams, 1983). Prevention of synthesis of PG constitutes the major means for the bac-

BACTERIAL CELL SURFACE

35

O-R' I

L

-Ala

@

I I

D -Glu

I

- Lys ( or DAPI-CROSSLINK D - Ala I L

+o

0

R = H or -C*-CH,

R'= H or LINKAGE to TElCHOlC or TEICHURONIC ACIDS FIG. 1. Peptidoglycan structure showing where bacteriolytic enzymes and autolysins 2, lysozyme; 3, N-acetylmuramyl-L-alanineamidase; may act. 1, N-Acetylglucosaminidase; 4, endopeptidase(s);5, D,L-carboxypeptidase. The extent of amino sugar modification depends on the bacterium. Most pathogens acetylate the amino sugars.

terial activities of cell wall antibiotics such as penicillin. If a bacterium is subjected to a cell wall antibiotic and continues to take up nutrients, then the bacterium will ultimately lyse. This is because no new supporting PG has been assembled to contain the enlarging cytoplasm. In addition to lysozyme (a P-1,4 muramidase), other enzymes, such as autolysins, may degrade preexisting PG. The target sites for autolysins are depicted in Fig. 1. Human serum also contains an N-acetylmuramyl-L-Ala amidase (Mollner and Braun, 1984), but its role as a host defense factor is not well understood. The gram-positive bacterium may contain macromolecules covalently bound to the PG. These include teichoic acids or teichuronic acids (Figs. 2 and 3). The teichoic acid is typically a structure composed of either poly(glycero1 phosphate) or poly(ribito1 phosphate). (It is of course known that some variations in the teichoic acid structure may occur, such as the inclusion of an amino sugar or a mannitol within the chain, but such details are beyond the scope of the present article.) The glycerol or ribitol residues may be substituted by a-or P-D-glucose, D-Ala, or a-or p-GlcNAc or other entities. The nature and extent of substitution de-

36

R.J. DOYLE AND E.M. SONNENFELD

I

( FATTY ACIDS 1 I -

-

-

R = a- or p- D glucose ; D a1anin.e ;OH

FIG.2. Structure of a lipoteichoic acid. (LTA). The fatty-acid end may be firmly bound to the cytoplasmic membrane. whereas the poly(glycero1 phosphate) units may penetrate the PG matrix and extend into the solvent phase. In some bacteria (pyogenic streptococci) the LTA molecules may be released from the cytoplasmic membrane and bind surfaceassociated proteins. Wall teichoic acids are covalently bound to muramic acid residues and are not attached to glycolipid or fatty acid.

pends on the bacterium. A teichuronic acid is typically composed of disaccharide or trisaccharide repeating units containing a uronic acid. The composite cell wall structure of PG and teichoic or teichuronic acid may constitute ~ 4 0 %of the dry weight of a gram-positive cell. The teichoic or teichuronic acids may serve as antigens (Ag), phage receptor sites, proton- or metal-binding sites, wall volume expanders, or autolysin-binding sites, or they may have additional roles in the physiology of the bacterium. Figure 4 shows a thin section of the pathogen B. anthracis. The cell wall is easily recognizable along the cell cylinder and the cell septum. The gram-positive cell surface may also contain LTA. These are

I

01

c=o

0-

H CH

n

FIG 3. Segment of a cell wall-associated teichuronic acid. This teichuronic acid is from Micrococcrrs IvsodeiLricus (Hase and Matsushima, 1972).


37

FIG.4. Thin section of a Bacillus anthrucis cell showing surface array, PG, capsule, and plasma membrane. Courtesy T. J. Beveridge.

poly(glycero1phosphate) molecules that possess a hydrophobic end (Fig. 2). The hydrophobic group is embedded in the plasma membrane, whereas the poly(glycero1 phosphate) may penetrate through the PG to reach the cell periphery. Antibodies (Ab) directed against poly(glycero1 phosphate) frequently agglutinate gram-positive bacteria known to be devoid of wall poly(glycero1 phosphate) (Wicken and Knox, 1975). Other surface components of the gram-positive bacterium may include noncovalently bound polysaccharide (sometimes a group-specific polysaccharide), a surface array, and capsular materials. All of these structures, except for the surface array, have been shown to have a role in pathogenesis (Table I). Flagella may be regarded as virulence determinants because they may cause bacteria to be propelled to nutrient-secreting mucosal sites. Bacillus anthracis is an interesting pathogen from the viewpoint of bacterial cell surfaces (Figs. 4-7). The bacterium contains a PG-polysaccharide wall. There is no teichoic acid, nor does an anti-poly(glycero1 phosphate) Ab agglutinate the bacteria. Bacillus anthracis contains a surface

38

R.J. DOYLE A N D E.M. SONNENFELD

IMPOKTANCE OF

TABLE I BACTERIAL CELL SURFACES

Surface structure Fimbriae (pili) Wall teichoic acid LTA

ffi Protein LPS Secreted polysaccharide and capsular materials Flagella

Surface array Teichuronic acid

IN

PATHOGENICITY

Role in virulence Adhesion and colonization Adhesion, prominent Ag Adhesion, modulates immune response, Ag, pyrogen Modulates immune response, induces inflammation, pyrogen, adjuvant Adhesion, Ag, toxin Toxic, Ag, modulates immune response. pyrogen. adjuvant Adhesion, protection against Ab; sequesters nutrients; retards phagocytosis and subsequent intracellular digestion Propel bacteria t o site of attachment (mucosal surfaces secreting nutrients) None known at present Ag; role in pathogenesis unknown

array (Figs. 4-7) and may secrete a poly(D-glutamic acid) capsule. The capsule seems to be required for virulence. The bacterium also secretes several proteins, some of which may be intercalated within the PG matrix. The protective antigen (PA), for example, can be shown to be surface-bound by use of direct- or indirect-fluorescence methods. The PA is one component of a tripartite toxin essential for the virulence of B. anthrucis. Proteins designated as EA-I and EA-I1 (“extractable antigens”) are closely associated with the PG layer. It is possible that the EA proteins may be subunits of the surface array materials. The gram-positive bacterial surface is relatively thick, resulting in the ability of the bacteria to retain the Gram stain (Beveridge and Davies, 1983). The thickness of a Bacillus subtilis wall is of the order of 25 nm (Beveridge and Murray, 1979). Most cell walls of gram-positive bacteria are solvent-exposed, making them capable of interaction with Ab, enzymes, and environmental molecules (Fig. 8). The wall is reasonably permeable, making it possible for antibiotics, nutrients, and low molecular weight proteins to be taken up or secreted. The gram-negative cell surface is more complex than that of the gram-

FIG.5. Negative stain of capsular material and surface array of Bacillus anthracis. Micrograph courtesy of T. J. Beveridge.

WALL MATRIX

SURFACE ARRAY (EA- I Ag 1 ( E A - I I Ag) LlPOTElCHOlC ACID PA -POLYSACCHARIDE POLY ( D GLUTAMIC ACID)

-

CYTOPLASMIC MEMBRANE FIG.6. Model depicting surface structures of Bacillus anthracis. EA, Extractable antigen; PA, protective antigen. Polysaccharide is covalently attached to PG. Lipoteichoic acid does not penetrate through PG because anti-poly(glycero1 phosphate) Ab will not agglutinate the bacteria. The PG and polysaccharide are turned over during growth to the extent of -50% per generation.

FIG.7. Computer-enhanced image analysis of the surface array of Bacillus anthroris. The apparent symmetry is PI, suggesting that the subunits are assembled free of contact with other subunits. Courtesy of T. J. Beveridge and M. Stewart.

FIMBRIAE (or Fl8RlLLAE 1 TEICHURONIC

-

PEPTIDOGLYCAN LTA PROTEIN CYTOPLASMIC MEMBRANE

41

BACTERIAL CELL SURFACE PROTEIN FlMBRlAE (PILI) LIPOPOLYSACCHIAA:IDE

c

0 and R - A g LIPID A

POPROTE IN EPTIDOGLYCAN

--CYTOPLASMIC MEMBRANE

FIG.9. Cross-sectional representation of the gram-negative cell surface. The PG matrix is relatively thin, of the order of one to three glycan strand equivalents in thickness. The PG may turn over. but in gram-negative rods the turned-over materials are largely reutilized. In many gram-negative bacteria, a lipoprotein is covalently attached to the PG. The fattyacid portion of the lipoprotein is anchored in and stabilizes the inner leaflet of the outer membrane. The outer membrane forms a passive permeability barrier, thereby protecting the PG and cytoplasmic membrane from lysozyme and other potentially deleterious molecules. Small molecules (nutrients) may penetrate the outer membrane through protein channels (porins). The LPS is anchored in the outer leaflet of the outer membrane via hydrophobic interactions. The oligosaccharide structure is completely solvent-exposed and available for interaction with Ab or bacteriophages. Proteins may be embedded in the outer membrane and serve as Ag. Fimbriae may penetrate the PG and outer membrane and extend into the solvent. Lectins at the very tip of the fimbriae may serve to tether the bacteria to complementary surfaces.

positive cell. The gram-negative bacterium possesses a thin layer of PG, possibly equivalent to one to three layers of PG. The wall is crosslinked in a manner similar to that of the gram-positive bacterium and contains the same kinds of amino sugars and amino acids as the gram-positive PG. There is no teichoic acid or teichuronic acid, however. The gram-negative PG is not solvent-exposed, but is surrounded by the outer membrane (Fig. 9). The outer membrane is morphologically similar to the plasma membrane, but it has a much different composition than the inner membrane. The outer membrane contains proteins, phospholipids, a lipoproFIG.8. Representation of a cross section of a gram-positive bacterium. The PG nearest the cytoplasmic membrane is not so stressed as the PG on the surface. When PG turnover occurs, it is usually the wall most stressed and removed from the plasma membrane. In the typical gram-positive cell, there may be 25-35 layer equivalents of glycan to make up the wall thickness. Molecules of LTA may penetrate the PG matrix in some species. Proteins may be embedded in the cell wall or surface-exposed. Teichoic or teichuronic acids are covalently attached- to the muramic acid residues of PG.

K.J.D O Y L E A N D E.M. S O N N E N F E L D

42

0

0 P - A r a N KDO I I KDO

( F A ) GlcN

-

- KDO -

I (FA) GlcN I

@

t

o

r

a p C H O +-

H2 N

D

I

Glc

- Gal

9" "i 9" 'i'

t

C

Gal

-

Re

P -P E T

LIPID A

P-ET I P Hep I I Hcp HepI P

specific pCHO

wo

Qo

-

/ c

P P - ET

NH

(4-Ara

N)

I

0 I

NH 1

FA FA

FA

FA

(Glc N )

(Glc N )

FIG.10. Structures for LPS (A) and lipid A (B). These generic structures show the 0 and R antigens, as well as mutations in the R saccharide. KDO, 2-keto-3-deoxyoctonate; CHO, carbohydrate; ET, ethanolamine; Hep, heptose; Glc N , glucosarnine; FA, fatty acids: Ara N. arabinosamine.

tein (which may be covalently bound to PG, but with the lipid end extending into the outer membrane), trace metals, and an LPS (Fig. 10). The outer membrane may serve to protect a bacterium from environmental molecules or host defense factors. The LPS component of the outer membrane may be the sole virulence determinant for some gram-negative bacteria, as it possesses a spectrum of toxic qualities (Table I). For some gram-negative bacteria, injury to the outer membrane results in increased sensitivity to antibiotics, Ab, or lysozyme. The gram-negative bacterium may secrete capsular materials or protein toxins. In addition, the gramnegative bacterium may possess proteinaceous structures called fimbriae or pili, which may be involved in adhesion of the cells to substrata (Fig. I I). Gram-positive cells may also have surface structures called fimbriae, but these appear to be aggregated proteins rather than the highly ordered structures found in the gram-negative cells. Much of the remainder of this review deals with the biological properties of bacterial cell surface components. In addition, the role of bacterial adhesion and the role of cell wall turnover in bacterial pathogenesis will be discussed.


43

FIG. 11. Fimbriae associated with Escherichia coli. Arrow points to fimbrial structures possessing MS lectin activity (type 1 fimbriae). Bar = 100 nm. Reprinted by permission of T. I. Beveridge and Academic Press (Krell and Beveridge, 1987).

111. Biological Reactions to Peptidoglycans

In 1957, it was noticed that a prolonged inflammatory reaction occurred in the skin of rabbits after injection of crude streptococcal extracts (Schwab and Cromartie, 1957). Cardiac lesions similar to those seen in rheumatic fever were noted in mice following the intraperitoneal injection of a sterile extract of sonically disrupted group A streptococcal cells (Cromartie and Craddock, 1966). It was thought that the toxic material responsible for the lesions may have been cell wall fragments in both the skin and heart lesions. During this time it was also recognized that bacterial cell walls might contribute to the etiology of rheumatoid arthritis (Hamer-

44

R.J. DOYLE A N D E . M . SONNENFELD

man, 1966) and that similarities between the polysaccharides of human connective tissue and bacterial polysaccharides might be an important factor in the pathogenesis of the disease. Many studies have been completed over the years (Table II), and from these there emerge two important properties associated with a cell wall's capacity to induce a chronic arthritis in laboratory animals: ( 1 ) the molecular weight of the inducing fragments (Chetty et al., 1982) and (2) their ability to persist in host tissue (Schwab and Ohanian, 1967; Lehman et ul., 1985; Stimpson et al., 1986). Cell wall fragments with MW 5.3 x 10' induced only an early acute joint inflammation (Chetty et al., 1982; Fox P t ul., 1982), whereas larger fragments of MW 500 x 10' induced a chronic arthritis of late onset. Intermediate-sized fragments of MW 50 x 10' induced a chronic and acute arthritis. In seeming contradiction to these results, Kohashi et al. (1976) found that arthritogenicity was associated with PG subunits of two or more disaccharides in length and that an attached polysaccharide containing rhamnose had no effect. The vehicle for their cell wall preparations was an oil emulsion as opposed to an aqueous suspension, and it was injected into rats directly in the inguinal lymph nodes as opposed to intraperitoneal injection. Their purpose was to study the nature of adjuvant-induced arthritis (Pearson, 1956). These workers also found that removal of the GlcNAc moiety from a disaccharide-heptapeptide-disaccharide resulted in loss of arthritogenicity. Persistence of cell wall is directly proportional to its resistance to lysozyme (Schwab and Ohanian, 1967). Resistance to lysozyme is enhanced by 0-acetylation TABLE 11 BIOLOGICAL PROPERTIES OF PG A N D DERIVATIVES Property

Reference

Arthritogenicity Pyrogenicity lmmunogenicity Adjuvanticity Stimulation of IL- I production Somnogenic Enhanced resistance to pathogens Stimulation of phagocytic cells Increased production of collagenase and prostaglandin Cytolysis of neoplastic cells Immunosuppression by inhibition of 1L-2 Augmentation of serum interferon production Polyclonal B-cell activator Mitogenic

Fox et ul. (1982) Takada and Kotani (198.5) Heymer et ul. (1976) Ellouz et a / . (1974) Vermeulen and Gray (1984) Krueger ei ul. ( 1982a) Fraser-Smith et ul. (1982) Fraser-Smith et ul. (1982) Wahl ei (11. (1979). Taniyama and Holden (1979) Leclerc and Chedid (1983) lkeda el ul. (198.5) Specter el a/.( 1977) Damais ei ul. (1977)


4s

of muramic acid residues (Giesbrecht et al., 1982), the presence of a rhamnose-containing polysaccharide attached to the PG (Schwab and Ohanian, 1967), and lack of N-acetylation of glucosamine residues (Hayashi et al., 1973). Resistance of cell wall to degradation by macrophages and monocytes may contribute to its ability to act as a persistent Ag, thereby creating the necessary milieu for the incitement of a chronic arthritis. In fact, it has been shown that streptococcal cell wall will persist in macrophages (Rickles et al., 1969) that demonstrate a cytotoxic effect for mouse L cells (Smialowicz and Schwab, 1977). These authors speculate that the chronic tissue destruction exemplified by streptococcal cell wall in vivo may be the result of this cytotoxic ability of macrophages. The most extensively studied organism with regard to arthritis and cell wall has been Streptococcus pyogenes, which is both resistant to lysozyme and contains a group-specific polysaccharide containing rhamnose. Group A streptococcal cell walls were sensitive to lysozyme when the walls were chemically N-acetylated, but removal of the group-specific carbohydrate before N-acetylation rendered the walls even more susceptible to lysozyme (Gallis et al., 1976). It is thought that the group polysaccharide sterically hinders degradation by lysozyme. In staphylococci, autolytic enzymes appear to be inhibited by anionic polyelectrolytes (Ginsburg et al., 1985) and activated by cationic proteins found in lysosomes. Therefore, the accumulation of sulfated polysaccharides at sites of inflammation may tend to cause persistence of undigested cell wall material within macrophages. It has been noted that a higher relative risk for some forms of arthritis is related to genetic background. It is now known that rheumatoid arthritis is associated with human lymphocyte antigens HLA-DW4 (Rodnan and Schumacher, 1983) and HLA-DR4 (Albert and Scholz, 1987). However, the association with bacterial cell walls, though appealing, is still tentative. It is a tempting premise that similarities exist between cell wallinduced arthritis in laboratory animals and certain forms of arthritis that plague humans (Hadler, 1976). This is partially substantiated by findings demonstrating that rheumatoid factors, developed in animals immunized with streptococci, recognize both the PG of the bacteria and the Fc component of IgG (Bokisch et al., 1973). A variety of tissues, including those from the joint capsule (Rodnan and Schumacher, 1983), appear to be antigenically similar to cellular components of streptococci. This finding is emphasized by the observation that patients with rheumatic fever, juvenile rheumatoid arthritis (Heymer et al., 1976), and rheumatoid arthritis (Braun and Holm, 1970) possess Ab that cross-react with PG. It may be significant to note here that arthritis is a very common symptom often associated with rheumatic fever (Freeman, 1979). A complex interaction

46

R.J. DOYLE AND E.M.SONNENFELD

between Ab to streptococcal group A cell wall components and host tissue seems to be involved in the pathogenesis of this disease (Rotta, 1969). Cell wall material from a wide variety of bacteria is known to induce arthritis in rats and guinea pigs (Hadler and Granovetter, 1978). Some of these organisms would be considered indigenous microbiota. Reiter’s syndrome, a form of reactive arthritis that is HLA-B27-associated (Aho, 1987). has been linked to prior infections caused by Yersinia (Ahvonen et al., 1969), Shigella (Good and Schultz, 1977), Salmonella (Trull et al., 1986), Chlamydia (Keat et al., 1987), Campylobacter (Inman, 1986), and Neisseria gonorrhoeae (Rosenthal et al., 1980). Reactive arthritis involves joints that are anatomically distant from a site of prior infection (Campa, 1983) that is typically enteric or venereal in origin (Aho, 1987). The antecedent infection usually occurs 1-3 weeks before the onset of an arthritis (Aho, 1987) that does not respond to antibiotics. The arthritis is thought to be mediated by persisting microbial Ag (Toivanen et al., 1987) that may involve bacterial cell wall (Bennett, 1978), or perhaps LPS (Yaron et al., 1980). Endotoxins from Escherichia coli, Shigellaflexneri, Salmonella typhosa, and Vibrio cholerae were able to stimulate human synovial and foreskin fibroblasts to secrete prostaglandin E (Yaron et a f . , 1980). Shigella endotoxin was shown to cause secretion of prostaglandin E and hyaluronic acid (Yaron et al., 1980). In addition, LPS is capable of binding to eukaryotic cell membranes (Wicken and Knox, 1977), and this may enhance its ability to persist in host tissues. There may even be an interplay of molecular mimicry, in which HLA Ag crossreact with microbial Ag (Inman, 1986). Another example of a reactivetype arthritis is the arthritis associated with rheumatic fever. It is likely that streptococcal cell wall is involved in its etiology. In the early 1970s, adjuvanticity, which was found originally in oil-water emulsions of killed Mycobacterium tuberculosis organisms (White et al., 1958), was determined t o be a general feature of PG (Nauciel et al., 1973; Nguyen-Dang et al., 1973; Adam et al., 1974), and in 1974 the smallest molecule that still maintained this feature was determined to be a synthetic N-acetylmuramyl dipeptide, or MDP (Ellouz et al., 1974). Figure 12 shows the structure of MDP, which possesses adjuvant properties and will also induce an acute (as opposed to chronic) arthritis in rats treated with repeated subcutaneous injections of MDP in saline (Zidek et al., 1982). Most of the material was rapidly removed from the body, but a small fraction was suspected t o remain present for a longer time. These experiments demonstrated that an oil emulsion (Kohashi et al., 1980) was not a necessary vehicle for MDP in the production of an acute arthritis. Other properties of MDP include its ability to enhance resistance in mice to a wide variety of organisms (Fraser-Smith et al., 1982). Treatment


47

CH20H

H

I 0 I

CH3- CH

N-$-CH3

H O

- CII - NH - CFH3- C - HN - HCI - CI1 - OH 0

6

CH2O I

C-OH It

0 FIG.12. Structure of MDP (N-acetyl-D-rnurarnyl-L-alanyl-D-isoglutarnine).

of macrophages with MDP produces an entire myriad of effects (FraserSmith et al., 1982), some of which are most likely mediated by interleukin 1 (IL-1). In fact, it is possible that IL-1 is responsible for some of the joint destruction seen in arthritis (Wood et al., 1983a). It is interesting that mouse macrophages will “process” cell wall material from B. subtilis and concomitantly will secrete IL-1 (Vermeulen and Gray, 1984) and glycopeptides that are structurally related to MDP. The ability of MDP to act as adjuvant probably also is a result of IL-1 production by macrophages (Wood et al., 1983b), but this has not been proven conclusively (Fig. 13). In addition to the properties already discussed, MDP also possesses a sleep-inducing effect (Krueger et aL, 1982a). The natural peptide, which is found in urine and cerebrospinal fluid, induces excess slow-wave sleep in rats, rabbits, and cats (Krueger et al., 1982b). The phenomenon of sleep inducement is not species-specific in that the peptide collected from the cerebrospinal fluid of goats could induce sleep in cats that lasted 12-24 hours (Pappenheimer et al., 1967). Factor S, as it was called, was originally thought to contain glutamic acid, alanine, DAP, and muramic acid in molar ratios of 2 : 2 : 1 : 1. Although the composition resembled bacterial PG, the “extra” glutamic acid could not be explained on that basis. In addition, the original collection of cerebrospinal fluid was thought to be free from the possibility of exogenous bacterial contamina-

48

R.J. DOYLE A N D E.M. SONNENFELD

MDP

or

-

PEPTIDOGLYCAN

MACROPHAGE

\ IL- I SENSITIZED CELLS

I

IL-2 EXPANSION of T-HELPER CELL POPULATION

i

ENHANCED HUMORAL and CELL-MEDIATED IMMUNITY Ftc;. 13. Hypothesized action of MDP or PG in the enhancement of humoral and cellmediated immunity.

tion (Pappenheimer rt a/.,1967). Nevertheless, the possibility that the sleep factor was derived from bacterial products absorbed from the gut (Maugh, 1982) could not be refuted because biosynthesis of muramic acid had never been shown in mammalian tissues. In fact, the actual presence of muramic acid tended to support the contention that the substance originated from bacteria (Karnovsky, 1986). Today, there is even some thought that muramyl peptides are similar to vitamins, since they may be required but cannot be synthesized by the host (Krueger, 1986). Some bacterial products can antagonize sleep, supporting the contention that these peptides play an important role in the regulation of sleep (Maugh, 1982). Normal human brain has been shown to contain muramic acid (Zhai and Karnovsky, 1984), while DAP, thought to be of bacterial origin, was found as a normal component of human urine in 1980 (Krysciak, 1980). Also, the L-alanine-D-isoglutamate-containingcompound, as it is found in PG, is biologically active, whereas the L-L or D-D isomers are not (Karnovsky, 1986). All of these data support the assertion that sleep factors are indeed of bacterial origin, and may even be responsible for sleepiness associated with infectious disease (Krueger, 1986). Using fast atom bombardment-mass spectrometry, the structure of the urinary sleep-promoting factor was determined (Fig. 14) (Martin et al., 1984). The major component was found to be a disaccharide containing GlcNAc and N-acetylanhydromuramic acid in linkage with L-alanine, D-

49

BACTERIAL CELL SURFACE CH2 -OH

0

II

C-OH

FIG.14. Structure of the urinary sleep-promoting factor N-acetyl-glucosaminyl-N-acetylanhydromuramyl-L-alanyl-D-glutamyl-diam~nop~melyl-D-alan~ne, as determined by fast atom bombardment-mass spectrometry (Martin et a / ., 1984).

isoglutamine, mesodiaminopimelate (DAP), and D-alanine. The muramyl form of this compound also possesses biological activity (Krueger et al., 1984), but it is less potent than the anhydro form (Krueger et al., 1986). Somnogenic activity is lost when the a-carboxylate of glutamic acid or the ecarboxylate of DAP is amidated (Krueger er al., 1984). Rosenthal and Krueger (1987) found that three types of PG fragments from Neisseria gonorrhoeae were capable of enhancing slow-wave sleep in rabbits. The fragments were N-acetylglucosaminyl- 1,6-anhydro-N-acetylmurarnyl-alanyl-glutamyl-diaminopimelyl-alanine, the corresponding anhydro-N-acetylmuramyl disaccharide with an additional alanine at the C terminus, and anhydro-N-acetylmuramyl tetrapeptide. It is interesting that IL-1 can also induce sleep (Krueger et al., 1984), since as stated earlier, PG can stimulate macrophages to secrete IL-1. This allows one to speculate on the possibility that muramyl peptides may function as somnogens via a mechanism involving IL-1 (Krueger, 1986).

IV. Bacterial Cell-Surface Amphiphiles Amphiphiles may contribute to the pathogenic potential of many bacteria. Both gram-positive and gram-negative bacteria possess surface amphiphiles that have been shown to display a variety of biological properties. In some cases, the amphiphile is toxic to the host, whereas in others, the amphiphile may provoke an immune response. Table I11 lists the most common amphiphiles encountered among bacterial pathogens. The LPS molecules of the Enterobacteriaceae have been one of the most widely studied groups of microbial structures. The LPS molecules


50

TABLE 111 1 M W K T A N T BACTERIAL. CELL S U R F A C E

Aphiphile Forssman Ag Enterobacterial common Ag

Lipomannan

LPS

Lipoprotein

LTA

AMPHIPHILES"

Comments A membrane component of the pneumococci. Structure consists of short-chain saccharide attached to lipid. Surface polysdccharide of most members of Enterobacteriaceae. Fatty acids may be linked to polysaccharide consisting of GlcNAc and a uronic acid. From membrane of members of the genus Micwcoccirs. Mannose polymer (partially succinoylated) containing terminal glycolipid. Component of outer membrane of some gram-negative bacteria. Consists of a complex heteropolysaccharide and lipid A (fatty acid, usually P-hydroxymyristic acid, covalently bound t o glucosamine). Covalently bound to PG of some gram-negative bacteria. May confer stability to outer membrane. Membrane component of many gram-positive bacteria. Usually consists of a polymer of glycerol phosphate, with fatty acids esterified a s glycolipids or phosphat idylglycolipids.

"Partially compiled from Wicken and K n o x (1981a.b).

have traditionally been prepared by the Boivin or Westphal procedures (Table IV). The preparations are generally contaminated with other molecules, such as RNA, glycogen, or protein. Regardless of the extent of contamination, the preparations retain their biological properties. The term endotoxin was originally used by Richard Pfeiffer in 1892 while working with cholera vibrios (Westphal et al., 1977). This term seemed appropriate because he observed that this particular toxic material was not excreted from living bacteria, as an exotoxin, but rather was released when the bacteria underwent lysis. Today the term endotoxin is still used, often synonymously with LPS, although this is not exactly correct. Endotoxin is actually LPS with attached protein complexes (Morrison and Ulevitch, 1978). In 1952, Westphal and Luderitz applied the phenol-water extraction method to isolate protein-free LPS (Westphal et ul., 1977). Protein is removed by the phenol, whereas the LPS is recovered from the aqueous phase. Chemically, LPS consists of three sections (Fig. 10): a hydrophilic region, a central acid core, and a lipid-rich region (Osborn et af., 1974). The hydrophilic head contains a heteropolysaccharide chain known as the 0-specific antigenic unit. The 0 Ag refers to the repeating carbohydrate linkages, which impart immunological identity to


51

TABLE IV LPS PREPARATION^ Preparation Boivin Morrison-Leive Leive-Morrison Hybrid-free Westphal

Freeman Ag

Lipid A

Ethylether

Procedure/properties Supernatants from whole cells extracted with trichloroacetic acid; contaminated with polysaccharide, RNA, acid-soluble proteins Butanol extraction yields a preparation with small amounts of contaminating protein. EDTA releases -50% of the LPS in a high molecular weight form. Hypertonic solution-extracted LPS preparations were more toxic and homogeneous than phenol-water extracts. When whole cells are extracted with 44% phenol at 64°C. a single phase forms. Upon cooling, the aqueous phase containing the LPS may be separated by centrifugation. Preparations are generally contaminated with proteins. When Boivin or Westphal LPS preparations are mildly hydrolyzed with acid, the Freeman Ag (polysaccharide) may be obtained by precipitation with alcohols. It is useful in determining epitopes of LPS preparations. Deep rough mutants may be extracted to yield lipid A (a toxophore), which can then be further purified by hydrophobic chromatography. These preparations are useful in studying the toxicity and biological properties of LPS molecules. Lipopolysaccharide is released in a soluble form from broken cell suspensions by ethylether.

“From Morrison and Leive (1975). Leive and Morrison (1972). Raynaud et d . (1973). Ribi et d.(1959). Jawetz e t a / . (1984). Westphal et a/. (1977). Morrison and Ulevitch (1978). and Rietschel et d. (1982).

the polysaccharide portion of the LPS. These carbohydrates generally occur as tetrasaccharide repeating units, although trisaccharide units are not uncommon. Mutations leading to loss of the repeating 0-Ag sequences result in a structure called the “R” Ag. The R-Ag name is derived from the word rough, which characterizes the colonies of the mutants. The R-Ag polysaccharide core may also be modified by additional mutations, leading to LPS molecules with various core sizes (Fig. 10). The polysaccharide portion of the LPS may be removed from the rest of the molecule by mild acid hydrolysis (Table IV). The released polysaccharide, called the Freeman Ag, is used to study the antigenic specificity of an LPS molecule without the complications arising from its lipid portion. As far as is known, all of the toxic qualities of an LPS molecule can be attributed to its lipid A moiety (Fig. 10). Luderitz ct al. (1966) showed that LPS preparations from various mutants in R- or 0-Ag saccharides were just as toxic as the complex LPS molecule. Some of the reported biological activities of LPS molecules are outlined in Table V. These

52

R.J. DOYLE AND E.M. SONNENFELD TABLE V BIOLOGICAL PROPERTIES OF LIPIDA Property Pyrogenicit y Adjuvanticity Mitogenicity (B-cell polyclonal activator) Elicits Shwartzman reaction Geiation of Limulus lysate Activates alternate pathway for complement Shock Aggregates platelets Antigenic (the lipid A portion is normally immunosilent) induction of hypoglycemia Leukopenia, followed by leukocytosis Release of prostaglandins from macrophages Abortion Activation of Hageman Factor X11 impairment of oxidative phosphorylation Enhancement of lysosomal enzyme activities Necrosis of tumor cells

AND

LPs Reference

Siebert (1952) Freeman (1979) Gery et a / . (1972) Shwartzman (1982) Levin and Bang (1964) Morrison and Kline (1977) Freeman ( 1979) Des Prez et a / . (1961) Westphal (1975) Wolfe et a / . (1977) Athens er a / . (1961) Rietschel et a / . (1982) Freeman (1979) Morrison and Cochrane (1974) Bradley (1979) Martini (1959) Bradley (1979)

range from the well-known pyrogenic property, to B-cell mitogenicity, to the activation of complement, and others. The pyrogenicity of LPS is due to the release of endogenous pyrogens from phagocytic cells following uptake of the LPS. The endogenous pyrogens are low molecular weight proteins that act on the hypothalamus gland. A febrile condition may occur as a result of viral or gram-positive bacterial infections as well, but the pyrogenic response is a characteristic of most gram-negative infections. Pyrogen (LPS) can be detected in very small quantities by use of Limulus amebocyte lysates. The lysates readily gel in the presence of LPS at 0.1 kg/ml. This level of sensitivity is useful not only in detecting LPS in various kinds of solutions and suspensions, but also in the laboratory diagnosis of meningitis. Spinal-tap fluids yielding a rapid gelation of the Limirlus amebocyte lysate suggest a gram-negative infection. The biological effects of LPS are mediated after it adheres to host tissue. Lipopolysaccharide attaches firmly to erythrocyte membranes via a lipoglycoprotein receptor (Springer et al., 1974). This receptor is specific for LPS in that it can reversibly block LPS from binding to red blood cells, (RBC), and it will not bind other bacterial Ag. Additionally, the fixation to RBC was inhibited by the receptor whether the LPS was from smooth or rough cultures. The activity of the receptor was confined to the protein moiety because removal of lipid or carbohydrate did not decrease


53

activity of the receptor. In addition to RBC, platelets, granulocytes, and mononuclear leukocytes also have high affinity for LPS, but it seems that this binding is due to lipid rather than a lipoglycoprotein (Springer and Adye, 1975). It is probable that hydrophobic interactions are important in the binding of LPS to RBC membranes and platelets and white cells. There is convincing evidence demonstrating that the lipid A part of LPS is responsible for its attachment to host tissue (Luderitz et al., 1973). The phenomenon of lectinophagocytosis (Ofek and Sharon, 1988), whereby lectins on the surfaces of phagocytes recognize specific sugars or saccharides to promote microbial phagocytosis, has not been studied with purified LPS molecules. One of the biological properties of LPS is its ability to activate both the classical and alternate pathways of the complement cascade (Morrison and Kline, 1977). The lipid A region of LPS is responsible for activation of the classical pathway and is antibody-independent, although the extent of the activity is not identical for all species of LPS (Luderitz et al., 1973). The properdin-induced, or alternate pathway, is activated by the polysaccharide portion of LPS. Solubilized lipid A, rendered soluble by alkali treatment or serum albumin, was found to be highly active as an anticomplement agent, in addition to promoting mouse lethality, pyrogenicity, bone marrow necrosis, and Limulus gelation (Luderitz et al., 1973). From these results it was concluded that lipid A exists as the “biologically active center” in LPS. This is further substantiated by results of the effects of synthetic lipid A, which is free of contaminating bacterial components yet retains biological activity (Rietschel et al., 1987). It appears that the polysaccharide section acts to solubilize lipid A in the intact molecule, and polysaccharide can be replaced by serum albumin (Luderitz et al., 1973). Removal of fatty acids (Nowotny, 1987), alkylation (Chedid et af., 1975), or dephosphorylation(monophosphoryllipid A) (Johnson et al., 1987) can lead to detoxification of lipid A, in which the altered molecule retains many of its beneficial aspects (Nowotny, 1987). Lipopolysaccharide can act as an adjuvant, is mitogenic for B cells, and is considered a polyclonal B-cell activator. These characteristics are also attributable to lipid A (Chiller et al., 1973). Another effect of LPS is the Shwartzman phenomenon, which may be considered a type of disseminated intravascular coagulation (DIC) (Jawetz et af., 1984). In this reaction, an animal receives an intradermal injection of endotoxin and the following day receives endotoxin intravenously. Necrosis of the initial skin site occurs in a few hours. When endotoxin is given intravenously on each of 2 successive days, DIC occurs. The initial dose of endotoxin can be replaced by carbon particles or other material that has the effect of obstructing the reticuloendothelial system. The effectiveness of the Shwartzman reaction

54


is proportional to the quantity of lipid A in the sensitizing substance (Ohta et al., 1985). Responses of individual types of cells to LPS are varied. The LPS may bind to platelets via its lipid A (Morrison and Oades, 1979) and cause aggregation and release of mediators, such as 5-hydroxytryptamine (Des Prez rt al., 1961). The lytic response appears t o be dependent on the alternate pathway of complement (Momson and Oades, 1979). The mitogenic response of lymphocytes to LPS seems to occur after its administration in vivu or following its mixture with cells in v i m . The B-cell response, limited to mice, results in the synthesis and secretion of immunoglobulins (Ig) from immunocompetent cells. The mitogenic activity of LPS, along with the T-cell mitogenic activities of concanavalin A and other lectins, led t o a much better understanding of the roles of lymphocytes and plasma cells in immunity. Behling et al. (1976) found that when fatty acids were substituted on the amino group of glucosamine, the resulting glycolipids were potent B-cell mitogens, suggesting that the lipid A moiety of LPS is responsible for the mitogenic effects. Treatment with LPS results in alterations in the metabolism of cells, including a decrease in activity of mitochondria1 dehydrogenases and an increase in the incorporation of thymidine, uptake of glucose, formation of lactic acid, and activity of lysosomal hydrolases (Bradley, 1979). Injected into experimental animals, LPS can induce a myriad of nonspecific effects, including fever, hypotension, aberrations in white blood cell counts, DIC, irreversible shock, and death (Rietschel et al., 1982). In contrast, the host may actually require the continual exposure to LPS in the gut for proper maturation of the immune system, while from the bacterium’s point of view, LPS is a protective outer coating, aiding in its ability to resist phagocytosis (Rietschel et al., 1982). Lipopolysaccharide molecules appear t o have several effects on polymorphonuclear leukocytes (PMN). Injection of LPS causes a leukopenia followed by a leukocytosis (Mechanic et al., 1962). The leukopenia may depend on the presence of lipid A (Corrigan and Bell, 1971). The LPS of rhizobia plays a significant role in their ability to interact with legumes during nitrogen fixation. The association is through the 0 polysaccharide and the lectin of the legume. It is selective for each species of nitrogen-fixing rhizobia-legume pair that is found to enter into a symbiotic relationship in nature, and it explains, at least in part, the high degree of specificity exhibited by this relationship (Wolpert and Albersheim, 1976). The LTA molecule (Fig. 2) contains a highly polar poly(glycero1 phosphate) chain and a hydrophobic end, consisting primarily of fatty-acid esters. The fatty-acid composition of an LTA molecule generally reflects


55

the fatty-acid composition of the bacterial membrane, as shown for analyses of several preparations from different genera. Probably the most useful method for LTA preparation involves extracting a bacterial suspension with 44% phenol at 62"-65"C (Fischer et d . , 1983). A single phase forms at the elevated temperatures. Upon cooling, the aqueous and phenol phases separate and the LTA can be recovered from the aqueous layer. Prepared in this way, LTA is contaminated with nucleic acids, protein, and in certain cases, polysaccharide. Nucleases and proteases can be used to degrade the contaminating materials. Finally, the highly purified LTA is obtained by gel exclusion chromatography of the digest on crosslinked agarose. Silvestri et al. (1978) found that small quantities of LTA could be obtained in highly purified form by extraction with liposomes. An especially promising method for LTA purification has been described by Josephson et al. (1986). After producing LTA from phenolwater extracts of cells, it was purified using a salt gradient elution from DEAE-cellulose. Prepared in this manner, LTA resolved into two main components that differed in fatty-acid content. In order to study the biological properties of LTA, it is necessary to be able to identify and remove potential contaminants. Table VI summarizes some of the reported effects of LTA on biological systems. (Another section details the role of the LTA in adhesion of gram-positive cocci to mucosa.) It is interesting that many of the same effects have been observed for LPS molecules as well. Both types of molecules are amphiphiles. Both have long-chain hydrophilic repeat units, TABLE VI SUMMARY OF SOMEREPORTED BIOLOGICAL PROPERTIES OF BACTERIAL LTA Property

Reference or review paper

Adhere to red cells and other cell types Complex with M protein of streptococci Mitogenic Antigenic Activates macrophages Shwartzman reaction Activates complement Complexes with FN Inhibits streptococcal glucosyltransferase Inhibits autolysins of bacteria Stimulates bone resorption Stimulates nonspecific immunity Solubilizes glucans in alcohol Gelation of Limulus lysate

Hewett et a / . (1970) Ofek et a / . (1982) Mishell et a / . (1981) Wicken and Knox (1975) Wicken and Knox (1980) Wicken and Knox (1980) Fiedel and Jackson (1981) Courtney et a/. (1983) Kuramitsu er a / . (1980) Cleveland et a / . (1975) Wicken and Knox (1980) Wicken and Knox (1980) Cowan et a/. (1988) Kessler (1983)

56


and both have hydrophobic ends. Both types of molecules form micellar aggregates and both bind to various kinds of membranes. The reported toxicities of LPS, however, are far greater than those generally acknowledged for LTA. The mitogenic effects of LTA have been studied by several investigators. Mishell et al. (1981) have reported that LTA preparations are B-cell mitogens. Beachey et a / . (1979) previously had reported that LTA was a weak T-cell mitogen. Earlier, Miller et al. (1976) could find no evidence for LTA-induced mitogenic effects. Hamada et al. (1985) have provided strong evidence to show that LTA preparations from several streptococci were good nonspecific B-cell mitogens of murine spleen cell lymphocytes. Mouse thymocytes were not activated by the LTA. In addition, deacylated LTA was a far weaker mitogen than the fully acylated preparations (Hamada et al., 1985). Most of the LTA preparations employed by Hamada et al. were effective mitogens at 0.1-10 pglml. Some of the discrepancies in results for LTA-induced mitogenicity may be due to lack of knowledge of state of purity, extent of acylation, or different concentrations employed by various investigators. Lipoteichoic acid may have a dual effect on immunocompetent cells. Miller and Jackson (1973) found that LTA (and deacylated LTA) from pyogenic streptococci reduced the B-cell response as measured by Ab production to sheep RBC. Miller ef al. (1976) later observed that the LTA could potentiate Ab production against an LPS Ag. Hamada et al. (1985) found that when LTA and sheep RBC were injected simultaneously via the intravenous route, an enhanced immune response occurred. Similarly, both materials added to cell cultures produced an elevated response against the sheep cells. There may be effects of LTA that are as yet poorly understood in terms of immune regulation. A proper balance of immune-cell activation and suppression may be required to mount a normal immune response against bacterial pathogens. It has been known for some time that LTA could initiate the enzymatic events leading to gelation of the Limulus amebocyte lysate. Kessler (1983) studied the structural requirements of LTA for eliciting the gelation reactions. Lipoteichoic acid molecules with shortened poly(glycero1 phosphate) chains were superior to longer chain LTA. In addition, substitution of the glycerol residues by D-Ala or by saccharides resulted in preparations with higher specific activities. The results seem to suggest that hydrophobic LTA are superior to relatively hydrophilic LTA in initiating the gelation reactions. Because the LTA is much less effective, compared to LPS molecules, in promoting the lysate gelation (Kessler, 1983), clinical usage of the reaction has been limited in detecting gram-positive infections or contaminants.


57

The fatty-acid groups of LTA are also required to sensitize RBC to agglutination by anti-poly(glycero1 phosphate) (Hamada et al., 1985). There has been some degree of controversy surrounding the binding of LTA by RBC. Chorpenning et al. (1979) and Cooper et al. (1978) concluded that lipid-free teichoic acids could readily bind to RBC membranes. Results of Hamada et al. (1979, 1985) and Chiang et al. (1979) support the view that lipid acyl groups are required for the spontaneous association between the RBC membranes and teichoic acid preparations. Chiang et al. (1979), Hewett et al. (1970), and Ofek et al. (1975) have noted that deacylated LTA molecules seem to possess little affinity for animal cell membranes. It is unlikely that the membranes possess specific LTA-binding proteins. Present results suggest that the lipid moiety of the LTA can bind directly to hydrophobic sites in the membranes, causing the poly(glycero1phosphate) chains to protrude outward, where they may interact with Ab or lectins. Lipoteichoic acid has been reported to be a regulator of autolytic activity for several gram-positive bacteria. Cleveland et al. (1975) were the first to show that an LTA preparation could inhibit a bacterial autolytic enzyme. Autolysins from Staphylococcus aureus (Suginaka et al., 1979), Streptococcus pneumoniae (Holtje and Tomasz, 1975), Streptococcus faecalis (Cleveland et al., 1976), and B. subtilis (Rogers et al., 1984) have been reported to be inhibited by LTA preparations. The results of the foregoing researchers were obtained by use of in vitro measurements. How LTA could regulate an autolysin in vivo is unknown. The lipid moiety seems to be a structural requirement for inhibition of autolysins in vitro. Autolysis of whole cells and cell wall turnover seem to be good indicators of autolytic activity in vitro. When Jolliffe et al. (1981) added LTA to growing cultures of B. subtilis, there was no reduction in the rate of cell wall turnover, suggesting that LTA was not interacting with the autolysins. Furthermore, although susceptibility of B. subtilis to nafcillin is proportional to cell wall turnover, there was no effect of LTA on the bacteria when mixed with the antibiotic (Jolliffe et al., 1982). If LTA interacts with autolysins in vivo to regulate autolytic activity somehow, it would be expected that the LTA could dissociate from the enzyme. To date, however, association-dissociation equilibria or kinetics have not been described for LTA-autolysin interactions. Cowan et al. (1988) observed that various LTA preparations could promote the solubilization of D-glucans in concentrated ethanol solutions. A few micrograms of LTA could form alcohol-soluble complexes with nearly 1 mg of glucan. The glucan is normally insoluble in the alcohol, but LTA prevents its precipitation. The only structural requirement for the solubilization reaction is the poly(glycero1phosphate) chain (Doyle et

58


al., 1975). Lipid-free teichoic acids also form alcohol-soluble complexes with polysaccharides. Interestingly, Cowan et al. observed that cell-free whole saliva (but not parotid saliva) could also promote the solubilization reaction, using a high molecular weight a4,6 glucan as the polysaccharide. The salivary component capable of complexing with the glucan was microbially derived LTA. When whole saliva was treated with sucrose or penicillin, LTA (or deacylated LTA) was released from the normal microbiota, thereby enhancing the glucan solubilization by the saliva. Furthermore, when saliva was added to a Sephacryl column equilibrated with 80% ethanol, LTA and a 60-kDa protein were retained, only to be eluted by water. It appears that the LTA in saliva not only binds glucans in ethanol, but at least one salivary protein as well. Parrish and Doyle (unpublished observations) have used the alcohol-Sephacryl-water elution system to purify LTA from Streptococcus sobrinus and B . subtilis. The biological significance of LTA-glucan or LTA-protein complexes in alcohols is unknown, but it may reflect interactions between LTA and hydrophilic molecules in hydrophobic milieus. Cowan et al. (1988) suggested that these kinds of interactions may be important in the formation of dental plaques. Macromolecular complex formation, aided by stabilizing hydrophobic interactions, has been considered a driving force in the adhesion of oral streptococci to teeth (Doyle et al., 1982).

V. Surface Adhesins of Bacteria and Pathogenesis Most bacterial diseases require that the bacteria colonize a site in a host so that a sufficiently large number of cells be available to discharge toxic materials or induce inflammatory responses. The colonization must be preceded by adhesive events before bacterial mass can increase significantly. Sometimes the adhesion of bacteria to host cells involves only a few bacteria. Once adherent, the bacteria can then undergo cell division. There are various surfaces in the human body onto which bacteria may adhere. These include keratinized and nonkeratinized epithelial cells, endothelia, bone, and saliva-coated teeth. Many bacteria adhere to all of these surfaces, but may be found only in certain parts of the body. An E. coli for example, would normally be considered as a bacterium residing near or on gut mucosa, but the organism can adhere to many types of cells. An understanding of the apparent tropism of E. coli for gut mucosa must take into account other factors, such as nutrient supply, temperature, and host defense molecules (e.g., lysozyme, Ig). Adhesion of a bacterium to a substratum requires that the bacterium approach a surface closely enough so that complementary molecules can


59

form a complex (Figs. 15 and 16). Most bacteria are highly negatively charged on the cell surfaces, but they also possess clusters of positive charges on their surfaces. In addition, most bacteria possess hydrophobic molecules (hydrophobins) near or on their cell walls. The presence of oppositely charged groups and hydrophobins make it possible for two highly negative surfaces to form a stable union. Oral streptococci, for example, adhere avidly to negatively charged pellicle proteins even though the bacteria have net negative surface charges. For most pathogenic bacteria, adhesion to some kind of surface is an ecological advantage. Adhesion provides a means for the bacteria to be more effective in obtaining nutrients. Escherichiu coli bound to tissue culture cells is able to divide more rapidly than nonadherent E. coli and exhibits a shorter lag time in division than nonadherent cells (Zafriri et al., 1987). Various surface components of a bacterium may participate in adhesion (Tables I, VII). Fimbriae, for example, possess lectins or lectinlike proWEAK ASSOCIATION (REVERSIBLE 1

u u

%8Q

K,

K-I

COLONIZATION

\ IN VAS I0N

FIG. 15. Steps leading to firm adhesion of a bacterium to a substratum. The bacterium initially approaches the substratum and is bound loosely with only a few reversible interactions ( K ,rate). The bacterial ligands may subsequently combine with complementary receptors, creating an “irreversible” adhesion ( K , rate). The rate constant K.? is very low, showing that dissociation is a critical step in the formation of a stable cell-substratum union. Once firmly adherent, the bacteria may divide (colonize) and may produce symptoms of disease (invasion). (See also Gristina et a / . , 1985; Gristina, 1987).

60


111

e-

*

BACTERIA

Ill

BLOOD FLOW CILIARY ACTION COUGHING DES Q UA M AT I 0N EXCRETION PERISTALSIS SECRETIONS SNEEZING

-

Ill AIR,FLUID FLOW

UNCOLONIZED MUCOSA

C0LONIZ E D MUCOSA

FIG. 16. Factors involved in the adhesion of bacteria to mucosal surfaces. Even if the bacterium can interact with complementary receptors on the mucosa, assurance of adhesion may not be realized because of air or fluid flow. Fluids may contain antibodies, complement, lysozyme. lactofenin, and other agents that could interfere with adhesion to epithelial or endothelial surfaces. Addition of sugars or saccharides to the system may also block adhesion. Adapted from Ofek and Beachey (1980b).

teins that enable the bacteria to adhere to carbohydrates on substrata. The specificities of some of the fimbrial and surface lectins are reviewed in Table VIII. Type I fimbriae of the Enterobacteriaceae complex with mannose (Man) or Man derivatives, whereas type P fimbriae bind to Galcrl-4Gal residues. Other fimbriae may interact with GlcNAc, sialic acids, or Gal-containing structures. Nonfimbrial proteins also possess lectin activities. Streptoeoccrrs cricetirs binds a-1.6-linked isomaltooctaosesdecaoses (Drake et al., 1988a,b). In some cases, LTA and surface proteins contribute to adhesion. Secreted polysaccharides may also play a role in the tethering of some pathogens to substrata. The importance of adhesion to infectivity is shown in Table IX. Gramnegative bacteria possessing fimbriae are more infectious than their nonfimbriated variants. In most of these cases, the fimbriae bear lectins specific for sugars or saccharides. In E. coli, the most widely studied fimbriae are specific for Man and Galal-4 Gal. If fimbriae are involved in virulence, then carbohydrates complementary for the lectins may prevent infections. Results from several laboratories are summarized in Table X showing that carbohydrates can reduce experimental infections due to E.

61

BACTERIALCELLSURFACE TABLE VII SURFACE ADHESINS OF SELECTED BACTERIAL PATHOGENS Bacterium Staphylococcus aureus

Streptococcus pyogenes

Adhesin(s) Wall teichoic acid Surface protein Hydrophobins M protein LTA

Streptococcus agalactiae

Streptococcws pneumoniae Streptococcus sanguis

Staphylococcus suprophyricus Staphylococcus epidermidis

Escherichia coli

LTA, with surface proteins LTA Surface protein Lectin Hydrophobins and pol yanions LTA Lectin Extracellular polysaccharide (PCHO) H ydrophobins Type 1 (fimbriae) Type P (fimbriae)

Reference Aly and Levit (1987) Kuusela (1978); Ryden et ul. ( 1983) Mamo er a / . (1987) Tylewska et a / . (1988); Ellen and Gibbons (1972) Beachey and Ofek (1976); Ofek et a/. (1975) Jacques and Costerton (1987) Teti ef a/. (1987); Nealon and Mattingly (1984) Miyazaki et a/. (1988) Anderson et al. (1983; 1986) Nesbitt et a / . (1982); Cowan er a/. (1986a,b); Busscher and Weerkamp (1987) Teti e f a / . (1987) Gunnarson et a / . (1984) Christensen et a / . (1982)

Hogt et a / . (1983) Mirelman and Ofek (1986) Lefler and Svanborg-Eden (1980)

coli, Klebsiella pneumwiae, and Sh. flexneri. The presence of Man or a mannoside prevented the infections associated with E. coli bearing type 1 fimbriae (see also Table XI). The premise that infections may be prevented or inhibited by carbohydrates is attractive (Fig. 17). To date, however, practical considerations have precluded any significant clinical tests. For example, Man added to fluids for intake would be rapidly metabolized or excreted in the human. Some saccharides may be toxic, whereas others are not available in sufficient quantities to institute appropriate human experimentation. Another avenue for prophylaxis may take advantage of the immune response against fimbriae. Several reports have shown that Ab directed against fimbriae afford protection against infection in various experimental animals (Table XII). Possibly, antilectin immune responses are responsible for the observed protection, although this is not known with certainty. The lectin molecule is thought to reside at the very tip of the fimbrium and to comprise only a small percentage of fimbrial protein (DeGraaf and Mooi, 1986). Most antifimbrial Ab would

TABLE VlII LECTINS OF BACTERIAL PATHOGENS"

SPECIFICITIES OF S U R F A C E

Sugar or saccharide

Bacterium

N - Acet yllactosamine

Leptotrichia buccalis. Eikenella corrodens Pasteurella multocida, Escherichia coli, Chlamydia trachomatis Vibrio cholerae E. coli Staphylococcus saprophyticus Actinomyces spp. E. coli Neisseria gonorrhoeae Streptococcus pneumoniae Bartonella bacilliformis Streptococcus cricetrrs E. coli Bacteroides spp., Mvcoplnsma pneumoniae E. coli

N-Acet y Igl ucosamine L-Fucose Gala( 1-4)Gal GalP( 1-4)GlcNAc P-Galactosides Gal, Galp( 1-3)Gal Gal-GalN Ac-Gal GlcNAcP( 1-3)Gal D-Glucose lsomaltooctaose-isomaltodecaose Man and mannosides Sialic acid Sialic acid-Gal

"Derived from Drake er a/. (1988a): Mirelman and Ofek (1986): Wadstrorn and Tmst (1984): Beutheral. (1987j:Gunnarsonef nl. (1984); Uhlenbruck.(l987); Holrngren cr nl. 11983): and Ofek er a/. (1917).

TABLE IX ADHESION OF GRAM-NEGATIVE BACTERIAin Relative adhesion Bucterium

in vitro

ViWO AND

INFECTIVITY"

Relative infectivity

Bacterial variants ~

~~

Poor

High Low

Fimbriate Nonfimbriate

Escherichia coli

Good Poor

High Low

K88 * K88-

E. coli

Good Poor

High Low

CFA' CFA-

Neisseria gonorrhoeae

Good Poor

High Low


Proteus mirabilis

Good Poor

High Low


Salmonella typhimrrrium

Good Poor

High Low


Border ella pertussis

(enterotoxigenic strain)

Good

"Adapted from Beachey cr ol. (1982).

62

TABLE X PREVENTION OF INFECTIONS BY FIMBRIAL LECTININHIBITORS Bacterium Escherichia coli (type P fimbriae)

Animal and site of infection"

Inhibitof

Reference

Mouse, UT

Globotetraose

Leffler and SvanborgEden ( 1980) Roberts et a / . (I 984)

Monkey, UT

Gala4GalpOMe

E. coli (type I fimbriae)

Mouse, UT

MeaMan

Aronson et a / . ( 1979)

E. coli (type I fimbriae)

Mouse, GI

Man

Goldhar et a / . ( 1986)

Shigella jlexneri (type I fimbriae)

Guinea pig, eye

Man

Andrade (1980)

Klebsiella pneumoniae (type I fimbriae)

Rat, UT

MeaMan

Fader and Davis (1980)

"UT. Urinary tract; GI, gastrointestinal tract. *Me. Methyl.

TABLE XI OF INHIBITION OF ADHESIONBY SUGARS OR SACCHARIDESAND MODIFICATION BACTERIALINFECTION'

Tissue

Number of animals

Escherichia coli E. coli

Mouse bladder Mouse bladder

101

E. coli

Mouse bladder

35

Klebsiella pneumoniae K . pneumoniae

Rat bladder Rat bladder

5 5

K . pneumoniae Shigella jlexneri

Rat bladder Guinea pig eye Guinea pig eye

5 20

Bacterium

Sh. j7exneri

99

20

Inhibitor None Methyl-a-w mannoside Methyl-a-Dglucoside None Methyl-a-Dmannoside D-G~UCOSe D-Man (right eye) D-Glucose (left eye)

Percentage colonized or infected

11 22

64 80 20 100

20 70

"Results derived from Andrade (1980); Aronson et a / . , (1979); and Fader and Davis (1980). All the bacteria possessed type 1 fimbriae.

63

64


A

Bacterium

FIG. 17. Blocking of bacterial adhesion by adhesin or receptor analogs.

therefore be expected to be directed against nonlectin protein. The use of fimbriae or fimbrial lectin as a vaccine against various infectious agents holds promise. It will probably be necessary t o clone the fimbrial lectin before enough of the protein is available for use as a vaccine. Bacteria, like all life forms, have a tendency to adapt to their environment. There are many examples to show that bacteria must face changing environments, however. A Pseudomonas aeruginosa, normally an inhabitant of plants, may find itself occasionally (in evolutionary history) on an animal cell surface. The Pseudomonas may require different surface structures to adapt from plant to animal. Similarly, an E. coli residing near gut mucosa, rich in Man-containing proteins, characteristically possesses receptors for type 1 fimbriae. On the other hand, bladder and kidney tissue is rich in glycolipids containing the Galcx1-4Gal sequence. It is not surprising therefore that most gut-associated E. coli express type 1 fimbriae and most genitourinary tract E. coli express type P fimbriae. It is as if the E. coli has evolved a means to sense that an environmental change may occur. It now seems clear that the phenomenon of phase

PROTECTION AGAINST Animal

TABLE XI1 Escherichiu co/i PROVIDED

INFECTION WITH

Site“

Fimbriae

Rats Rats Pigs Monkeys

UT GI GI UT

Type Type Type Type

Mice

UT

Type P

Pigs

GI

Type K99

“UT. Urinary tract: GI, gastrointestinal tract

1 1

I

P

BY

ANTIFIMBRIAL AB Reference

Silverblatt and Cohen (1979) Guerina er a / . (1983) Jayappa er a / . (1985) Kaack er a / . (1988); Roberts er a / . (1984) O’Hanley er al. (1985); Schmidt et al. (1988) Isaacson et al. (1980)

65


transition (Fig. 18) occurs in order for a bacterium to diversify its habitat. Phase transition (Eisenstein, 1981) reflects a change in the expression of a surface component (lectin, etc.) at a predictable rate. Phase transitions seem to occur at the rate of 1 in 102-105,much higher than for a mutation. A phase variant would give rise to a new phase variant at the same rate. Thus, E. coli possessing Man-sensitive (MS) lectins give rise to variants that are devoid of the lectins, but these variants later give rise to MS' phenotypes. Phase transitions occur regularly and often independently of growth conditions. Table VII shows that several bacteria have been reported to adhere to surfaces by multiple mechanisms. Some bacteria, such as Staphylococcus epidermidis, can be found associated with various environments. The presence of S . epidermidis adherent to catheters, implants, and so on, may be a result of the adhesion of a phase variant producing an exopolysaccharide (Christensen et al., 1982, 1987). Most clinical isolates of S . epidermidis do not produce exopolysaccharide, but polysaccharide-producing variants seem to colonize biomaterials selectively (Hogt et al., 1983, 1986).

0 000

0 1

0

GENERATIONS

FIG. 18. Phase transitions in bacteria. In this example, a bacterium possessing an MS lectin undergoes several divisions, and a Man-resistant phenotype ultimately appears. The Man-resistant phenotype may undergo several divisions before it produces a MS phenotype. These kinds of transitions are not mutations, but occur in -1 of 10'-105 cells.

66


The view that multiple adhesins may be involved in microbial adhesion is shown in the literature on P . aeruginosa (Table XIII). Various adhesins have been ascribed to the bacterium, including MS type I fimbriae, hydrophobins, exopolysaccharides, sialic acid-specific lectins, GlcNAc-specific lectins, N-acetylmannosamine-bindinglectins, and recently, ganglioside-specific proteins. It is unlikely that phase variation could account for the reported diversity of adhesins for P . aeruginosa. Garber et al. (1985) reported that several isolates containing various surface characteristics (mucoid, rough, etc) were devoid of Man-specific lectin activities. In agglutination reactions using intact P . aeruginosa and papain-treated RBC, no sugars or saccharides were found capable of hemagglutination inhibition. Reagents such as tryptophan and p-nitrophenol were effective inhibitors of hemagglutination, prompting Garber ef al. to conclude that hydrophobic interactions were critical in adhesion. Pseudomonas aeruginosa is known to have the ability to survive under nutrient limitations in a variety of habitats. It may be that the members of the presently defined TABLE XI11 SOMEPROPOSED MECHANISMS FOR THE ADHESIONOF P. ueruginosa ANIMALCELLSO Mechanism or observation Protein-protein complex is enhanced by removal of FN by trypsin. Adhesion of the bacterium may occur more effectively on injured epithelia than on normal epithelia. Adhesin is specific for sialic acid on cellular surfaces or in mucins. Pseudomonas aeruginosa adheres better to injured tracheal cells than to normal tracheal cells. Adhesion to ocular tissue is dependent on pili. Mucins act as receptors for the bacteria. Possible involvement of sialic acid or GlcNAc. An adhesin is specific for Man. Free pili inhibit adhesion to injured tracheal epithelia. Hydrophobic interactions promote hemagglutination Bacterial hydrophobicity appears to be unrelated to adhesion to tracheal epithelial cells of mink Extracellular factors in growth medium enhance adhesion of P. ueruginosa. Mucoid exopolysaccharide is thought to be adhesin for

TO

References Woods e/ a / . (1981) Stern e/ a / . (1982) Ramphal and Pyle (1983a) Ramphal and Pyle (1983b) Uhlenbruck ef a / . (1983) Vishwanath and Ramphal (1985) Speert e/ a / . (1984) Rarnphal el a / . (1984) Garber e / a / . (1985) Elsheikh et a / . (1985) Ogaard et a / . (1985) Ramphal and Pier (1985)

P. aeruginosa.

Multiple adhesins may be involved in attachment of P. aeruginosa to buccal cells. Hydrophobic interactions may mediate nonopsonic phagocytosis.

McEachran and Irvin (1985) Speerl et (it. ( 1986)

67


TABLE XI11 (conf.) Mechanism or observation Sialic acid-containing receptors may be present in ocular epithelia. Hydrophobic interactions may take place via lipidbinding adhesin. Exopolysaccharide of the bacterium may complex with mucins of host. Adhesins capable of binding to oral bacteria may also bind to animal cell surface. The bacterium appears to bind via its cell pole area to human cilia. Multivalent alginate from the bacterium agglutinated both human buccal cells and tracheal epithelial cells. Sialic acid may be a receptor for P. aeruginosa. Corneal ulceration associated with soft contact lenses may depend on polysaccharide-mediated adhesion of P. aeruginosa to the contact lens. Lectins specific for sialic acids attach the bacteria to various cell types. N-Acetylmannosamine may be an ocular receptor for P .

References Hazlett e t a / . (1986) Ramphal et a/.(1986) Ramphal et a/. (1987) Komiyama et a/. (1987a) Franklin et al. (1987) Doig et a/. (1987) Komiyama et a / . (1987b) Slusher et a/. (1987) KO et a / . ( I 987) Hazlett et a/. (1987)

aeruginosa.

Pseudomonas aeruginosa adhesion to contact lenses was enhanced by mucins and several proteins. The bacterium may bind to gangliotetraosylceramide or gangliotriaosylceramide. _____

~

~~

Miller ef a/. (1988) Krivan et at. (1988) ~~

~

“Stanley (1983) also has provided evidence to suggest that P. aeruginosa adheres to inanimate surfaces by multiple mechanisms.

species of P . aeruginosa possess unique adhesins, making the species more heterogeneous than heretofore thought. In gram-positive bacteria, adhesion has been extensively studied in the genera Streptococcus and Staphylococcus. Lectins are not nearly as prominent in gram-positive bacteria as they are in the gram-negative prokaryotes. Some oral streptococci have been reported to possess glucanbinding lectins (Drake et al., 1988a,b; McCabe et al., 1977; Gibbons and Fitzgerald, 1969). Staphylococcus suprophyticus possesses a surface lectin specific for Ga@(1-4)GlcNAc residues (Gunnarson et al., 1984). Members of the genus Actinomyces possess Gal- and GalNAc-specific surface lectins (Cisar, 1986). Drake et al. (1988a) reported that the glucan-binding lectin of S . cricetus was specific for only glucans containing high contents of a-l,6 anomeric linkages (Table XIV). Furthermore, when inhibition studies were performed, it was observed that the combining site of the lectin optimally accommodated 8-10 hexose residues (Fig. 19 and 20).

68

R.J. DOYLE AND E.M. SONNENFELD TABLE XIV a-1.6 GLUCOSIDIC BONDI N T H E AGGLUTINATIONOF S~reproeorciiscriretits A H T " ROLE OF THE

Glucan

a-I ,6 (%)

B- I208 B- 1225 B- 1255 B- I298 8-742 B- I299 B-I35S(s)

95 90 82

Other linkages (9%)

Decrease in absorbance (95)

5

76 68 71 16

10

64 57

18 36 43

50

so

45

55

0

0 I

"Reprinted courtesy of the authors (Drake P I 01.. 1988a) and the American Society for Microbiology. "Glucans with known molar percent anomeric linkages were used to determine the specificity of the glucan-binding lectin. GBL. Each glucan was prepared in PBS at a final assay concentration of 10 &ml. Glucans such as lichenan ((al,4):, a-1.31. pullulan (a-1.4.a-1.6).amylose (a-1.4).laminarin (p-1.3, p-1.6). rnaltoheptaose (a-1.4). glycogen (a-1.4, a-1.6).along with carbohydrates such as glucose. maltose. isomaltose. isomaltopentaose. isomaltooctaose. nigeran (a1.3. a-I.41.and fructose were incapable of promoting agglutination.

z 0

!c3z

W

U

c3

(3

a

LL

0

z

E! b-

30 20 10

40

0.I

MALTOHEPTAOSE

02

0.3 0.4

c 0.6

0.8

1.0

2.0

!

3.0 4.0

!

:! ! A 1 6.0 8.0 10.0

GLUCAN (rng/rnl) FIG. 19. Inhibition of glucan T2000-mediated aggregation of Streptocorcrrs cricerus by isomaltosaccharides. Suspensions of S. crirerris AHT were incubated with the prospective inhibitors and assayed for glucan T2000-induced aggregation. IM-8, lsomaltooctaose; 1M-6, isomaltohexaose; IM-5, isomaltopentaose. Reprinted courtesy of the authors (Drake ei d., 1988a) and the American Society for Microbiology.


3.0

-

2.5

-

E 2.0

-

p

69

-0

e .-

-u"\

=r

1.5

0 .c

-

0

E 1.0 L

0 0

5

0 0.5 c

-

\ \

0

0

a

b

\

0 ~ ' " ' ' ' " " ' 0

I

2 3

4 5

\

6 7 8 9 10 I I 12 13 14 15 16 17 18 19 20

55

GLUCOSE RESIDUES FIG. 20. Glucan concentration required for 50% inhibition of glucan T2000-mediated agglutination of Streptococcus cricetus. Inhibition data obtained with glucan TI0 and the isomaltosaccharides were plotted in terms of the concentration of inhibitor needed to achieve a 50% level of inhibition versus the number of glucose residues of each inhibitor. Note that isomaltotriose (date not plotted) essentially represents infinity as no inhibition was observed even at concentrations s 3 3 mg/ml. Reprinted courtesy of the authors (Drake et a / . , 1988a) and the American Society for Microbiology.

This is an unusual combining site as most lectins bind monosaccharides or disaccharides (Goldstein and Hayes, 1978). Saccharides such as maltoheptaose were without effect on lectin activity. Streptococcus cricetus is one of the "mutans" streptococci and may be involved in dental caries. The glucan-binding lectin may enable the bacteria to adhere to tooth surfaces and subsequently initiate dental caries. It is known that sucroseconsuming populations demonstrate higher canes rates than non-sucroseconsuming populations. Sucrose is metabolized directly by extracellular glucosyltransferases to yield a-l,6glucans and fructose. The human diet is usually rich in a-1,4glucans (starches) but poor in a-1,6glucans, so it seems unlikely that most foodstuffs could inhibit colonization of the streptococci. Colonized streptococci could more effectively trap nutrients and could more effectively withstand low concentrations of Ab or lysozyme or other antibacterial agents.

70


The adhesion of pyogenic cocci to mucosa appears to involve multiple mechanisms (Table VII). Streptococcus pyogenes has several components on its cell surface (Fig. 21) that could participate in adhesion reactions. The normal habitat for pyogenic cocci is an animal host. In humans, the bacteria frequently colonize the throat and mucus membranes of the upper respiratory tract and are transmitted by droplets, direct contact, fom-ites, saliva, and so on. The carrier state involves the successful colonization of the bacteria without the appearance of symptoms. Streptococcus pyogenes appears to adhere to mucosa via a bridging mechanism involving fibronectin (FN) (Fig. 22). It is known that LTA molecules can form complexes with M protein via interactions between the poly(glycerol phosphate) and lysine residues on the protein (Ofek et al., 1982).This leaves the hydrophobic end of the LTA exposed to solvent and available for interaction with other proteins or amphiphiles. The F N molecule is thought to be able to bind the hydrophobic residues of the LTA near the amino terminus of the protein. The carboxy terminus, in contrast, anchors the FN molecule to the mucosal surface. The net result is that FN can tether the bacterium to the cell surface utilizing LTA as a ligand, so in reality, both LTA and FN serve as bridging molecules. Table XV reviews some of the evidence for involvement of both LTA and FN in the adhesion reactions. Antibodies against either LTA or FN reduce streptococcal adhesion. Loss of LTA from the bacterial surface also results in reduced adhesion. Tissue culture cells producing relatively low quantities of FN form relatively poor substrata for the streptococcal adhesion. The deacylated form of LTA does not serve as an inhibitor of adhesion, whereas hydrophobin-binding molecules such as serum albumin are good inhibitors. Furthermore, soluble LTA prevents the binding of soluble FN to S. pyogenes. The collective evidence is that both LTA and P E PT ID OG LY C A N HYALURONIC ACID L

M-PROTEIN

-

MEMBRANE

.GROUP SPECIFIC PO LY S A CCH A R IDE

FIG.21. Surface structures of Sfreptococcus pyogenes.

71


-PLASMA PEPTIDOGLYCAN

LlPOTElCHOlC M-PROTEIN

(m = FATTY ACID 1 EPITHELIAL

~~,

FIG.22. Adhesion of Streptococcus pyogenes depends on an FN bridge. The organism may secrete LTA during growth. The LTA binds to M protein via protein-glycerol phosphate interactions, leaving a solvent-exposed hydrophobic end. This hydrophobic end binds to complementary sites on the FN molecule. The carboxy-terminal amino acids of FN bind strongly to epithelial cells. The net result is that the bacteria can be specifically tethered to the epithelial cells via an FN bridge. The model requires participation of wall matrix, LTA, M protein, and FN. The M protein may contain a segment that interacts with the PG (Pancholi and Fischetti, 1988). The main features of the model were derived from Beachey and Ofek (1976), Beachey et a/. (1983); Courtney e t a / . (1986); and Ofek et a/. (1982).

TABLE XV CONTRIBUTIONS OF LTA AND FN TO THE ADHESIONOF Streptococcus pyogenes TO MUCOSA" LTA

FN

Soluble LTA prevents bacterial adhesion. Deacylated LTA is not an inhibitor of adhesion. Anti-LTA Ab reduces adhesion of S .

Soluble FN inhibits bacterial adhesion. LTA prevents binding of soluble FN to S . pyogenes. Anti-FN Ab reduce adhesion of S.

pyogenes.

Sublethal concentrations of penicillin cause loss of surface LTA from streptococci and result in decrease in adhesion of the bacteria. Molecules that complex with LTA, such as serum albumin, reduce streptococcal adhesion.

pyogenes.

Removal of FN from buccal cells decreases streptococcal adhesion.

Tissue culture cells expressing FN bind streptococci better than cells producing little FN.

"Derived from Beachey (1981): Beachey and Courtney (1987): Beachey et al. (1983): Ofek and Beachey (1980a.b); Ofek et a/. (1982): Simpson et a/. (1980, 1987). and Simpson and Beachey (1983).

72


FN contribute to the adhesion of S . pyogenes to mucosa. Other results have suggested that mechanisms in addition to the M protein-LTA-FN interactions may be important in streptococcal adhesion. Tylewska et al. (1988) have suggested that M protein itself may possess lectinlike activity, utilizing fucose or galactose receptors on pharyngeal cells. Streptococcus agalactiae (group B streptococci) is a pathogen for infants and the immunocompromised. There remain several questions about its adhesion to animal cells. Inhibition by anti-LTA, LTA [but not poly(glycero1 phosphate)], and proteases, suggest a surface protein-LTA involvement similar to that of S . pyogenes. The bacterium has a similar surface to S . pyogenes in that it contains solvent-exposed proteins. Mattingly and Johnston (1987), however, showed that LTA is not readily released from the bacterial plasma membrane during growth. Jelinkova et al. (1986) observed that the adhesion of group B streptococci was unrelated to the type-specific protein. Bulgakova et al. (1986) have suggested that capsule may be important in the adhesion of group B streptococci. Some interesting results reported by Cox (1982) provide convincing evidence for a role of LTA in pathogenesis of the group B streptococci. Cox applied LTA to the oral cavity, perineum, and nape of three day-old mice. The mother was then painted with a suspension of S . agalactiae over her nipples. None of the animals receiving the LTA treatment were found to be positive for the streptococci after a 3-day period, whereas 47% of the control, untreated mice were culture-positive. It is difficult to escape the conclusion that LTA is involved in colonization of group B streptococci. Streptococcus pneurnoniae is a normal inhabitant of most humans. The bacterium typically colonizes the upper respiratory tract. The S . pneurnoniae may cause infections of the lungs, throat, ear, and central nervous system. It appears that S . pneurnoniae is one of the few streptococci to possess surface lectins (other than the oral streptococci as discussed earlier). Andersson et al. (1983) found that oligosaccharides bearing Gal-GlcNAc residues were effective inhibitors of adhesion to pharyngeal cells. Beuth et al. (1987) found that GlcNAc-Gal-containing glycolipids would sensitize guinea pig RBC to hemagglutination by S . pneurnoniae. Furthermore, glycolipids in human milk were shown to be effective inhibitors of the bacteria to pharyngeal cells (Andersson et al., 1986), suggesting a role for the glycolipids in natural protection against infection. Like streptococci, staphylococci are normal microbiota of the human. The skin, eyes, intestines, and nasopharyngeal areas provide substrata for the colonization of both S . ai~reiisand S . epidermidis. Staphylococcus aureus is typically a more aggressive pathogen than S . epiderrnidis (Pulverer et cd.. 1987). Most clinical isolates of S . aureus are coagulase-positive, whereas clinical isolates of S . epiderrnidis are coagulase-negative.


73

Neither S . aureus nor S . epidermidis is known to express surface lectins. Both species of staphylococci have surface proteins and both species possess teichoic acids covalently linked to PG. Staphylococcus aureus clinical isolates frequently express an Ig-binding protein called protein A. Molecules of LTA may also be surface-exposed in S. aureus (Jonsson and Wadstrom, 1984). Staphylococcus aureus has a surface protein that complexes avidly with FN (Froman et al., 1987). Wadstrom et al. (1987) have suggested that the invasiveness of S. aureus clinical isolates is directly proportional to the ability of the bacteria to bind FN. In this regard, Proctor et al. (1983) found that when S . aureus was treated with sublethal concentrations of penicillin, the density of FN-binding sites increased on the bacteria, along with an increased tendency to adhere to FN-coated substrata. Vaudaux et al. (1984) found that anti-FN Ab reduced the adhesion of S . aureus to biomaterial implants. The combined results lead to the conclusion that FN is a receptor for S . aureus. The chemical basis for the recognition of FN by surface protein(s) of S. aureus remains to be worked out. Staphylococcus aureus, similar to other pathogens (Table VII), may also have various means of adhesion. The bacterium is known to bind collagen (Holderbaum et al., 1985), gelatin (Carret et al., 1985), and laminin (Mota et al., 1988). Furthermore, cell wall teichoic acid containing poly(ribito1 phosphate) has been suggested to contribute to the adhesion of S. aureus to nasal epithelial cells (Aly and Levit, 1987). Staphylococcus epidermidis possesses an LTA and a cell wall poly (glycerol phosphate) teichoic acid. The bacterium also expresses surfacebound proteins. The bacterium, unlike S . aureus, does not secrete known toxins. Staphylococcus epidermidis exhibits classical phase variations on its cell surface. Christensen et al. (1987) showed that a single isolate could yield three types of clones with distinct surface characteristics. The wildtype clone adhered well to plastic and synthesized an extracellular polysaccharide. A weakly adherent phenotype was selected from the wildtype clone. From the weakly adherent phenotype, an adherent clone was isolated. The results suggest that S . epidermidis can adhere to surfaces via mechanisms that may be controlled by phase variations. Hogt et al. (1986) believe that hydrophobic phenotypes may selectively adhere to plastics and implants. Once adherent, phenotypes expressing extracellular polysaccharide may become dominant. One member of the genus Staphylococcus, S . saprophyticus, has been shown to express a surface lectin. A common cause of urinary tract infections in females, it is thought to bind to animal cells via a GlcNAc-specific lectin (Gunnarson et al., 1984; Beuth et al., 1987). However, LTA may also contribute to the adhesion of the bacterium because soluble LTA (but not deacylated LTA) inhibited adhesion to uroepithelial cells (Teti et

74


al., 1987). In addition, serum albumin was also observed to reduce the binding of S . saprophyticus to urinary cells (Teti et al., 1987). The bacterium may therefore have multiple adhesins on its surface. In fact, multiple adhesins seem to be the rule, rather than the exception, for bacteria that bind to animal cells.

VI. Turnover of Cell Wall and Pathogenesis

Cell wall turnover refers to the excision or shedding of PG and PGassociated polymers during cell metabolism (Doyle et af., 1988). In some cases, the turned-over wall components are not reutilized for new growth. For example, B. subtilis may lose ~ 5 0 % of its wall per generation because of turnover events (Mobley ef al., 1984). There is no evidence that any of the wall turnover products are reutilized or reincorporated into the wall. Other bacteria, such as E. coli or S . typhimurium, exhibit a significant cleavage of preexisting wall during growth, but most of the excised components are reincorporated into new wall (Goodell and Higgins, 1987). Wall turnover occurs as a result of the actions of bacteriolytic enzymes on the PG. These bacteriolytic enzymes may act on the glycan chain (muramidases or glucosaminidases), the linkage between the muramic acid and L-alanine (N-acetylmuramyl-L-alanine amidases, usually referred to simply as amidases), or peptides within the wall structure (peptidases) (see Fig. I). During growth, the autolysins appear to be well regulated. In some species, wall turnover accompanies growth, whereas in others wall turnover may occur in the stationary phase. In B. subtifis, autolysins appear to be regulated by the influence of the energized membrane (Fig. 23) (Jolliffe et al., 1981; Doyle and Koch, 1987). As long as the bacteria are producing protons, the wall is thought to assume a relatively low pH, thereby preventing autolysin activity near the membrane. When the protonic potential approaches zero, the cells begin to lyse. Organisms such as S. aureus have autolysins that seem to be regulated by LTA (Fischer and Koch, 1983; Fischer et al., 1983). Other bacteria, such as the pyogenic streptococci, d o not exhibit wall turnover. The reasons for wall turnover are unclear, but in B. subtilis at least, wall turnover appears to be linked with growth (Koch and Doyle, 1985; Kemper et al., 1988; Doyle et al., 1988). Pooley (1976a,b) found that when the radioactive wall precursor GlcNAc was pulsed into exponential B. subtilis cells, approximately one generation was required before turnover commenced. Furthermore, the rate of turnover was independent of the length of the pulse. Pooley concluded that the newly inserted wall

z

75


INSIDE

OUTSIDE

ENERGIZED MEMBRANE

0

@%

PROTEASE 831 NACTNATION OF AUTOLYSINS

0

INSIDE

OUTSIDE DEENERGIZED MEMBRANE

0 @% PROTEASE

&OFINACTIVATION AUTOLYSINS 0

Enzyme Inactivated by Energized Membrane

0

Active Enzyme

63 Enzyme Inactivated by Protease

FIG.23. Influence of energized membrane and proteases on autolysins of Bacillus subri/is. Autolytic activity is reduced by soluble proteases, but the proteases do not act on autolysins as they traverse the wall matrix. Wall-bound autolysin is more resistant to proteolysis than soluble autolysin. When protonmotive force is dissipated, autolysins assume an active conformation and cellular autolysis occurs. In B. subrilis, the protonmotive force can be dissipated by uncouplers of oxidative phosphorylation, by ionophores, by starvation for a carbon source, and by anaerobiosis. Restoration of the membrane potential results in the termination of uncontrolled autolysis. From Doyle and Koch (1987). Reprinted by permission of the authors and CRC Press.

76


material migrated from the inside of the wall face t o the outside of the wall face, where it became susceptible to turnover. The migration of wall from the inside to the outside took about one generation. The oldest wall contains PG components that are most removed from the energized membrane and are therefore susceptible to surface autolysins. In gram-positive bacilli, this inside-to-outside wall growth causes the cell to elongate. When PG components are added to preexisting wall and crosslinked, the wall then becomes stressed as a result of cellular turgor (Koch and Doyle, 1985). Addition of more wall results in the pushing out and stretching of older wall, thereby causing surface expansion (Fig. 24) (Kemper et a!., 1988).

Figure 25 shows a typical turnover experiment for a gram-positive rod. A culture of B. anthrucis was pulsed with [3H]GlcNAc for either 0.06 or 1.0 generations and then chased with 500 K , values of nonradioactive GlcNAc. Samples were taken during exponential growth and assayed for radioactivity. It is observed that, regardless of pulse time, the rate of loss of label from the cells was -50% per generation. The culture pulsed for

FIG.24. Model depicting the elongation of the side wall of Bucillus subtilis. A cell wall precursor is secreted by the cytoplasmic membrane and then crosslinked into the wall matrix facing the membrane (A). The wall segment is then pushed out by more recently added wall. This causes the wall segment to stretch. By the time it reaches the outer-wall face (about one generation), it has become highly susceptible to the actions of autolysins. An autolysin cleaves one site on the segment, thereby alleviating tension (B), and another autolysin finally cleaves the other bond holding the segment onto the wall matrix (turnover) (C). Addition of new wall with subsequent stretching will result in the elongation of the cell cylinder. Autolysin-deficient cells may have only enough enzyme to clip the most highly stressed bonds. Turnover in these cells will therefore be minimal and their surfaces may be "rough". From Kemper el ul. (1988). Courtesy of the authors and the American Society for Microbiology.

77


\

:el .-

U K

5'

0

I

I

I

2 GENE RAT I0N S

I

3

I

3.5

FIG.25. Turnover of cell wall of Bacillus anthracis. Cultures were pulsed with ['HIGlcNAc for either 0.06 (0)or 1.0 ( 0 ) generations. See text for details.

0.06 generations exhibited the typical delay before cell wall turnover began. Table XVI lists the pathogens known to turn over their cell walls. The role of wall turnover in pathogenesis is poorly understood. In B. anthrucis, -40% of the cellular dry weight is wall material. Assuming that 50% of the wall is turned over per generation, then growth in vivo would TABLE XVI PATHOGENIC BACTERIA EXHIBITING CELLWALL TURNOVER Bacterium Bacillus anthracis Bacillus cereus Escherichia coli Listeria monocytogenes Neisseria gonorrhoeae Proteus mirabilis Salmonella typhimurium Staphylococcus aureus Streptococcus mutans

References E.M. Sonnenfeld and R.D. Doyle (unpublished 1988) Chung (1971) Chaloupka and Strnadova (1972) Doyle et al. (1982) Hebeler and Young (1976a,b) Grneiner and Kroll (1981a,b) Goodell and Higgins (1987) Rogers (1967) Lesher et al. (1977)

78


result in a significant challenge to the immune system by the muramic acid-containing materials. Any Ab directed against wall components, such as teichoic acids, teichuronic acids, or PG, would probably offer little protection to the host, a s long as the wall components are being turned over. Turnover may therefore be one means of helping certain pathogens evade the immune system of the host. Some pathogens d o not turn over their cell walls. In fact, the lack of an active autolytic system may cause the bacteria t o persist in tissues (Fig. 26). Wall remnants of pyogenic streptococci may persist in tissues for months, because of their resistance t o lysosomal enzymes (Ginsburg, 1988). Ginsburg (1988) believes that cationic proteins in neutrophils and macrophages may activate autolysins of certain bacteria, resulting in the elimination of the microbes. On the other hand, proteolytic enzymes (Jolliffe ef a f . , 1982) may reduce the levels of autolysins so as to prevent bacteriolysis in vivo. Work by Wecke er al. (1986) showed that “liquoid,” an anionic polyanethole sulfonic acid, inhibited the autolysins of S. u ~ r e u sreducing , turnover and preventing cell separation. Ginsburg ( I 988) has suggested that polymers, such as heparin or glycosaminoglycans, may inactivate autolysins in vivo and thereby prevent rapid removal of whole or damaged bacteria. Peptidoglycan and muramyl peptides are thought t o play a role in periodontal diseases (Barnard and Holt, 1985). The immunomodulating activity of wall and wall degradation products may play a role in inflammation of gingival tissues leading to periodontal disorders. At this time, it is unknown which oral bacteria exhibit cell wall turnover. As far as is known, turnover in such periodontopathogens as Actinobacillus, Actinomyces, Eikenella, and Baoteroides has not been examined. Mychajlonka el al. (1980) found that in six strains of Streptococcus mutans and eight strains of S . sangrris, there was no evidence for turnover in exponential growth. In addition, wall synthesized during benzylpenicillin o r tetracycline treatment was not susceptible to turnover following removal of the antibiotics. FIG.26. (A) Sfaphylocorrus ~

T C N S incubated

in phosphate-buffered saline (PBS) for

15 hours at 37°C. The cells appear intact. EM x 62.700. (B) Staphylococci incubated for 15

hours at 37°C with leukocyte cationic proteins. Note both the massive destruction of the bacterial cell walls (bacteriolysis) and the dissolution of the inner cytoplasmic structure (plasmolysis). EM ~ 6 2 , 7 0 0 .(C) A macrophage in the synovium of a rat 3 days after the intraarticular injection of viable staphylococci. Both intact and plasmolysed staphylococci are present within the cytoplasm of a macrophage. but no sign ofcell wall breakdown (bacteriolysis) is evident. EM x 15,390. (D) A portion of a macrophage in rat synovium 10 days after the intraarticular injection of viable staphylococci. Note the presence of numerous empty intact shells compatible with plasmolysed cell walls, and the presence of intact cell walls within lysosomelike structures. EM x 15.390. Courtesy of 1. Ginsburg (Ginsburg, 1988) and Blackwell Scientific Publishers.


79

80


FK 26.

(con(.)

BACTERIALCELLSURFACE

FIG.26. (cont.)

81

82

R . J . DOYLE A N D E.M. SONNENFELD

FIG.26.

(cont.)


83

Lesher et al. (1977) found that subinhibitory concentrations of fluoride anion could induce wall turnover in Streptococcus mutans. The use of fluoride as an additive in drinking water to reduce dental caries is common. It would be interesting to determine whether the decline in dental caries has been marked by an increase in periodontal disease in fluorideconsuming communities.

VII. Concluding Remarks It is now clear, with only a few exceptions, that the bacterial cell surface is essential for pathogenesis. In recent years, the structures of most bacterial surface molecules have become better understood. This has led to a better description of the pathogenic mechanisms of some bacteria. In this article we have tended to emphasize three major features of the bacterial surface: First, the surface is dynamic, resulting in the sloughing off or turnover of wall envelope materials into the surrounding medium. Second, wall components exhibit a remarkable diversity of biological properties, and third, bacterial adhesion to substrata may depend on multiple adhesins. An area related to turnover of walls that has been overlooked is the role of muramic acid-containing materials in the pathogenesis of staphylococci, Neisseria, and B. anthracis. Wall turnover in each of these bacteria (types or species) is greater than that of E. coli or other gram-negative rods. If turnover is a natural consequence of cell division and if turnover products are important in pathogenesis, then a reasonable approach to antibiotic treatment would be to inhibit wall metabolism. It is known that protein synthesis inhibitors such as chloramphenicol cause wall thickening in bacteria. Most protein synthesis inhibitors in bacteria arrest cell division, but their effects on wall degradation in situ are unknown. An important area for future study may be related to the regulation of cell wall-degrading enzymes, such as autolysins. All the major surface structures exhibit multiple effects on biological systems. The recent observations concerning the role of muramic acidcontaining PG components in immune regulation demand that cell wall turnover events be characterized in situ. In addition, the prospect that muramyl peptides may modulate sleep in mammals requires that cell wall turnover be studied using conditions approximating those found in the intestine. It seems strange that a bacterial wall product could be a “vitamin” associated with sleep, but there is now enough literature to convince most that a more than casual relationship exists between normal microbiota and normal sleep. The findings of Lesher et al. on streptococ-

84


cal wall turnover in the presence of fluoride anion suggest that certain metabolic challenges may induce wall turnover. This poses interesting questions with regard to rheumatoid arthritis, in which streptococcal wall remnants have been implicated in inducing the symptoms. It is not at all certain whether insoluble wall o r slowly solubilized muramyl peptides are involved in the pathogenesis. Bacterial surface components involved in adhesion to animal cells have been identified in many cases. Furthermore, the molecular bases for adhesive events are known for some bacteria. It is surprising the research on the inhibition of adhesion has not resulted in a new means of preventing infections. A vaccine containing the Man-specific lectin of E. coli may prove to be useful in preventing gastrointestinal infections caused by the bacterium, as preliminary results have suggested. As soon as microbial lectins become available in sufficient quantities (cloning will probably be required for most surface lectins), there will no doubt be a significant effort to develop the proteins as vaccines. A complication in developing vaccines directed against adhesins is the fact that most pathogens can adhere to mucosa by more than one adhesin. It is likely that adhesinbased vaccines will require multiple components in order to provoke an immune response destined t o prevent microbial colonization.

ACKNOWLEDGMENTS Work relating to bacterial cell surfaces has been supported by NIH-NIDR DE-07 199, the Ohio Valley chapter of the March of Dimes, the U.S. Army Research and Development Command. and the N S F (PCM 7808903). The authors thank Prof. T . J. Beveridge for micrographs and discussions, Prof. 1. Ofek for discussions and data, and Prof. Me1 Rosenberg for hospitality and stimulating conversations. Dr. J. Ezzell was instrumental in our understanding of the B. unrhrucis cell surface.

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INTERNATIONAL REVIEW OF CYTOLOGY, VOL. 118

Cellular Studies on Marine Algae AHARONGIBOR Department of Biological Sciences, University of California, Santa Barbara, California 93106

I. Introduction The rapid progress of biology in the past few decades was accomplished by exploiting a few selected experimental organisms, of which Escherichiu coli, yeast, corn, and the fruit fly are outstanding examples. However, progress via the use of a limited number of model systems soon became quantitative in nature, and great qualitative steps forward were accomplished with the introduction of new model organisms. The same experimental organisms can sometimes be exploited for new areas other than those for which it was originally chosen. For example, the fruit fly Drosophila, the salivary glands of which contributed so much to classical genetics, is now being exploited for studies on the role of genes in embryonic development of multicellular animals. In this chapter I describe three of the enormous variety of algae. These three are fascinating and offer a promise for studies on basic biological problems. I would like to present these as challenges to the reader, hoping that ingenious experimenters will be able to exploit these organisms further. The simplest members of the plant kingdom are classified as algae. This subkingdom includes an enormous variety of organisms, from simple single cells to complex giant seaweeds. Among this vast number of plants, some proved to be very useful as model organisms for studies of specific phases in the lives of cells in general and of plant cells in particular. Classical examples are the use of single-cell green algae such as Chlorellu and Scenedesmus in studies on photosynthesis (Bassham, 1962; Lewin, 1962); similarly, Valoniu and Nitella were selected for studies on membrane phenomena and cytoplasmic streaming (Blinks, 1936; Osterhout, 1936). My objectives in writing this chapter are not to review such studies: instead, I describe several algae that are perhaps less famous but that I 93 Copyright 0 1989 by Academic Press. Inc. All rights of reproduction in any form reserved.

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have found to be interesting, because they possess properties that make them suitable for studies of important aspects of biology. One of these, Acetabirlaria, was used for many years for studies on nuclear-cytoplasmic relationships (Hammerling, 1963). These classic studies do not require a rereview, and I prefer to emphasize new or neglected aspects in the life of this alga that possess potential for further fruitful studies. I also chose to consider the green alga Boergesenia and the red alga Porphyra. The choice of the three organisms is purely subjective. With two of them. Acetabularia and Porphyra, I have had a long acquaintance and considerable first-hand experience, whereas with Boergesenia I have had only a short and limited interaction. I like these organisms not only because they have unique biological properties but also they are visibly pleasing and enjoyable to work with.

11. Acetabularia

Acetabularia are green algae, the genus belonging to the family Dasycladaceae of the order Siphonales. The family is characterized by a growth pattern in which the main axis of the plant develops whorls of branches. Some or all of these branches become the reproductive organs. In the genus Acetabularia, branches of the terminal whorl differentiate to become the spore-bearing fruiting body. Different species differ in the degree of association of the branches of the fertile whorl. They form, for example, an “umbrella”-shaped structure in A . mediterranea, while in others such as A . polyphesa they form disconnected individual fruiting branches, similar to a bunch of bananas. Intermediate-type structures are found in other species. Another important attribute of this genus is that their vegetative nucleus is invariably located in the rhizoidal section. Acetabuhria are distinguished by the fact that regular mitosis of the cell nucleus is delayed until the plant completes its vegetative growth and a fully differentiated fruiting whorl has formed. Until then the plant could be regarded as a single cell. However, once nuclear divisions start the plant is to be regarded as a coenocytic organism, similar to all other Siphonales algae. The secondary nuclei divide repeatedly and migrate into the branches of the fruiting body, where they become spaced regularly. Eventually the protoplasm around each nucleus becomes segmented and surrounded by a plasma membrane; a cell wall is then secreted, thus forming the reproductive cysts. Each cyst when formed contains a single nucleus, numerous chloroplasts, and mitochondria. In subsequent development of the cyst the single nucleus undergoes many divisions, eventually to produce many gametes. A gamete is a biflageilated naked cell with

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one nucleus and one chloroplast, resembling a Chlamydomonas cell. After their release, the gametes fuse in pairs to form a zygote, which grows to form the familiar vegetative cell. The gametes from a single cyst do not mate with each other. This suggests that the secondary nuclei are the products of meiosis and thus products of each haploid cyst are of the same mating type. It is the delay in the onset of nuclear divisions in the life of the vegetative cell that made these organisms suitable for the many interesting studies on the role of the nucleus and relationships between the cytoplasm and the nucleus (Hammerling, 1963; Gibor, 1966). Other important properties of Acetabularia are its ability to withstand severe mechanical damage, heal its resulting wounds, and resume growth. Small segments of a cell are capable of regenerating an entire plant as long as they contain the primary nucleus. Enucleated cell segments can continue to grow and even develop for many weeks. Drops of cytoplasm squeezed out of the cell wall can maintain their photosynthetic activity and cytoplasmic streaming for many days in vitro (Gibor, 1966). By centrifugation it is possible to move most of the cell contents to either the rhizoidal or the apical end of the cell. Several hours after the termination of the centrifugation, the cytoplasm moves back and restores the normal green appearance of the cell. The properties of these plants were exploited in classic experiments by Hammerling (1963) to demonstrate the role of the nucleus in determining the phenotypic properties of different species. Transplantation of nuclei by grafting the rhizoids of one species onto the stalk of another was later followed by the transfer of washed nuclei of one species to stalks of another. Such transplantations resulted in the “hybrid,” developing a fruiting body morphologically similar to the nucleus-donor species. In the growing cell, the development of the primary nucleus and the induction of mitosis were found to be controlled by the physiological state of the cytoplasm. Acetabularia could thus serve well for studies on the relationship between the nucleus and the cytoplasm. These biochemical and physiological aspects in the life of Acetabularia were and are still being actively investigated (Berger et al., 1987). There are other aspects of the unique biological properties of Acetabularia that are still underexploited and that deserve further study. An interesting biological problem for which this plant is suitable as an experimental model is aging at the cellular level. Hammerling noted that by repeated amputation of the developing “umbrella,” a cell could be maintained for many years in culture. The primary nucleus of the cell enlarges with the growth of the cell and reaches a maximum size when the fruiting body develops to its maximum size. This occurs just before the onset of mitosis and production of secondary nuclei. The large pri-

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mary nucleus possesses a highly enlarged nucleolus with a characteristic shape. If, however, the bulk of the vegetative cell is amputated prior to the onset of mitosis, the primary nucleus shrinks in size proportionately. The regenerating cell would have a smaller primary nucleus that would commence to enlarge again with the regrowth of the cell. By repeating such amputations every time the cell approaches maturity, it is possible to maintain active growth of the cell for many years. The cell appears to be rejuvenated by the amputations, and theoretically it could be maintained by such manipulations indefinitely. Conversely, a mature fruiting body if grafted onto a young stalk will impose the onset of meiosis and mitosis on the relatively small primary nucleus. The onset of meiosis can be regarded as the end of the juvenile or vegetative phase of the organism and the beginning of a maturation phase. The detailed molecular aspects of the changes in the size and activities of the primary nucleus in response t o the changes in the cytoplasm are yet to be studied. The "physiological age" of the cell can be manipulated by the experimenter. The nature of the signals exchanged between the cytoplasm and nucleus that regulate the activities of the nucleus are not yet known. It was established that primary nuclei can be isolated and maintained alive in vitro. Such nuclei could be returned to enucleated cells, where they functioned normally. With such experimental manipulations it should be possible to expose nuclei to different substances derived from chosen cytoplasms and determine their physiological effects. The physiological activities of the whorls of sterile hair are also of potential interest for research on cellular aging. In laboratory cultures, a growing A . rnedirerruneu cell usually possesses several active whorls. The whorls are symmetrical structures made up, in the case of A. rnediterruneu, of 14 branches. Each branch undergoes further branching. We refer to each level of branching as a tier, numbered by a Roman numeral. Thus there are 14 tier 1 branches, each one of them producing 3 tier 11 branches. Tier I1 branches produce 3 tier 111 branches each, which in turn gives rise to 2 tier IV branches. Tier IV branches produce either 1 o r 2 tier V branches (Gibor, 1973a). Sectors of two whorls are shown in Fig. 1; visible are an upper live whorl with branches full of organelles and a lower, older whorl, appearing to be empty. Simple measurements indicate that the total surface area of all the branches of a single whorl are about equal to the total surface area of the main cell body (Gibor, 1973b). The live whorls are active in the uptake of soluble substances from the environment. This could be readily demonstrated by immersing cells in a solution of a vital dye such as neutral red. The thinnest branches, tier V and IV, appear to be stained in < I minute, followed by tiers 111, 11, and I. The main cell body eventually also accumulates the dye. The uptake


97

FIG.1. Sectors of two whorls. The upper whorl is alive, and the branches of tiers I and I1 contain many chloroplasts. The branches of the lower whori appear to be empty. Up to five tiers of branches are clearly seen in this whorl, and the first three are marked by arrows.' x40. From Gibor (1973a,b); Courtesy of Springer Verlag.

of dyes can be inhibited by lowering the temperature, or poisoning the cells with KCN. Older whorls located farther down on the main stem, which appear to be empty, do not accumulate the dyes. Thus the fine branching of the cells is similar in physiological function to the function of root hairs, namely increasing the surface area of the cell in contact with the environment (Gibor, 1973b). Another indication that the whorls function in nutrient uptake is the observation that the length of the branches increased when cultured in a medium deprived of a nitrogen source, while in the presence of nitrate or urea in the culture medium the whorls appeared shorter and more compact (Adamich et al., 1975) (Fig. 2).

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0

1

5

mm

10

2

FIG.2 . Variations in whorl development in cells grown o n different media. Cell I grew for 30 days on 530 pg atom nitrogen per liter. as nitrate. Cell 2 grew for the same length of time in the same concentration of urea. Cell 3 grew in a nitrogen-free medium. From Adamich el d. (1975): Courtesy of the Journal of Phycology.

The cells maintained in nitrogen-free medium also appear to retain a larger number of viable whorls. Up to seven whorls are seen in cell 3 of Fig. 2. Note that in the oldest whorls of this cell only tiers 1 and 11 with perhaps a few tier 111 branches are still viable. In the nitrate-fed cell (cell 1 of Fig. 2 ) . there are only three live whorls. The physiological significance of the sterile whorls to cell elongation was indicated by experiments in which portions of the live whorls, from one side of a cell, were cut off or damaged by U V irradiation. Such cells were found to elongate unequally, causing the cells to bend toward the side of the cell from which the branches were removed or damaged (Fig. 3). The sectors of the cell from which the branches were removed did not elongate as much as the unmanipulated sectors. The unequal elongation of the sides of the cell indicates that the different sectors of the cell periphery are relatively insulated. Apparently there is no free diffusion of substances laterally across the cell. even though no internal barriers (e.g., cell walls) are present in these coenocytic cells.


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FIG.3. Cells 3 days after shaving the branches of whorls from one side of the cell. The line drawn in the background indicates the initial appearance of the cell on February 28, before the operation. Broken lines indicate the shaven side. Shown on the left are two cells with their rhizoids intertwined. From Gibor (1 977a); Courtesy of Springer Verlag.

New whorls form regularly with the elongation of the cell, and only the younger whorls remain alive. The live branches of tiers I and I1 contain many chloroplasts. There are only a few thin, long chloroplasts in tier 111, and chloroplasts are rarely seen in tier IV and above. Examination of the older whorls farther down the main cell axis reveals that the branches appear to become empty of their cytoplasm with aging. This aging process begins with the emptying of tiers V, then IV, and so on until the entire whorl appears empty (Fig. 1). Subsequently, the empty branches are shed and characteristic scars remain on the cell wall marking the earlier location of a whorl. These cellular branches can therefore be thought of as deciduous organelles. The entire complement of whorls of a cell such as cell 3 in Fig. 2 represents a set of cellular structures, or organelles, of progressively increased physiological age. The topmost whorl is young and still growing, with its tiers not having completed their elongation. The seventh whorl of the same cell consists of aged and dying branches; only tier I branches of this whorl appear to be still alive. It appears that the cytoplasm retracts back into the cell body prior to abscission. It is yet to be determined whether components of the cell wall are also recycled back into the cell.

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The movement of the cytoplasm into the branches of a young and growing whorl and the subsequent retraction into the main axis of the cell upon aging are both intriguing phenomena. Such movements are probably related to the striking migration of the cytoplasm and secondary nuclei into the compartments of the mature fruiting body. What role components of the cytoskeleton and plasma membrane play in directing these movements is yet to be determined. We know of several environmental factors that affect the development and aging of the branching whorls of the cell. As mentioned before, the cells retain their whorls in an active state in nitrogen-poor medium, while in nitrate-rich medium the number of active whorls is reduced. Apparently the aging of whorls occurs faster in wellnourished cells. Another important observation is that when cells are incubated in the dark they lose all their whorls in 10 days, and thus aging of the whorls is accelerated in the dark. Cells maintained under red light stop elongating and do not develop new whorls. Exposure to short flashes of blue light immediately induces the resumption of elongation and the growth of whorls (Schmid et ul., 1987) (Fig. 4). An action spectrum for this photomorphogenetic effect indicates that it is similar to other blue-light effects that were described for plant morphogenesis (Schmid e f ul., 1987). The spatial distribution of the blue-light photoreceptors were also investigated by Schmid and co-workers. The apical growing portion of the cell was found to be most sensitive to the bluelight stimulus, but other segments of the cell, including the rhizoid. could also respond to the blue-light stimulus. I t was concluded that the primary photoreceptors for this blue-light effect are probably present throughout the cell length. The blue-light effect on the initiation of elongation and whorl development can be seen in 1000 dyes and systematically examining phagocytic cells in various tissues and organs. According to Kiyono, histiocytes and the 173 Copyright 0 1989 by Academic Press. Inc. All rights of reproduction in any form reserved.

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BIOLOGY AND KINETICS OF KUPFFER CELLS IN LIVER

175

cells of the reticuloendothelium had the same ability of taking up dyes, and it was supposed that they had a common origin. He equated the Kupffer cells with the liver histiocytes. Taking this concept into consideration, Aschoff (1924) proposed the “Reticulo-Endotheliales System,” or RES. This system consisted of (1) reticular cells in the lymphoid organs, (2) endothelial cells of the hepatic lobules (Kupffer cells), splenic sinus, and lymph nodes, (3) histiocytes in the connective tissue, (4) splenocytes, and (5) monocytes (endothelial leukocytes or blood histiocytes). At that time, Zimmermann (1928) demonstrated three kinds of cells in and around the sinusoids of the liver: (1) endothelial cells, (2) “endocytes” (Fig. 2), and (3) “pericytes” (Fig. 3). He denied the syncytial nature of the endothelial cells and concluded that only the endocytes were Kupffer cells. The endocytes had been described by Browicz in 1898 (cited from Browicz, 1900). Browicz described pear-shaped cells hanging in the lumen of the sinusoid with their processes attached on the inner surface of the sinusoidal walls. The concepts as conceived by Browicz and Zimmermann were proven by modern techniques and methods of investigation to be fundamentally correct. However, their ideas encountered strong criticism from devotees of the RES, who proposed that the three profiles of cells corresponded to various functional stages of one cell type. Under the fluorescence microscope, quick-fading vitamin A fluorescence is emitted from cells scattered in the hepatic lobules. These fluorescent cells were thought to be reticuloendothelial Kupffer cells. Investigations by Wake (1964, 1971), using Kupffer’s gold chloride method, fluorescence microscopy, and electron microscopy (EM) provided new evidence indicating that Sternzellen, stained by the original gold chloride method, were persinusoidally located and stored vitamin A. Refined techniques such as the application of bone marrow chimeras, parabiosis, and autoradiography extended our knowledge on macroFIG. 1 . Light micrograph of perisinusoidal cells in rat liver, stained with the classical gold chloride method, developed by Kupffer in 1876. Kupffer referred to these cells as Sternzellen (stellate cells). X 240. FIG. 2. Light micrograph of endothelial cells and Kupffer cells, labeled with intravenously injected india ink particles. Kupffer cells can be recognized by large aggregates of dark material; endothelial cells contain smaller amounts of the same material. x 500. FIG.3. Light micrograph of a Golgi-stained rat liver preparation. Silver precipitates are present on a cell, called a pericyte by Zimmermann in 1923, and today mostly designated as a fat-storing, or stellate cell. X 2000. FIG. 4. Autofluorescence of vitamin A as demonstrated by fluorescence microscopy, which inspired Kudo in 1938 to recognize these cells as reticuloendothelial cells. At present we know that this reaction is also typical of fat-storing cells. X 500.

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phages considerably after 1960. Van Furth et al. (1972) proposed the concept of the mononuclear phagocyte system (MPS). According to this system, Kupffer cells originate from blood monocytes. Concerning the origin of macrophages and Kupffer cells, however, van Furth’s concept has not been fully accepted. Wisse (1970, 1972, 1974a,b). demonstrated, using perfusion fixation of rat livers, that endothelial and Kupffer cells are different types of cells without transitional forms, even under conditions reported to enhance the frequency of such transitions, such as partial hepatectomy, blockade of the RES, and RES stimulation by zymosan or splenectomy. Both cells also differed in cytochemical properties and endocytosed particles of different sizes by different mechanisms. With these refinements, the study of Kupffer cells has developed rapidly and has been extended to problems concerning the origin, structure, and function of these cells in normal and pathological conditions. In this review we present an overview of the current morphological and biochemical data. Many of the major advances in our knowledge about Kupffer cells have resulted from the development of new methods, such as cell isolation, purification, and culture.

11. Morphology of Kupffer Cells

A. FIXATION AND MICROSCIPIC RECOGNITION OF KUPFFERCELLS

Studying the morphology of the different types of sinusoidal cells and distinguishing among them has been made possible by the combination of perfusion fixation (Fahimi, 1967; Wisse, 1970), transmission electron microscopy (TEM) (Wisse, 1970, 1972), enzyme cytochemistry (Widmann et al., 1972; Wisse, 1974a,b), and particle (mainly 0.8-pm latex) injections (Widmann et al., 1972). By using a proper combination of these methods, it is possible to characterize four different cell types as integral inhabitants of the sinusoidal wall: endothelial cells (Wisse, 1970, 1972), Kupffer cells (Fahimi, 1970; Wisse, 1974a,b), pit cells (Wisse et al., 1976), and fat-storing cells or perisinusoidal stellate cells (Ito and Shibasaki, 1968; Wake, 1971, 1980). It is important to note that perfusion fixation not only causes better fine-structural preservation of the cells but also preserves the histological arrangement, position, and shape of the sinusoidal cells. We might conclude that perfusion fixation has enabled us to disprove the theory that various sinusoidal cells are different functional stages of a single cell type. By using the specific staining techniques for peroxidase (Fahimi, 1970; Wisse, 1974a,b), in combination with a small dose of 0.8-pm latex parti-


177

cles, it is possible to visualize rat liver Kupffer cells unequivocally in light-microscopic (LM) sections, in ultrathin sections, in frozen sections, and in freshly isolated sinusoidal cell suspensions. After culturing, however, Kupffer cells lose their peroxidatic activity (Emeis and Planque, 1976). Other criteria for the characterization of Kupffer cells, such as (a) cell shape or the position in the sinusoid, (b) the uptake of small particulate colloids such as carbon, colloidal gold, and thorotrast, or (c) esterase or acid phosphatase activity or the presence of lysosomal dense bodies or other dense inclusions, are not conclusive evidence to characterize Kupffer cells in situ (Emeis and PlanquC, 1976).These considerations are valid for rat liver; the characterization of Kupffer cells in mouse liver is further complicated by the fact that some sinusoidal endothelial cells are also positive for peroxidase (Stohr et af., 1978). The situation in human liver is at present unclear. The recognition of Kupffer cells at low magnification in the light microscope facilitates the study of lobular distribution of Kupffer cells and allows the quantitation of Kupffer cells in different experimental situations by counting the number of cells within a given microscopic field (Bouwens et al., 1984; Bouwens and Wisse, 1985). Promising new possibilities for recognizing sinusoidal cells may be found in the development of antibodies, which has been made possible by isolation and purification methods for different types of sinusoidal cells. B. KUPFFER CELLSIN TEM Kupffer cells and pit cells have variable shapes and positions in the sinusoids, in contrast to endothelial and fat-storing cells. This aspect suggests that the latter cell types are true sessile components of the sinusoidal wall, whereas the former types might be more plastic and mobile. This is supported by the fact that Kupffer cells and pit cells move in experimental circumstances to different positions, such as the space of Disse or the space between parenchymal cells, or even take part in the formation of liver granuloma (Wisse, 1974a,b; Wisse et al., 1976). Nevertheless, both Kupffer cells and pit cells can be regarded as true inhabitants of the sinusoidal wall, because they anchor with microvilli in the space of Disse and they show local proliferation after partial hepatectomy ,just as endothelial and fat-storing cells do. The shape and the surface of Kupffer cells is irregular; the cells have elongated cytoplasmic processes that can stretch along or underneath the endothelium (Fig. 5). Kupffer cells lie upon, or are embedded in or covered by the endothelium. In some cases, they may traverse the sinusoidal lumen with a thick cytoplasmic process. The presence of fenestrae easily

I78


179

distinguishes endothelial processes from those of Kupffer cells. No special cell contacts exist between Kupffer cells and endothelial cells; they simply touch each other and lack gaps or specialized junctions of any kind. Kupffer cells also contact other sinusoidal cells, like fat-storing cells, pit cells, or collagen bundles in the space of Disse, but no special morphological structures result from these contacts. Kupffer cells do not contact other Kupffer cells, except during granuloma formation, such as is seen after zymosan injections (Wisse, 1974b). With special precautions or experimental approaches, a fuzzy coat can be visualized covering the Kupffer cell surface. Perfusion fixation with glutaraldehyde does not preserve this fuzzy coat. However, it can be found (a) 3 minutes after intravenous injection of colloidal particles or erythrocyte ghosts, which probably stabilizes the coat by attachment (Heifer, 1970; Wisse, 1974a);(b) in osmium-fixed frozen sections; and (c) in different intracytoplasmic structures such as wormlike structures (Toro et af., 1962) and fuzzy-coat vacuoles (Wisse, 1974a). The nature of this fuzzy coat is unknown and forms one of the intriguing problems of Kupffer cell biology in that phagocytosis starts with the attachment of particles during which the presence of receptors and opsonic proteins might play a decisive role. The further analysis of molecules in the fuzzy coat seems possible by using available visualization techniques such as immunogold staining of ultrathin frozen sections. Kupffer cells play a major role in the uptake of endotoxin, altered blood cells, or foreign particles. To accomplish endocytosis, Kupffer cells possess several morphologically recognizable mechanisms that have been shown to contain or to transport material. The mechanisms involved are bristle-coated micropinocytosis, fuzzy-coat vacuoles (a kind of macropinocytosis), wormlike structures, and phagocytosis by engulfment and further internalization. Interestingly, these structures (with the exception of phagocytosis) can be seen in normal Kupffer cells of animals that have received no injection or treatment, implying that these endocytotic mechFIG. 5 . Transmission electron micrograph of a Kupffer cell in rat liver. The cell can be specifically recognized by peroxidase staining, resulting in electron-dense reaction product in the RER and the nuclear envelope. Further details in the picture represent dense bodies or lysosomes and ruffles at the cell surface. L, Sinusoidal lumen; Pc, parenchymal cell; bc, bile capillary; SD, space of Disse. X 4526. FIG. 6. Scanning electron micrograph of a Kuppfer cell in siru. The surface of the cells shows numerous folds and plicae. The cell body bulges into the lumen, and interaction with blood cells is to be expected. The sinusoidal wall contains endothelial fenestrae (arrows). The space of Disse is deprived of endothelial lining, and shows the microvillous sinusoidal surface of the parenchymal cells (*). C, A small bundle of collagen is situated at the parenchymal cell surface. X 15,425.

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anisms may be constantly operational and active (Wisse, 1977). Apart from vacuoles of different sizes, a heterogeneous population of dense bodies (lysosomes) can be found in the cytoplasm. The size, density, and shape of these bodies are highly variable, but their abundance and relative volume (Blouin el a / . , 1977) clearly indicate a considerable capacity of Kupffer cells in vivo for endocytosis and digestion. The absence of recognizable structures in these lysosomes under normal circumstances suggests a rapid digestion and/or the uptake of molecules rather than particles or (parts of) cells. Fat droplets, autophagic vacuoles, or multivesicular bodies have not been described for Kupffer cells in siru. Kupffer cells possess a normal set of cellular organelles. The nuclear envelope, together with the rough endoplasmic reticulum (RER) and the mysterious annulate lamellae, is the site where the peroxidatic activity is located. The shape of the nucleus is different from other sinusoidal cells; flat or blunt endings can give the nucleus an almost rectangular appearance in some sections. The presence of a number of RER cisternae and Golgi apparatuses suggest the involvement of Kupffer cells in protein secretion. For further morphological descriptions of Kupffer cells, the reader is referred to Fahimi (1970, 1982), Wisse (1974a,b, 1977), and Wisse and Knook (1979).

c. KUPFFERCELLSIN SCANNING ELECTRON MICROSOPY(SEM) Most Kupffer cells are easily recognizable in SEM specimens taken from perfusion-fixed livers (Fig. 6). In these preparations, natural surfaces of cells and sinusoids become available for SEM observation by applying freeze-fracture, critical-point drying, and gold sputtering. The intactness of the sinusoidal wall, mainly the fenestrated endothelium, critically depends on the application of a “physiological,” low-pressure perfusion fixation (De Zanger and Wisse, 1982), which results in the sinusoids being cleared of cells and plasma. Kupffer cells are infrequently present and take positions at bifurcations or larger periportal sinusoids. Unfortunately, not all Kupffer cells can be recognized with 100% certainty in SEM, because the two reliable TEM criteria (i.e., peroxidatic activity and ingestion of latex particles) have limited value in SEM. In SEM, Kupffer cells have variable surface irregularity (Motta, 1977). In some cases, the cell surface bears some small and slender microvilli, in other cases microvilli are accompanied by different pseudopodia and larnellipodia. Sometimes, regions with a surface irregularity corresponding to wormlike structures can be observed. The characteristic irregularity of the Kupffer cell surface differs drastically from the smooth surface of endothelial cells, or the branched, flat luminal surfaces of fat-storing


18 1

cells, occasionally seen through large gaps in the endothelial lining. Kupffer cells branch, and this phenomenon can give the cells a stellate appearance. In SEM, one has a better three-dimensional impression of the shape and magnitude of Kupffer cells whereas LM or TEM sections represent only a finite two-dimensional sample. Thinner projections might radiate from the Kupffer cell surface to the endothelial wall. These thin processes resemble the guy ropes of a tent. In Motta’s opinion (1977), such processes attach the Kupffer cell to the endothelial lining and might cause the appearance of gaps (openings larger than fenestrae). At higher magnification small pits can be observed that correspond to the presence of coated pits. At these places, no attachment of material can be observed. In the case of particle injections, SEM preparations vividly illustrate some of the events taking place at the surface of Kupffer cells. The surface can be seen to be covered with small colloidal particles or can be seen engulfing larger particles, such as latex. In all cases, the presence of a number of platelets surrounding the Kupffer cell during this phagocytic activity becomes apparent. The platelets seem to be attracted by an unknown factor, but they are not phagocytosed. Their presence can be confirmed by in vivo microscopy and sectioned material studied in LM or EM. Many hematologists have been questioned about the possible meaning of this phenomenon, but at present no obvious reason for this platelet clustering seems to exist.

111. Population Dynamics of Kupffer Cells

A. DISTRIBUTION AND POPULATION

SIZE

Kupffer cells are the most important category of fixed reticuloendothelial cells (Jones and Summerfield, 1982; Singer et al., 1969). They are thought to constitute the largest population of fixed macrophages in humans and various vertebrate species. In many diseases, an increase and sometimes a depletion of liver macrophages is known to occur. It is therefore of interest to investigate the change in numbers and distribution of Kupffer cells in normal and experimental situations, together with the mechanisms of their generation. A prerequisite to allow easy and reliable quantitation of Kupffer cells is an unequivocal identification of these cells at the LM level. Uptake of latex beads >O. 1 pm allows distinction between Kupffer cells and sinusoidal endothelial cells (see also Section II,A), but it has been demonstrated that not all Kupffer cells can be labeled by intravenous injection of

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such particles. Especially cells located in central parts of the liver lobule seemed less active in phagocytosis, even when these particles were perfused retrogradely through the hepatic vein (Sleyster and Knook, 1982). We found that after latex particle (0.8-km) administration in doses approaching overload condition, a maximum of only 65% of Kupffer cells, as recognized by peroxidase staining, were labeled in liver sections (Bouwens et al., 1989). The best method currently to stain rat Kupffer cells specifically is endogenous peroxidase staining. At the EM level, all cells that can be recognized as Kupffer cells by position and ultrastructural characteristics (Wisse, 1974a,b) contain peroxidase activity in the RER, nuclear envelope, and annulate lamellae. Moreover, no other sinusoidal cell type exhibits this peroxidase pattern (Wisse and Knook, 1979). The merit of this technique is that it also can be used at the LM level. Furthermore, the peroxidase marker is found in all cell intersections and it seems to be independent of the functional state or position of the Kupffer cells in the liver lobule. In this way, small Kupffer cells in the central region are not overlooked or confused with other sinusoidal cell types. Using peroxidase staining and morphometric methods, the total number of Kupffer cells was estimated to be -200 x lo6 for a rat weighing 250 g and a liver weighing 9 g (Bouwens ef a/., 1986a). After cell isolation, however, a recovery of 4-14 X 10" Kupffer cells per gram liver has been reported, representing -22% of all nonparenchymal liver cells (Knook et al., 1977). In situ, the distribution of Kupffer cells over the liver lobule is not homogeneous. When the liver lobule is divided in three regions from portal to central, the Kupffer cells have been reported to be distributed in an approximate ratio of 4 : 3 : 2, by means of morphological and endocytotic characterization (Kaneda and Wake, 1983; Sleyster and Knook, 1982). There is an obvious enrichment of Kupffer cells around portal veins, where these cells are larger and more active than those in the central region of the lobule. After cell isolation, two Kupffer cell subpopulations can be discerned that differ in size and several activities (Sleyster and Knook, 1982). This means that the Kupffer cell population is probably functionally heterogeneous. When the Kupffer cell population is stimulated to expand by a single intravenous injection of zymosan, its cell number was seen to increase 4fold within a period of 5 days (Bouwens et a!., 1984). This dense Kupffer cell population was distributed relatively homogeneously throughout the liver lobule, although there again existed a slightly higher density around portal veins, together with focal accumulations of macrophages in granulomas. A comparable increase of liver macrophages after stimulation by


183

a variety of inflammatory agents has been reported (Diesselhoff-den Dulk et al., 1979; Kelly et al., 1960, 1962; Kelly and Dobson, 1971; North, 1969, 1970; Warr and Sljivic, 1974). It has been demonstrated that the glucan-induced numerical increase of liver macrophages correlated directly with an increase in reticuloendothelial clearance of particles from the blood (Di Luzio et al., 1970). Such a Kupffer cell increase undoubtedly represents an important mechanism for host defense but can also contribute to the damage of parenchymal cell structure and function. The understanding of the mechanisms generating this population expansion is therefore of considerable interest for the understanding of the pathogenesis and therapeutic intervention of chronic inflammatory diseases. It is a commonly held belief that increased tissue macrophage numbers are the direct consequence of an enhanced influx of monocytes, the putative macrophage precursors, to the site of inflammation. Moreover, according to the current concept of the MPS, all tissue macrophages or resident macrophages, including Kupffer cells, should be considered as nondividing end cells derived from blood monocytes (Van Furth et al., 1972; Van Furth, 1982). Therefore, little attention has been paid to the possible role of an enhanced local replication of tissue macrophages after stimulation. Nevertheless, there is an increasing amount of evidence indicating that macrophages in several tissues retain their mitotic potential. The quantitative significance of such local proliferation of tissue macrophages as compared to the derivation from blood monocytes is the subject of considerable controversy. B. ONTOGENY

A replicating population of sinusoidal macrophages is established from day 11 of gestation in fetal rat liver (Deimann and Fahimi, 1978; Naito and Wisse, 1977; Pino and Bankston, 1979). This is well before bone marrow is formed (from day 19) and before monocytes have appeared in the circulation (from day 17). These fetal Kupffer cells are already phagocytic and have the characteristic peroxidase localization in RER and nuclear envelope, which differs from the granular peroxidase pattern encountered in monocytic cells. These observations exclude the monocytic origin of Kupffer cells at that stage of development and suggest that they have an early embryonic or extraembryonic origin, probably to be found in the yolk sac.

c. BONEMARROWDERIVATION VERSUS LOCALPROLIFERATION In the liver of sublethally irradiated recipients that have received bone marrow grafts, macrophages of the donor type were identifiable by means

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of sex chromosome staining in humans (Gale et a f . , 1978) or by immunological markers within rat-mouse chimeras (Shand and Bell, 1972). After human liver transplantation, recipient-type macrophages recognizable by karyotyping invaded the donor liver (Portmann et al., 1976) or even seemed to replace the original Kupffer cell population (Porter, 1969). This experimental evidence in favor of a bone marrow derivation of Kupffer cells, however, also demonstrated their mitotic potential in that karyotypic characterization required the occurrence of Kupffer cell metaphases. The origin of liver macrophages accumulating after RES stimulation by inflammatory agents such as zymosan, Corynebacteriurn parvurn, Listeria monocytogenes, bacterial endotoxin, and estrogens has been investigated by different experimental approaches. When tritiated thymidine was administered to animals before the application of the inflammatory stimulus, an influx of extrahepatically labeled macrophages was demonstrated to occur into the liver. Because free tritiated thymidine was no longer available at the time of stimulation, the observed increase in labeled liver macrophages could not be attributed only to local proliferation (Diesselhoff-den Dulk et a f . ,1979; North, 1970). However, local proliferation of resident macrophages that were shown to be already present in the liver before the time of stimulation also was reported under these experimental circumstances (Kelly and Dobson, 1971; North, 1969). The existence of local and extrahepatic Kupffer cell derivation has been observed after partial irradiation of animals with either the liver o r the bone marrow shielded (North, 1970; Warr and Sljivic, 1974). This dual origin of Kupffer cells also can be concluded from reports of apparently contradictory results with parabiotic experimental animals (Kinsky e f a f . , 1969; Volkman, 1976). Because of this dual origin, one may have to discriminate between two different Kupffer cell populations: those proliferating in situ and those recently recruited from bone marrow. Mitotic activity of Kupffer cells in siru has been reported in normal liver (Bouwens et al., 1989; Kelly et a f . , 1962; Widmann and Fahimi, 1975; Wisse, 1974b), in regenerating liver (Bouwens et a f . , 1984; Widmann and Fahimi, 1975; Wisse, 1974b), after liver stimulation with varying agents such as zymosan (Bouwens et al., 1984; Warr and Sljivic, 1974; Wisse, 1974b) estradiol (Kelly et a / . , 1960), L . monocytogenes (North, 1969), glucan (Deimann and Fahimi, 1979), and in fetal liver (Deimann and Fahimi, 1978; Naito and Wisse, 1977). These data demonstrate that Kupffer cells in different animal species have the potential of self-replication. Although this activity is low in steady-state conditions, cell division can be enhanced by different stimuli. In an attempt to estimate the relative importance of local production


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and extrahepatic recruitment of Kupffer cells during liver regeneration after two-thirds partial hepatectomy and after zymosan stimulation, the metaphase-arrest method was used to determine the potential doubling time of the expanding Kupffer cell population (Bouwens et al., 1984). These experiments revealed that during liver regeneration, the Kupffer cell population grew with a mean doubling time of 3.6 days and that local replication ensured a potential doubling time of 3.7 days. From these data it can be concluded that the total production of new Kupffer cells could be provided by local proliferation. In the case of zymosan-induced exponential growth, these data were 2.5 and 3.5 days, respectively, which means that -70% of the population expansion was due to local proliferation (Bouwens et al., 1984). In the latter experimental model, >90% of Kupffer cells could be labeled by repeated administration of tritiated thymidine over a period of 48 hours (Bouwens e? al., 1986a). This means that nearly all Kupffer cells were in the cell cycle. Furthermore, selective Xirradiation of the liver with a sublethal dose of 850 rad significantly inhibited Kupffer cell growth, whereas irradiation of the whole body with the liver shielded did not have this effect (Bouwens e? al., 1986b). In both experimental models-partial hepatectomy and zymosan stimulation-it could be demonstrated that the dividing Kupffer cells were mature macrophages that were already residing in the liver before the time of stimulation, and thus, were not recruited by the stimulus. This was supported by labeling the cells with latex particles several days before the stimulus (Bouwens et al., 1984). This leads us to conclude that Kupffer cells have a dual origin (i.e., local proliferation and extrahepatic recruitment), but the predominant mechanism underlying their population growth consisted of local proliferation of mature Kupffer cells. In other experimental situations, however, the relative importance of both mechanisms may vary depending on the conditions. D. THE KUPFFERCELLPRECURSOR It is established that Kupffer cells can originate from tissues outside the liver, and because the Kupffer cell precursor must be present in peripheral blood, the most likely candidate for this is the monocyte. Monocytes are the only leukocytes that can develop macrophage characteristics, both in vivo and in vitro, yet there is as yet no incontrovertible proof for the existence of differentiation from monocytes into Kupffer cells. In spite of extensive ultrastructural investigations on this problem (Bouwens et al., 1984; Bouwens and Wisse, 1985; Wisse, 1974a,b), there exists only one report claiming the existence of so-called transitional forms between monocytes and Kupffer cells (Deimann and Fahimi, 1979). Such transi-

tional forms occurring in low numbers after glucan stimulation in rats, were described as cells with the typical peroxidase pattern of Kupffer cells, with the additional presence of monocyte-specific granules. However, our observations on the ultrastructure of sinusoidal cells have revealed the following.

I . Kupffer cells can exhibit a large amount of different lysosomal and other granular structures with varying morphology and density. 2. Kupffer cells often contain erythrocyte debris that also reacts with the peroxidase substrate, and these phagosomes can be confused with peroxidase-positive monocyte granules. 3. Kupffer cells have catalase activity in microperoxisomes that may be confused with peroxidase-containing lysosomes. 4. Even in material that was not incubated for peroxidase demonstration, dense granular structures can occur in Kupffer cells. The occurrence of Kupffer cells containing some dense granules, or “monocyte-type” lysosomes with a halo, is therefore not reliable evidence for the existence of transitional forms between monocytes and Kupffer cells. Moreover, monocytes can be readily seen in sinusoids of normal or stimulated animals, and these cells were never observed developing ER peroxidase activity or other typical Kupffer cell features. On the contrary, after zymosan stimulation, large numbers of monocytes were seen in liver sinusoids developing into large, activated, inflammatory macrophages that remained distinct from the resident Kupffer cells (Bouwens and Wisse, 1985). Thus, it can be suggested that two phenotypically different macrophage types occur during liver inflammation: an “exudate” or monocytic type, and a “resident” type, like the Kupffer cell. This situation is comparable with the situation described in the peritoneal cavity (Daems ct al., 1976; Volkman, 1976; Volkman et al., 1983). In contradistinction to Kupffer cells, the rnonocyternacrophages, attracted by inflammatory stimuli, remain in the liver for a limited period and thus belong to a transient population. The direct Kupffer cell precursor has been described as a peroxidasenegative phagocytic cell that looks like an immature cell, containing few organelles and, in particular, few lysosomes and vacuoles (Bouwens and Wisse, 1985). Whether these latter cells belong to a monocyte subset that have lost their granular peroxidase activity, as is known to occur in monocytes in vitro (Breton-Gorius ct al., 1980), or whether these cells form a distinct macrophage sublineage is not yet established. However, colonies of macrophages, clonigenically derived from individual bone marrow precursors, have been reported to express different phenotypes, suggesting the existence of distinct macrophage lineages (Bursuker and Goldman,


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1983). The existence of separate, stable macrophage subsets differing in functions, phenotype, and origin, however, is still a controversial but fascinating subject. IV. Isolation, Purification, and Culture of Kupffer Cells

Several procedures are presently available for the preparation of isolated and purified Kupffer cells. Each procedure has its own effect on the final yield, the purity, and the functional characteristics of the isolated Kupffer cells. Generally, these procedures involve perfusion and incubation of the liver with dissociating enzymes. The use of collagenase results in a suspension that contains all types of liver cells (Brouwer et al., 1982a,b; Knook e f al., 1982; Nagelkerke et al., 1982).The use of pronase, which selectively destroys parenchymal cells, results in a suspension that consists almost exclusively of sinusoidal cells (Brouwer et al., 1982b). The advantages and disadvantages of these methods have been discussed elsewhere (Brouwer et al., 1982b). The various steps in the purification of Kupffer cells include the following: 1. Perfusion of the liver with dissociating agents. With small experimental animals like the rat and the mouse, perfusion of the total liver is performed. For larger animals and humans, generally only a part of the liver is perfused. 2. Incubation of liver tissue for further dissociation or destruction of parenchymal cells. 3. Further purification of Kupffer cells by centrifugation procedures or by selective attachment. The following is a summary of our present knowledge with regard to the various steps of isolation and the behavior of Kupffer cells during culture. If not stated otherwise, the description refers to the isolation, purification, and cultivation of Kupffer cells removed from the livers of rats. A. ISOLATION OF KUPFFERCELLS

Isolation starts with perfusion of the liver with a fluid containing dissociating agents such as collagenase, pronase, or a combination of both enzyme preparations (Table I). Only collagenase perfusion can be employed for the isolation of both parenchymal and sinusoidal cells from the same liver (Nagelkerke et al., 1982; van Berkel, 1982). The perfusion is generally performed at 37°C for optimal activity of

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TABLE I CHARACTERISTICS OF

KUPFFEKCELLS

ISOLATED B Y VARIOUS

Sinusoidal cell preparation s/r of

Isolation method Pronase 37°C

10°C Collagenase Pronasecollagenase

Kupffer cells

21 17 14 19

Yieldh of Kupffer cells (X

10")

34 35 39 46

METHODS"

Kupffer cell fraction' Compositiond

L

E

K

3 6 15

24 18 7

74 76 78 76

24

Protein content (&lo6 cells)

87.4 113.9 n.d. 114.2'

Viability' of Kupffer cells

90 90 85 90

"Average of last four experiments. Cells were isolated with pronase at 37°C (Knook and Sleyster. 1976). with pronase at 10°C (Praaning-Van Dalen and Knook. 1982). or with pronase and collagenase (Knook C f ul., 1982). 'Yield represents the number of cells per liver (mean weight 4.8 g ) of 3- to 4-month-old female Brown Nonvay/Billingham Rijswijk rats with an average body weight of 150 g. 'Standard procedures were applied for purification of Kupffer cells by centrifugal elutriation. The standard separation chamber was replaced by the Sandreson chamber for the purification of the Kupffer cells obtained by the pronase 10°C method. dLymphocytes ( L ) . endothelial (EJ. and Kupffer ( K ) cells are expressed as percentages of total cell number. 'Percentage of cells that exclude 0 . 2 5 8 trypan blue. Results were confirmed by EM studies. 'Not determined.

the dissociating enzymes. However, methods are now available that are performed at d 10°C. using either pronase (Praaning-Van Dalen and Knook, 1982) or collagenase (Nagelkerke et al., 1982). After perfusion, the hepatic tissue is incubated for further dissociation by mechanical or enzymatic means. In addition, selective destruction of parenchymal cells can be accomplished by pronase treatment (Knook et a/., 1982), extended collagenase treatment (Brouwer et al., 1982a), or incubation with enterotoxin (Berg et al., 1979). The incubation step can be omitted if one employs the "two-step perfusion" method, that is, an initial calcium-free perfusion, followed by perfusion with collagenase and calcium (Nagelkerke et ul., 1982), or the coldpronase method employing pronase at 10°C (Praaning-van Dalen and Knook, 1982). €3. PURIFICATION OF KUPFFER CELLS

The yield of Kupffer cells in sinusoidal cell preparations varies with the isolation method used (Table I). For further purification, most labora-


189

tories employ the method of Knook and co-workers, that is, the application of a discontinuous metrizamide gradient to eliminate red blood cells and parenchymal cell debris, followed by centrifugal elutriation to obtain highly purified Kupffer cells (Knook and Sleyster, 1976, 1980; Knook et al., 1977; Brouwer et al., 1984). Centrifugal elutriation has no detrimental effects on the Kupffer cells and results in relatively pure cell preparations (Table I). As an alternative method, selective adherence of Kupffer cells to culture dishes during overnight culture of sinusoidal cells has been employed (Munthe-Kaas et al., 1975). This method has several disadvantages, including the phagocytosis of cell debris by Kupffer cells and contamination with endothelial cells, which sometimes attach even faster than Kupffer cells (Brouwer et al., 1980, 1982a; De Leeuw et al., 1982). Some characteristics of isolated Kupffer cells are given in Table I. The yield of Kupffer cells in the various isolation methods does not differ much; the largest number, 10 X lo6 cells per gram liver, is obtained with the pronase-collagenase method. Kupffer cell viability usually exceeds 90%. Sinusoidal cells prepared by the collagenase method are contaminated with -46% parenchymal cells in spite of attempts to remove these cells by differential centrifugation. Prolonged differential centrifugation results in a considerable loss of Kupffer cells (van Berkel, 1982; Brouwer et al., 1982a). After separation of sinusoidal cells by centrifugal elutriation, the relative distribution of the cell types in the “Kupffer cell fraction” varies with the isolation method (Table I).

c. EVALUATION OF THE ISOLATION METHODS Endocytosis can be considered as one of the most important functions of Kupffer cells, and thus the quality and usefulness of a particular isolation method should be judged by the retention of endocytic properties in the isolated cells. However, each isolation method has its own advantages, depending on the cellular characteristics demanded and the specific aim of a study. If a high yield of pure Kupffer cells is needed, the method of preference is the pronase-collagenase method (Table I). When the capacity of Kupffer cells to endocytose in vitro is to be determined, the cold-pronase isolation method (Praaning-Van Dalen and Knook, 1982) or the cold-collagenase method (Nagelkerke et al., 1982) appears to be suitable. Advantages of these low-temperature (10°C) methods include (1) prevention of degradation of substances ingested in situ, which allows the study of in vivo endocytosis; (2) prevention of the ingestion of cellular debris during the isolation procedure, which may lead to an incorrect re-

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K. WAKE

ET A[.

distribution of cellular components (e.g., enzymes); and (3) a substantial retention of membrane receptors, which allows in vitro studies on endocytosis, directly after Kupffer cell isolation. Several membrane receptors will be damaged by the use of pronase during the isolation procedure. Kupffer cells, freshly isolated by the coldpronase method, immediately bind and endocytose various substances. Receptors for mannose-terminated glycoproteins and for colloidal albumin are preserved to a large extent after cold-pronase treatment (Praaning-Van Dalen and Knook, 1982). Thus, Kupffer cells isolated by the cold-pronase method do not need a recovery period during short-term culture. Some lipoprotein receptors are destroyed by pronase probably even at low temperature. For studies on these receptors, the collagenase isolation method is to be preferred (Nagelkerke er al., 1982).

D. CULTURE OF KUPFFEK CELLS During a short-term culture (8-24 hours), the Kupffer cells show recovery from trauma inflicted during isolation (Brouwer et al., 1982a). Longterm maintenance cultures (2-10 days) of isolated Kupffer cells may be used for studying several of their functions, such as ( I ) interaction with other cells, microorganisms, and molecules; (2) synthesis and secretion of effector substances; (3) involvement in the immune processes; and (4) the tumoricidal and natural killer (NK) cell activity of cultured Kupffer cells. Kupffer cells in maintenance culture largely retain their differentiated characteristics with regard to morphology, enzyme cytochemistry, membrane receptors, endocytosis, and lysosomal functions for periods of 510 days (Brouwer et al., 1982a,b; De Leeuw et al., 1983). Cultures of Kupffer cells can be very useful in studying functional aspects that cannot be investigated in vivo or in situ. Although several investigators have used maintenance cultures of Kupffer cells for these purposes, a comparison of various functional capacities of cultured cells with those of cells in intact livers has not been sufficiently performed. It is encouraging that one of the few studies reporting such a comparison demonstrated that the maximal rate of uptake of colloidal albumin by Kupffer cells in maintenance culture was in the same order of magnitude as the rate of uptake by Kupffer cells in the intact liver (Brouwer et al., 1985). However, one should consider the possibility that both qualitative and quantitative changes in Kupffer cell function may arise as a consequence of culture conditions.


191

V. Metabolic Responses of Stimulated Rat Kupffer Cells in Vitro Kupffer cells provide a first line of defense against noxious material (bacteria, viruses, toxins, etc.), approaching the liver through the portal vein. As a result, the cells are expected to be in a more-or-less activated state, and this raises the problem of defining the resting versus the stimulated state of the Kupffer cells. In particular, the procedures of their isolation from livers with the help of hydrolytic enzymes, such as collagenase and pronase or mixtures thereof, will always bring them in contact with cell debris and might stimulate the cells to some extent. Using Kupffer cells, kept in primary culture for 2 days, one can observe strong reactions to various stimuli, such as opsonized erythrocytes, zymosan particles, immunocomplexes, endotoxin, or Ca2+ionophores. The intensity and the velocity of the responses to these stimuli are not uniform; the most conspicuous reactions are seen after the phagocytosis of different particles or exposure to endotoxin. A. METABOLICEFFECTS OF ENDOCYTOSIS Phagocytosis of particulate matter appears to involve several receptors of variable specificity. Binding sites for Fc, C3b, mannose (N-acetylglucosamine), galactose (N-acetylgalactosamine), and apolipoprotein B are well known (for a review, see Knook and Wisse, 1982). Receptors for fibronectin-containing particles (Rieder et al., 1982) and sites for the uptake of insulin and glucagon (Kreusch, 1980) have also been observed. Cell organelles like parenchymal cell mitochondria are endocytosed rapidly by cultured Kupffer cells; the typical enzymatic activities of these organelles can be detected in cell extracts for several hours after ingestion (Rieder and Decker, 1984). This finding supports the morphological observation that intact material is present for some time after internalization. One of the early events observed after stimulation by particles is the so-called oxygen burst that has been described for many phagocytosing cell types (for a review, see Klebanoff, 1980). In Kupffer cells, most of the extra 0, taken up can be accounted for as 0 ; in the extracellular fluid (Bhatnagar et al., 1981). The superoxide anion radical is formed by NADPH reduction of 0,, a reaction that is catalyzed by a membraneassociated NADPH oxidase: 20,

+ NADPH > 20; + NADP' + H'

The large amount of NADPH required during phagocytosis is provided

I92

K. WAKE

ET A L

by a strongly enhanced flux of glucose through the pentose phosphate pathway (Bhatnagar et a!., 1981). Kupffer cells neither contain nor synthesize glycogen, in contrast to alveolar macrophages (Hoffmann et al., 1978). Therefore, the instant requirement for glucose cannot be met by an intracellular store but must be satisfied by an enhanced uptake. The observation (Bhatnagar et al., 1981)that cytochalasin B prevents 0; formation is in line with this interpretation. This compound was shown to inhibit carrier-mediated hexose transport (O’Flaherty et al., 1984). The crucial role of the pentose phosphate pathway in this process may explain the high content of glucose-6-phosphate dehydrogenase (G6PD) in Kupffer cells, and this, in fact, accounts for most of the enzyme activity present in the liver (Knook et al., 1980). The suppression of 0;formation by SH-blocking agents, such as iodoacetamide, has been traced to the inhibition of G6PD (Bhatnagar et al., 1981). Superoxide is considered as an activated oxygen species participating in the oxidative destruction of invading particles, especially of the cell membrane of bacteria. In the presence of superoxide dismutase, superoxide is converted to H,O, and in the presence of the myeloperoxidase of leukocytes, the powerful -OH radical is formed. It also should be mentioned that the NADPH oxidase reaction produces, rather than consumes, intracellular protons. It is not known what role is played by the accumulation of protons near the site of the interaction between the oxidase and NADPH; countertransport with metal ions such as Na’ or Ca” is a possibility. The 0; production by phagocytosing Kupffer cells was found to depend on the presence of Ca” in the medium (Birmelin and Decker, 1983). B. THEROLE OF CALCIUM A rapid influx of Ca” into Kupffer cells occurs after the onset of particle phagocytosis (Birmelin and Decker, 1983) (Fig. 7). The uptake of 4SCaZ+ by rat Kupffer cells in primary culture is stimulated 7-fold over the basal exchange rate and lasts 15minutes. It is immediately followed by an enhanced active release of Ca” from the cells. It appears that the Ca” fluxes, rather than the concentration in the intracellular compartments, are responsible for several secondary effects. They include, in addition to the 0; production just mentioned, an activation of phospholipase A?, the synthesis and release of various arachidonic acid (AA) derivatives, and a transient rise of the intracellular levels of cyclic nucleotides. It is obvious, however, that the superoxide production and the synthesis of eicosanoids are not necessarily correlated (Dieter et al., 1986) (Fig. 8). These processes are provoked simultaneously by the phagocytosis of particles and by phorbol 12-myristate acetate (PMA). However,

-


193

30-

m22-

D

QX

Ep0

min

FIG.7. Effect of zymosan stimulation on the uptake of calcium ions by Kupffer cells cultivated for 72 hours. Calcium uptake was evaluated by adding a small quantity of radioactive calcium to the culture dishes. After washing, the uptake of the isotope by the cells was measured in a liquid scintillation spectrometer. Uptake in zymosan-treated cells ( ); untreated cells, (0),

+

prostaglandins (PG) are also released after the exposure of Kupffer cells to lipopolysaccharides (LPS) or to the calcium ionophore A 23187, but little 0 ; is released under those conditions. Furthermore, the formation of superoxide is not affected by pretreatment of the cells with dexamethasone, while the arachidonate cascade is strongly suppressed by glucocorticoids. In addition, the calmodulin antagonist calmidazolium (R 2457 1) also does not affect the zymosan-triggered formation of superoxide but inhibits the liberation of arachidonate (Birmelin el al., 1984).

C. ARACHIDONATE METABOLISM The intracellular availability of free arachidonate appears to be the limiting factor for eicosanoid synthesis by rat Kupffer cells. The phospholipase A, reaction serves both the cyclooxygenase and the lipoxygenase pathway by providing tetraenoic acid. Kupffer cells kept in primary culture for 48 hours respond to a phagocytic stimulus by enhancing the activ-

I94

K. WAKE

E'I'AL

A

1

bl, .:.. .:

..... ..... ..... ..... ..... .......... ..... ..... .....

T

.:....... .......

T

T h PAF

T

LPS

AA

FIG.8. Effect of various stimuli on PGE, release and superoxide production by Kupffer cells treated with dexamethasone. Dotted bars represent cells treated with dexamethasone ( 1 p M ) : open bars show the effect of 0.5 mg/ml zymosan (Zy). I p M phorbol myristate acetate (PMA). 20 p M A 23187. 5 nM platelet-activating factor (PAF) 30 pg LPS, or 30 pM AA. The release of PGE, was determined by radioimmunoassay. and the released superoxide was measured by cytochrome C reduction.


195

ity of phospholipase A,, which is specific for the addition of an arachidonoyl group in the 2-position of the phosphatidylcholine at a pH optimum of 8.1 (Birmelin et al., 1984). Calmodulin inhibitors prevent this rise of phospholipase A, activity if given to the cells prior to or together with zymosan. Calmodulin is present in cultured Kupffer cells in amounts (3.2 pg per lo6 cells as determined by CAMP phosphodiesterase assay) exceeding those of parenchymal cells and various other cells (Birmelin et al., 1984). Calmodulin inhibitors are without effect if added to the cellfree extract of stimulated cells. Furthermore, calmodulin added to the extract of unstimulated Kupffer cells does not increase phospholipase A, activity. It appears that the zymosan-triggered stimulation either is mediated by lipocortin or is brought about by a direct calmodulin-dependent interconversion of phospholipase A,. Kupffer cells are the most potent producers of prostanoids among the different liver cell types. Two kinds of stimulators are known to elicit eicosanoid release: (1) contact with phagocytosable material (e.g., zymosan, opsonized erythrocytes, mitochondria, heat-inactivated bacteria), and (2) inflammatory and immunomodulating agents (e.g., LPS, PMA) or calcium ionophores. While the arachidonate cascade (Fig. 9) starts immediately after contact of the Kupffer cells with the phagocytotic stimulus, reverting to a quiescent state after 1 hour, this response is much slower but lasts longer (324 hours) after exposure to LPS (Birmelin et al., 1986).

-

PraUcyclm

Prata$andim

Iffib1

(0.E.F SmOSI

1hmmbOl.M Ap

W E E

LNlIOh”9

FIG.9. Schematic survey of the formation of the difficult kinds of eicosanoids from AA.

196

K . WAKE ET AL

The major AA metabolite released after stimulation is PGD2, but PGE,, PGFZct, a PGA,-like compound, thromboxane (measured as TXB,) accompanied by 15-hydroxy-5,8,iO-heptatrienoicacid (HHT), small amounts of prostacylin (measured as 6-keto-PGF,, or 6-KPGF,, and HETEs are also released (Birmelin and Decker, 1984; Decker et ul., 1986) (Table 11). N o PGE, and very little peptidoleukotrienes are synthesized by stimulated rat Kupffer cells. Quantitative, perhaps even qualitative differences may exist in the eicosanoid patterns of Kupffer cells from different species. Interestingly, rat Kupffer cells only slightly inactivate prostanoids and leukotrienes (LT); they are able, however, to convert LTC, to LTD,, LTE,, and N-acetyl LTE,. Hepatocytes, on the other hand, do not produce but quite actively degrade added eicosanoids (Fig. 10) (Tran-Thi et d . , 1986). Neither phagocytosis nor synthesis of PGE, by Kupffer cells is influenced by added PGE, or PGE,, in contrast to what is observed with other mononuclear phagocytes, such as periotoneal macrophages (Oropeza-Rendon er d . , 1980). The amount of PGEz released by zymosan is the same as that elicited by treatment of Kupffer cells with Ca" ionophore. The effects of A 23187 and zymosan, however, are not additive. Ca'+ channel blockers like verapamil do not inhibit PGEz release. Ca" entry into Kupffer cells seems to use mechanisms different from those operating in heart muscle and other verapamil-sensitive tissues (Fleckenstein, 1980). A highly efficient inhibitor of PGEz synthesis that may also be of physiological significance is LTB, (Decker and Birmelin, 1984). This LT suppresses

TABLE I1 CAPACII Y O F LIVER CELLSTO SYNTHESIZE PROSTANWDS

Maximal rates of synthesis" (ng/ hour) by Cells

PGD,

PGE,

Hepatocyteb Endothelial cells Kupffer cell\

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