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0-Acetylated peptidoglycan: its occurrence, pathobiological significance, and biosynthesis
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ANTHONY J.
CLARKE' AND
CLAUDED U P O N T ~
Guelph- Waterloo Centre for Graduate Work in Chemistry, Department of Microbiology, University of Guelph, Guelph, Ont., Canada N I G 2 WI Received July 22, 1991 Revision received October 9, 1991 Accepted October 15, 1991 CLARKE, A. J., and DUPONT,C. 1992. 0-Acetylated peptidoglycan: its occurrence, pathobiological significance, and biosynthesis. Can. J . Microbiol. 38: 85-91. Bacterial cell walls and their structural units, particularly peptidoglycan, induce a vast variety of biological effects in host organisms. The pathobiological effects of peptidoglycan are greatly enhanced by various modifications and substitutions to its basic composition and structure. One such modification is the presence of acetyl moieties at the C-6 hydroxyl group of N-acetylmuramyl residues, and to date, 11 species of eubacteria, including some important human pathogens, such as Neisseria gonorrhoeae, Proteus mirabilis, and Staphylococcus aureus, are known to possess 0-acetylated peptidoglycan. This review addresses the influence of 0-acetylation of peptidoglycan on its resistance to degradation both in vitro and in vivo, the clinical importance of the modification, and the currently held views on the pathway for its biosynthesis. Key words: peptidoglycan, murein, 0-acetylation, lysozyme, arthritis. CLARKE,A. J., et DUPONT,C. 1992. 0-Acetylated peptidoglycan: its occurrence, pathobiological significance, and biosynthesis. Can. J . Microbiol. 38 : 85-91. Les parois cellulaires bacteriennes et leurs unites structurales, notamment le peptidoglycan, induisent une grande variete d'effets biologiques sur les organismes hates. Les effets pathobiologiques du peptidoglycan sont accentues grandement par les modifications et les substitutions variees de sa composition de base et de sa structure. La presence de radicaux acetyles a la position du groupe hydroxyl en C-6 des residus N-acetylmuramyles constitue l'une de ces modifications. Jusqu'a present, 11 espkces d'eubacteries, incluant quelques pathogknes humains importants comme Neisseria gonorrhoeae, Proteus mirabilis et Staphylococcus aureus, posskdent un peptidoglycan 0-acetyle. Cette revue traite de l'influence de ce type d'acetylation du peptidoglycan sur sa resistance a la degradation in vitro et in vivo, de l'importance clinique de la modification ainsi que des opinions qui ont cours actuellement concernant le sentier de sa biosynthkse. Mots elks : peptidoglycan, mureine, 0-acetylation, lysozyme, arthrite. [Traduit par la redaction]
Introduction Peptidoglycan in a heteropolymer of distinctive composition and structure and is uniquely associated with prokaryotic cell walls. Its chemistry and biosynthesis have been reviewed (Park 1987; Shockman and Barrett 1983). Basically, it is composed of 0-1,4-linked N-acetylglucosamine and N-acetylmuramic acid residues, the latter being modified by a tetrapeptide composed of both L and D amino acids. In most cases, these peptide side chains are involved in the direct cross-linking between adjacent glycan strands, but in some instances more complicated arrangements exist. For example, a pentaglycine peptide bridges the neighboring peptide side chains within the peptidoglycan sacculus of Staphylococcus aureus. In Gram-positive bacteria, the highly cross-linked peptidoglycan strands are arranged in a thick .three-dimensionalconcentric array (Snowden and Perkins 1990), whereas those of Gram-negative bacteria are probably no more than three layers deep (Glauner et al. 1988; Labischinski et al. 1991). The continuous peptidoglycan layer that covers the inner (cytoplasmic) membrane provides shape and rigidity to ' ~ u t h o rto whom all correspondence should be addressed. 2 ~ r e s e n taddress: Department of Chemistry, Carlsberg Laboratorium, Gamle Carlsbergvej 10 Valby, Copenhagen, DK2500, Denmark. Printed in Canada / Irnprirne au Canada
bacterial cells and, more importantly, contains the turgor pressure exerted by the cytoplasm. Peptidoglycan composition and structure are now known to be complex, as exemplified in a recent study by Glauner et al. (1988). From a digest of Escherichia coli peptidoglycan, 30 different types of muropeptides were isolated by HPLC and characterized. The diversity of structures is due to the free combination of seven different types of side chains with two types of cross-bridges between the peptides of adjacent strands. In this review, we discuss the effect and biosynthesis of one clinically significant modification to peptidoglycan, that of the 6-0-acetylation of muramyl residues.
Biological properties of peptidoglycan Following the lysis of invading microorganisms, mammalian tissues clear cellular debris, including peptidoglycan, very rapidly through the action of the hydrolytic enzymes of either phagocytic cells or serum. However, in some instances, high molecular weight fragments of peptidoglycan have been observed to persist and circulate in the host organism. These large peptidoglycan fragments have been shown to induce diverse pathobiological and pathophysiological effects, many of which have been recently reviewed (Seidl and Schleifer 1985). Some of these include the induction of slow-wave sleep (Johannsen et al. 1989, 1990), com-
CAN. J. MICROBIOL. VOL. 38, 1992
TABLE1 . Bacteria reported to possess peptidoglycan containing 6-0-acetylmuramyl residues Species
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Gram positive Streptococcus faecalis Micrococcus luteus Staphylococcus aureus Lactobacillus acidophilus Lactobacillus fermentum Gram negative Moraxella glucidolytica Pseudomonas alcaligenes Neisseria perflava Neisseria gonorrhoeae
Proteus vulgaris Proteus mirabilis
Strain
% 0-acetyl
Reference
ATCC 9790 nr Copenhagen SG5 11Berlin H(NC1B 6571) 63 AM Gasser ATCC 9338
Abrams 1958 Brumfitt et al. 1958 Ghuysen and Strominger 1963 Burghaus et al. 1983 Snowden et al. 1989 Coyette and Ghuysen 1970 Logardt and Neujahr 1975
nr nr nr I 1260 FA19 FA 140 FA171 FA136 F62 FA 102 CL1 BR87 1342 7502 2686 7405 CS7 609 624 8035 8038 129 1 JW3 1 P18 19 19 Perkins Perkins GB8 ATCC 7002 ATCC 29245 ATCC 33583 ATCC 4307 1 ATCC 25933 ATCC 33659 ATCC 12453 ATCC 14273 TGH 9041 TGH 7341 F 16 F49 1
Martin et al. 1973 Martin et al. 1973 Martin et al. 1973 Blundell et al. 1980 Blundell et al. 1980 Swim et al. 1983 Swim et al. 1983 Swim et al. 1983 Swim et al. 1983 Swim et al. 1983 Swim et al. 1983 Swim et al. 1983 Swim et al. 1983 Swim et al. 1983 Swim et al. 1983 Swim et al. 1983 Swim et al. 1983 Swim et al. 1983 Swim et al. 1983 Swim et al. 1983 Swim et al. 1983 Swim et al. 1983 Swim et al. 1983 Fleck et al. 197 1 Martin and Gmeiner 1979 Dupont and Clarke 1991a Blundell and Perkins 198 1 Dupont and Clarke 1991a Dupont and Clarke 1991a Dupont and Clarke 1991a Dupont and Clarke 1991a Dupont and Clarke 1991a Dupont and Clarke 1991a Dupont and Clarke 1991a Dupont and Clarke 1991a Dupont and Clarke 1991a Dupont and Clarke 1991a Dupont and Clarke 1991a Dupont and Clarke 1991a Dupont and Clarke 1991a Dupont and Clarke 1991a
NOTE:nr, not recorded.
plement activation (Kawasaki et al. 1987; Verbrugh et al. 1980), pyrogenicity (Johannsen et al. 1990; Masek et al. 1968), and arthritogenicity (Cromartie 1981). As early as 1966, Hamerman proposed that oligosaccharide or glycopeptide fragments derived from bacterial cell walls or capsules may be the inciting agents for rheumatoid arthritis in mankind (Hamerman 1966). Although extrapolation of recent findings to mankind is still premature, their is a plethora of data in the literature to indicate that such occurs in animal models (Chedid et al. 1978; Cromartie 1981; Esser et al. 1985; Fleming et al. 1986; Fox et al. 1982;
Koga et al. 1985; Koga and Pearson 1976; Ginsburg and Sela 1976; Rosenthal and Fleming 1985; Stimpson et al. 1986). That high molecular weight fragments of peptidoglycan are required to induce arthritic conditions was clearly demonstrated in studies by Fox et al. (1982) and Janusz et al. (1984, 1986). Purified peptidoglycan digested with mutanolysin (a muramidase isolated from Streptomyces globisporus) was shown to lose its arthropathic potential. In a separate experiment, a single intravenous injection of mutanolysin into peptidoglycan-induced arthritic rats prevented the development of recurrent, chronic erosive arthritis. In vivo studies
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0 II
o \*
Stage 1
l o
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NHCCH, II 0
0 NHCCH, CH3CH II 0 C=O beptide
Stage 2
FIG. 1. Structure of the glycan portion of the repeating subunit of 0-acetylated peptidoglycan. with the pathogens Staphylococcus aureus and Neisseria gonorrhoeae have indicated that the phenomenon of peptidoglycan persistence in a host is directly attributable, in-these instances, to the presence of numerous 0-acetyl substituents on the glycan backbone (Blundell et al. 1980; Fleming et al. 1986; Rosenthal et al. 1982; Rosenthal and Fleming 1985; Swim et al. 1983).
6-0-Acetylated peptidoglycan 0-Acetylated peptidoglycan was first observed independently in Steptococcus faecalis by Abrams (1958) and in Micrococcus luteus by Brumfitt and co-workers (Brumfitt et al. 1958) 30 years ago. Since then, 0-acetylated peptidoglycan has been observed in a total of 47 strains of 11 species of both Gram-positive and Gram-negative bacteria, including some important pathogens (Table 1). It is quite conceivable that many other bacteria possess 0-acetylated peptidoglycan but have escaped detection as a result of the base lability of the modification. 0-Acetylation of peptidoglycan occurs at the C-6 hydroxyl group of N-acetylmuramyl residues producing the corresponding 2,6-diacetylmuramyl derivative (Fig. 1). The extent of naturally occurring 0-acetylation in microorganisms ranges between < 10 and 70%, while a spontaneous mutant of Micrococcus luteus cultured in the presence of hen egg-white lysozyme was reported to have a molar ratio of N-acetylmuramic acid : 0acetyl of 1:l (Brumfitt et al. 1958). Despite intensive investigations by a number of groups, the physiological function of this modification to peptidoglycan as it pertains to .the bacterium remains unclear. However, the role of 0-acetylation in conferring resistance to the hydrolytic activity of hen egg-white lysozyme was discerned soon after the initial discovery of the modification (Brumfitt 1959). Subsequent studies have confirmed the resistance of most muramidases to 0-acetylated peptidoglycan, but it should be noted that the efficacy of others, such as the N,Odiacetylmuramidases of Chalaropsis (Hash and Rothlauf 1967) and Streptomyces globisporus (mutanolysin) (Hamada et al. 1978), is not affected by 0-acetyl groups. As with other types of modifications to peptidoglycan, such as the presence of teichoic acids at the C-6 atom of murarnyl residues of Gram-positive bacteria, the 0-acetylation of the peptidoglycan of a pathogenic microorganism thus provides protection from the hydrolytic action of host muramidases. Correlations have been made between the strain-related differences in muramidase sensitivity and the extent of 0-acetylation of gonococcal peptidoglycan for both the hen egg-white (Blundell et al. 1980; Rosenthal et al.
-0l , -0 -0 -0 - q - f, A
-~-o-o-o-o~o Stage 3
-0-0-0-0 A
-fi-o-o-fio - ~ - ~ stage~ 4 -
FIG. 2. Proposed scheme of peptidoglycan biosynthesis in P. mirabilis. , pre-existing ("old") 0-acetylated subunits; 0, newly incorporated 0-acetylated subunits; , newly incorporated non-0-acetylated subunits; -, 0-1,4 glycosidic linkages between subunits; A , peptide cross-links between glycan strands. (Adapted from Gmeiner and Kroll, 1981.) 1982; Swim et al. 1983) and human sera and polymorphonuclear (Rosenthal et al. 1983) enzymes. The extent of muramidase resistance to the solubilization of peptidoglycan by both human and hen egg-white enzymes was also shown to be dependent upon the degree of 0-acetylation in 14 strains of Proteus mirabilis (Dupont and Clarke 1991a). In the latter case, an increase in the overall change in the standard Gibbs free energy of activation (A(AG)) as a consequence of increasing 0-acetylation was observed. This was shown to result from the weaker affinity of the enzyme for the modified substrate, presumably through steric hinderance since the C-6 hydroxyl moieties of the substrate directly participate in its binding to the active-site cleft of the enzyme (Blake et al. 1967; Imoto et al. 1972). The consequences of this phenomenon are thus considerable in view of the detrimental effects exerted by circulating peptidoglycan fragments in a mammalian host organism.
Biosynthesis of 6-0-acetyl peptidoglycan Whereas its role in resistance to muramidases and the consequences of such have been well documented, very little is known concerning the biosynthetic sequence of reactions leading to the assembly and 0-acetylation of peptidoglycan. There is substantial evidence to suggest that 0-acetylation occurs after nascent peptidoglycan strands have been both attached and cross-linked to the pre-existing sacculus (Dougherty 1983a; Gmeiner and Kroll 1981; Gmeiner and Sarnow 1987; Lear and Perkins 1983, 1986, 1987; Snowden
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CAN. J. MICROBIOL. VOL. 38, 1992
et al. 1989). By pulse-chasing radiolabelled N-acetylglucosamine into the peptidoglycan of P. mirabilis, Gmeiner and co-workers (Gmeiner and Kroll 1981; Gmeiner and Sarnow 1987) showed that only non-0-acetylated peptidoglycan subunits are incorporated into the growing polymer, and they are subsequently 0-acetylated following the crosslinking of peptide chains by transpeptidases. In their model (Fig. 2), newly inserted subunits are attached to "old" 0-acetylated subunits by peptide cross-linkage, and the new chain is extended by non-0-acetylated subunits. A second non-0-acetylated chain is assembled and linked to the previously incorporated chain. This implies that both 0-acetylated and non-0-acetylated subunits may serve as acceptors in the transpeptidation reactions. The new peptidoglycan region subsequently becomes 0-acetylated chain by chain. Employing similar experimental strategies, analogous results have been obtained by others with N. gonorrhoeae (Lear and Perkins 1983, 1986, 1987, Dougherty 1983a). The 0-acetylation of gonococcal peptidoglycan was also observed to be a slower process than crosslinking, indicating that subunits already incorporated into the pre-existing peptidoglycan must then undergo O-acetylation (Lear and Perkins 1983). However, rather than observing a time lag between incorporation and 0-acetylation of new peptidoglycan subunits as reported for P. mirabilis, the acetylation appeared to proceed immediately after their cross-linkage to the growing polymer. The presence of a significant degree of 0-acetylation after a very short time of labelling raised the possibility of a cytoplasmic or pretranslocated 0-acetylation of the subunits. Searches for lipid (bactoprenvl) - linked N-acetylglucosaminyl-N,O-diacetylmuramyl pentapeptide precursors in either the cytoplasm or the cytoplasmic membrane, however, proved futile (Lear and Perkins 1986). The short-term 0-acetylation characteristic of the gonococcal species was also observed in the Gram-positive S. aureus, and again no 0-acetylated peptidoglycan bactoprenyl intermediates could be detected (Snowden et al. 1989). Taken together, these observations imply that O-acetylation of peptidoglycan takes place outside the cytoplasmic membrane and that an acetyl transferase, in addition to an acetyl donor, must be present external to the cytoplasm. Indeed, 0-acetylation of peptidoglycan has been observed to continue in a cell-free biosynthetic system of P. mirabilis peptidoglycan (Martin 1984). The fact that the required enzyme(s) and cosubstrate are not lost in the preparation of the permeabilized cells suggests that the components remain firmly cell bound, presumably to the cytoplasmic membrane. Further details on both the biosynthesis and physiological role of peptidoglycan 0-acetylation have been obtained from experiments with various antibiotics. The bacteriostatic antibiotic chloramphenicol has been shown to enhance the degree of 6-0-acetylation of the peptidoglycan of both N. gonorrhoeae (Rosenthal et al. 1985) and S. aureus (Johannsen et al. 1983). Increases in 6-0-acetyl content from 46 to 70 and from 35 to above 85% were reported for N. gonorrhoeae and S. aureus, respectively. The chloramphenicol-induced increase in the degree of 6-0acetylation of the N. gonorrhoeae peptidoglycan was observed to immediately follow the cessation of protein synthesis and that subsequent acetylation was limited to newly incorporated peptidoglycan. Therefore, while the process of
0-acetylation occurs outside the cytoplasm, it would appear that it is regulated by agents that act directly on cytoplasmic targets. Of therapeutic concern, the increased 6-0-acetyl content of cell walls in bacteria treated with this antibiotic serves to increase its resistance to the lysosomal muramidases of the phagolysosomes (Ginsburg and Sela 1976) and thereby enhances the potential for the induction of rheumatic arthritis in mammalian joints. A sharp decline in the degree of peptidoglycan O-acetylation has been observed in N. gonorrhoeae (Blundell and Perkins 1981; Dougherty 1983b, 1985), S. aureus (Burghaus et al. 1983), and P. mirabilis (Martin and Gmeiner 1979) upon treatment with penicillin G. Comparison of the penicillin-binding protein (PBP) profiles of both wild-type (FA 19) and penicillin G sensitive (FA 19101 ( penA)) strains of N. gonorrhoeae with the extent of peptidoglycan 0-acetylation led Dougherty (1983b, 1985) to implicate PBP-2 as having a specific and possibly direct role in 0-acetylation. At levels below minimal inhibitory concentration (MIC), penicillin G binds PBP-2 and PBP-3 of the wild-type strain and causes a substantial decrease in the 0-acetyl modification of the peptidoglycan. With the strain PA 19101, however, penicillin G only binds PBP-3, and there is no apparent change in 0-acetyl content. High-affinity binding to PBP-2 was subsequently found to inhibit D,Dcarboxypeptidase activity, leading to increased pentapeptidecontaining subunits in the wall (Garcia-Bustos and Dougherty 1987). Since 0-acetylation is a postsynthetic modification of the peptidoglycan, it was proposed that pentapeptide subunits are a poor substrate for this reaction and, thus, the failure to process incorporated disaccharide pentapeptide to tetrapeptide via D,D-carboxypeptidase(presumably PBP-2) would indirectly inhibit further O-acetylation of the peptidoglycan. An analogous mechanism for the penicillin G inhibition of 0-acetylation in P. mirabilis was previously postulated by Martin and Gmeiner (1979). The P. mirabilis peptidoglycan sacculus was viewed as comprising areas of the polymer that differ in their composition and thought that penicillin G selectively inhibits the synthesis of only those areas that accommodate the 0-acetyl modification. In a more recent study, Labischinski and co-workers correlated the decrease in 0-acetylation of S. aureus peptidoglycan with the onset of penicillin G induced autolysis (Sidow et al. 1990). Changes in the extent of 0-acetylation were detected before the onset of autolysis, suggesting that for S. aureus, decreased 0-acetylation may be a prerequisite for bacteriolysis. Their observations on cross-linking and 0-acetylation of peptidoglycan under the influence of penicillin G also provided some insight into possible alterations of the portion of the peptidoglycan sacculus synthesized before addition of the antibiotic. Material synthesized before the addition of penicillin G was only marginally affected regarding its degree of cross-linking but strongly affected with respect to 0-acetylation. This is consistent with the interpretation of others regarding the peptidoglycan chemistry of P. mirabilis (Gmeiner and Kroll 1981) and N. gonorrhoeae (Lear and Perkins 1987) in that the 0-acetyl groups are attached after assembly of the peptidoglycan sacculus. Until recently, nothing specific was known about the pathway of peptidoglycan 0-acetylation other than it must be a postsynthetic or maturation event. A putative acetyl transferase(s) was assumed to exist, but even the source of
MINIREVIEW / MINISYNTH~?~E
acetate was unknown. Through both in vivo and in vitro labelling experiments with P. mirabilis, we have recently provided evidence to suggest that acetate is transferred from the N-2 position of either N-acetylglucosaminyl or N-acetylmuramyl residues to the C-6 hydroxyl group of the latter via an N--0 acetyl migration during peptidoglycan turnover (Dupont and Clarke 1991b, 1991c). Radioactive 0-linked acetate was recovered from the purified pe tidoglycan of cells grown in the presence of [acetyl- H]Nacetylglucosamine, whereas the inclusion of other potential sources of transferable acetate, such as pyruvate, acetylphosphate, or acetate, did not interfere with radiolabel transfer. No such transfer of radiolabel was observed in peptidoglycan isolated from either Escherichia coli incubated with the same compound or P. mirabilis grown with [l , 6 - 3 ~N-acetylglucosamine ] or [1- 1 4 ~ ] g l u c o s a m i n e (Dupont and Clarke 1991b). The specific transfer of radioactive acetate was also observed with a cell-free biosynthetic system of peptidoglycan when supplemented with UDP[acetyl-1- 14c]N-acetylglucosamine (Dupont and Clarke 1991c), providing further evidence for a N-- 0 transacetylation as the pathway for the 0-acetylation of peptidoglycan. The activity of the putative enzyme involved is thus thought to be similar to N-arylhydroxarnic acid N,O-acetyltransferase (AHAT) (EC 2.3.1.56), which transfers the N-acetyl group of some aromatic acethydroxamates to the 0-position of some aromatic hydroxylamines. AHAT has recently been proven to exist as a unique and independent enzyme in a variety of mammalian tissues (Smi,th and Hanna 1986). In view of the absence of readily available activated acetate (e.g., acetyl CoA or acetyl phosphate) in the milieu external to the cytoplasm, it is conceivable that P. mirabilis has developed a method for utilizing the conserved bond energies stored within the peptidoglycan sacculus to achieve 0-acetylation in a manner analogous to transpeptidation. The fact that free amino sugars are not detected in the peptidoglycan sacculus but have been observed in the spent culture medium as turnover products (Dupont and Clarke 1991b) indicates that the process of peptidoglycan turnover and 0-acetylation could be associated. Although there is a dearth of information concerning the details of peptidoglycan turnover in P. mirabilis, extensive studies have been pursued with E. coli and members of the genus Bacillus (for a recent review see Doyle et al., 1989). With E. coli, components of -the peptidoglycan polymer, including oligopeptides which comprise -thepeptide moiety of ,the muramyl side chain, are recovered by the cell for reutilization after their hydrolysis from the sacculus (Goodell and Higgins 1987). This observation of recycling further lends support to the hypothesis of N - 0 acetyl transfer during peptidoglycan turnover. That both peptidoglycan autolytic (Blundell and Perkins 1985; Chapman and Perkins 1983) and N - 0 transacetylation activities are retained in a cell-free system led to the speculation that, by analogy with some of the penicillin-binding proteins of E. coli (for a review see Waxman and Strominger, 1983), the two activities maybe catalyzed by a single bifunctional enzyme (Dupont 1991). However, much further intensive investigations are required to bear out ,this postulate. The phenomenon of N- 0 acyl transfer has been widely investigated since 1924, when Bergman and Mickeley demonstrated the migration of acyl groups from N-acylamino alcohol to form an 0-acyl analog under the influence of
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anhydrous acidic reagents (Bergman and Mickeley 1924). It was shown by White (White 1938) and later by Fodor and 0tvos (Fodor and 0tvos 1956) that the N-acetyl group of N-acetylglucosamine derivatives easily migrates under the influence of acid to the oxygen atom at C-3. Hence, it appears feasible that N--0 acetyl transfer may occur in peptidoglycan, especially if it is intimately coupled to the release of glucosamine during the process of turnover. Acknowledgements These studies were supported by operating grants to A.J.C. from both the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Medical Research Council. C.D. was a recipient of an NSERC postgraduate scholarship. Abrams, A. 1958.0-Acetyl groups in the cell wall of Streptococcus faecalis. J. Biol. Chem. 230: 949-959. Bergman, M., and Mickeley, A. 1924. Rearrangements of peptidelike substances. 111. Derivatives of dl-serine. Z. Physiol. Chem. 140: 128-145. Blake, C.C.F., Johnson, L.N., Mair, G.A., et al. 1967. Crystallographic studies of the activity of hen egg-white lysozyme. Proc. R. Soc. London, Ser. B, 167: 378-388. Blundell, J.K., and Perkins, H.R. 1981. Effects of P-lactam antibiotics on peptidoglycan synthesis in growing Neisseria gonorrhoeae, including changes in the degree of 0-acetylation. J. Bacteriol. 147: 633-641. Blundell, J.K., and Perkins, H.R. 1985. Selectivity for 0-acetylated peptidoglycan during endopeptidase action by permeabilized Neisseria gonorrhoeae. FEMS Microbiol. Lett. 30: 67-69. Blundell, J.K., Smith, G.J., and Perkins, H.R. 1980. The peptidoglycan of Neisseria gonorrhoeae: 0-acetyl groups and lysozyme sensitivity. FEMS Microbiol. Lett. 9: 259-261. Brumfitt, W. 1959. The mechanism of development of resistance to lysozyme by some Gram-positive bacteria and its results. Br. J . Exp. Pathol. 40: 441-451. Brumfitt, W., Wardlaw, A.C., and Park, J.T. 1958. Development of lysozyme resistance in Micrococcus lysodeikticus and its association with an increased 0-acetyl content of the cell wall. Nature (London), 181: 1783-1784. Burghaus, P., Johannsen, L., Naumann, D., et al. 1983. The influence of different antibiotics on the degree of 0-acetylation of staphylococcal cell walls. In The target of penicillin. The murein sacculus of bacterial cell walls, architecture and growth. Edited by R. Hakenbeck, J. Holtje, and H. Labischinski. Walter de Gruyter and Co., New York. pp. 317-322. Chapman, S.J., and Perkins, H.R. 1983. Peptidoglycan degrading enzymes in ether-treated cells of Neisseria gonorrhoeae. J . Gen. Microbiol. 129: 877-883. Chedid, L., Audibert, F., and Johnson, A.G. 1978. Biological activities of muramyl dipeptide, a synthetic glycopeptide analogous to bacterial immunoregulating agents. Prog. Allergy, 25: 63-105. Coyette, J., and Ghuysen, J.M. 1970. Structure of the walls of Lactobacillus acidophilus strain 63 AM Gasser. Biochemistry, 9: 2935-2943. Cromartie, W. J . 198 1. Arthropathic properties of peptidoglycanpolysaccharide complexes of microbial origin. In Arthritis models and mechanisms. Edited by H. Deicker and L.C. Schultz. Sprinter-Verlag, New York. pp. 24-38. Dougherty, T. J. 1983a. Synthesis and modification of the peptidoglycan in Neisseria gonorrhoeae. FEMS Microbiol. Lett. 17: 51-53.
Dougherty, T. J . 1983b. Peptidoglycan synthesis in Neisseria gonorrhoeae strains sensitive and intrinsically resistant to P-lactam antibiotics. J. Bacteriol. 153: 429-435.
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Dougherty, T.J. 1985. Involvement of a change in penicillin target and peptidoglycan structure in low-level resistance to /3-lactam antibiotics in Neisseria gonorrhoeae. Antimicrob. Agents Chemother. 28: 90-95. Doyle, R. J., Chaloupka, J., and Vinter, V. 1989. Turnover of cell walls in microorganisms. Microbiol. Rev. 52: 554-567. Dupont, C. 1991. Characterization of peptidoglycan 0-acetylation in Proteus mirabilis. Ph.D. thesis, University of Guelph, Guelph, Ont. Dupont, C., and Clarke, A.J. 1991a. Dependence of lysozymecatalysed solubilization of Proteus mirabilis peptidoglycan on the extent of 0-acetylation. Eur. J. Biochem. 195: 763-769. Dupont, C., and Clarke, A. J. 1991b. Evidence for N - 0 acetyl migration as the mechanism for the 0 acetylation of peptidoglycan in Proteus mirabilis. J. Bacteriol. 173: 43 18-4324. Dupont, C., and Clarke, A.J. 1991~.In vitro synthesis and 0 acetylation of peptidoglycan by permeabilized cells of Proteus mirabilis. J. Bacteriol. 173: 4618-4624. Esser, R.E., Stimpson, S.A., Cromartie, W.J., and Schwab, J.H. 1985. Reactivation of streptococcal cell wall-induced arthritis by homologous and heterologous cell wall polymers. Arthritis Rheum. 28: 1402-1411. Fleck, J., Mock, M., Minck, R., and Ghuysen, J.-M. 1971. The cell envelope of Proteus vulgaris P18. Isolation and characterization of the peptidoglycan component. Biochim. Biophys. Acta, 233: 489-503. Fleming, T.J., Wallsmith, D.E., and Rosenthal, R.S. 1986. Arthropathic properties of gonococcal peptidoglycan fragments: implications for the pathogenesis of disseminated gonococcal disease. Infect. Immun. 52: 600-608. Fodor, G., and ~ t v o s L. , 1956. Die Raumstruktur der Aminodeoxyzucker. 11. Mitteil: Die Konstellation des D-Glucoamins. Chem. Ber. 89: 701 -708. Fox, A., Brown, R.R., Anderle, S.K., et al. 1982. Arthropathic properties related to the molecular weight of peptidoglycanpolysaccharide polymers of streptococcal cell walls. Infect. Immun. 35: 1003-1010. Garcia-Bustos, J.F., and Dougherty, T. J. 1987. Alterations in peptidoglycan of Neisseria gonorrhoeae induced by sub-MICs of /3-lactam antibiotics. Antimicrob. Agents Chemother. 31: 178-182. Ghuysen, J.M., and Strominger, J.L. 1963. Structure of the cell wall of Staphylococcus aureus strain Copenhagen. 11. Separation and structure of disaccharides. Biochemistry, 2: 1 1 19-1 125. Ginsburg, I., and Sela, M.N. 1976. The role of leukocytes and their hydrolases in the persistence, degradation and transport of bacterial constituents in tissues: relation to chronic inflammatory processes in staphylococcal, streptococcal and mycobacterial infections and in chronic periodontal disease. CRC Crit. Rev. Microbiol. 4: 249-332. Glauner, B., Holjte, J.V., and Schwarz, U. 1988. The composition of the murein of Escherichia coli. J. Biol. Chem. 263: 10 088 - 10 095. Gmeiner, J., and Kroll, H.P. 1981. Murein biosynthesis and 0-acetylation of N-acetylmuramic acid during the cell division cycle of Proteus mirabilis. Eur. J. Biochem. 177: 171-177. Gmeiner, J., and Sarnow, E. 1987. Murein biosynthesis in synchronized cells of Proteus mirabilis. Quantitative analysis of 0-acetylated murein subunits and of chain terminators incorporated into the sacculus during the cell cycle. Eur. J. Biochem. 163: 389-395. Goodell, E.W., and Higgins, C.F. 1987. Uptake of cell wall peptide by Salmonella typhimurium and Escherichia coli. J. Bacteriol. 169: 3861-3865. Hamada, S., Torii, M., Kotani, S., et al. 1978. Lysis of Streptococcus mutans cells with mutanolysin, a lytic enzyme prepared from a culture liquor of Streptomyces globisporous 1829. Arch. Oral Biol. 23: 543-549. Hamerman, D. 1966. New thoughts on the pathogenesis of
rheumatoid arthritis. Am. J. Med. 40: 1-9. Hash, J.H., and Rothlauf, M.V. 1%7. The N,O-diacetylrnuramidase of Chalaropsis species. 1. Purification and crystallization. J. Biol. Chem. 242: 5586-5590. Imoto, T., Johnson, L.N., North, A.T.C., et al. 1972. Vertebrate lysozymes. In The enzymes. Vol. 7. 3rd ed. Edited by P.D. Boyer. pp. 666-868. Janusz, M.J., Chetty, C., Eisenberg, R.A., et al. 1984. Treatment of experimental erosive arthritis in rats by injection of the muralytic enzyme mutanolysin. J. Exp. Med. 160: 1360-1 374. Janusz, M.J., Esser, R.E., and Schwab, J.H. 1986. In vivo degradation of bacterial cell wall by the muralytic enzyme mutanolysin. Infect. Immun. 52: 459-467. Johannsen, L., Labischinski, H., Burghaus, P., and Giesbrecht, P . 1983. Acetylation in different phases of growth of staphylococci and their relation to cell wall degradability by lysozyme. In The target of penicillin. International FEMS Symposium on the Murein Sacculus of Bacterial Cell Walls. Edited by R. Hakenbeck, J . Holtje, and H . Labischinski. Walter de Gruyter and Co., Berlin. pp. 261-266. Johannsen, L., Rosenthal, R.S., Martin, S.A., et al. 1989. Somnogenic activity of 0-acetylated and dimeric muramyl peptides. Infect. Immun. 57: 2726-2732. Johannsen, L., Toth, L.A., Rosenthal, R.S., et al. 1990. Sommnogenic, pyrogenic and hematologic effects of bacterial peptidoglycan. Am. J. Physiol. 259: R182-R186. Kawasaki, A., Takada, H., Kotani, S., et al. 1987. Activation of the human complement cascade by bacterial cell walls, peptidoglycans, water-soluble peptidoglycan components, and synthetic muramylpeptides. Studies on active components and structural requirements. Microbiol. Immunol. 31: 554-569. Koga, T., and Pearson, C.M. 1976. Induction of polyarthritis in the rat with a water-soluble component of Mycobacterium tuberculosis wax D and with various bacterial cell walls or their watersoluble components. In Rheumatic fever and rheumatoid arthritis. Edited by Y. Otaka. Igaku Shoin Ltd., Tokyo. pp. 242-259. Koga, T., Hirofuji, T., Kotani, S., et a/. 1985. Acute joint inflammation in mice after systemic injections of the cell wall, its peptidoglycan, and chemically defined peptidoglycan subunits from various bacteria. Infect. Immun. 50: 27-34. Labischinski, H., Goodell, E. W., Goodell, A., and Hochberg, M.L. 1991. Direct proof of a "more-than-single-layered" peptidoglycan architecture of Escherichia coli W7: a neutron small-angle scattering study. J . Bacteriol. 173: 75 1-756. Lear, A.L., and Perkins, H.R. 1983. Degrees of 0-acetylation and cross-linking of the peptidoglycan of Neisseria gonorrhoeae during growth. J. Gen. Microbiol. 129: 885-888. Lear, A.L., and Perkins, H.R. 1986. 0-Acetylation of peptidoglycan in Neisseria gonorrhoeae. Investigation of lipid-linked intermediates and glycan chains newly incorporated into the cell wall. J. Gen. Microbiol. 132: 2413-2420. Lear, A.L., and Perkins, H.R. 1987. Progress of 0-acetylation and cross-linking of peptidoglycan in Neisseria gonorrhoeae grown in the presence of penicillin. J . Gen. Microbiol. 133: 1743-1750. Logardt, I.M., and Neujahr, H.Y. 1975. Lysis of modified walls from Lactobacillus fermentum. J. Bacteriol. 124: 73-77. Martin, H.H. 1984. In vitro synthesis of peptidoglycan by spheroplasts of Proteus mirabilis grown in the presence of penicillin. Arch. Microbiol. 139: 371 -375. Martin, H.H., and Gmeiner, J. 1979. Modification of peptidoglycan structure by penicillin-action in cell walls of Proteus mirabilis. Eur. J. Biochem. 95: 487-495. Martin, J.P., Fleck, J., Mock, M., and Ghuysen, J.M. 1973. The wall peptidoglycans of Neisseria perflava, Maraxella glucidolytica, Pseudomonas alcaligenes and Proteus vulgaris strain P18. Eur. J . Biochem. 38: 301-306. Masek, K., Raskova, H., and Rotta, J. 1968. The mechanism of
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the pyrogenic effect of streptococcus well wall mucopeptide. J . Physiol. (London), 198: 345-353. Park, J.T. 1987. Murein synthesis. In Escherichia coli and Salmonella typhimurium: cellular and molecular biology. Vol. 1 . Edited by F.C. Neidhart. American Society for Microbiology, Washington DC. pp. 663-671. Rosenthal, R.S., and Fleming, T. J. 1985. Arthropathic properties of gonococcal peptidoglycan. In The pathogenic neisseria. Edited by G. Schoolnik. American Society for Microbiology, Washington, DC. pp. 352-359. Rosenthal, R.S., Blundell, J.K., and Perkins, H.R. 1982. Strainrelated differences in lysozyme sensitivity and extent of O-acetylation of gonococcal peptidoglycan. Infect. Immun. 37: 826-829. Rosenthal, R.S., Folkening, W. J., Miller, D.R., and Swim, S.C. 1983. Resistance of 0-acetylated gonococcal peptidoglycan to human peptidoglycan-degrading enzymes. Infect. Immun. 40: 826-829. Rosenthal, R.S., Gfell, M.A., and Folkening, W.J. 1985. Influence of protein synthesis inhibitors on regulation of extent of 0-acetylation of gonococcal peptidoglycan. Infect. Immun. 49: 7- 13. Seidl, P.H., and Schleifer, K.H. (Editors). 1985. Biological properties of peptidoglycan. Walter de Gruyter, New York. Shockman, G.D., and Barrett, J.F. 1983. Structure, function and assembly of cell walls of gram-positive bacteria. Annu. Rev. Microbiol. 37: 501 -527. Sidow, T., Johannsen, L., and Labischinski, H. 1990. Penicillininduced changes in the cell wall composition of Staphylococcus aureus before the onset of bacteriolysis. Arch. Microbiol. 154: 73-8 1 .
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Smith, T. J., and Hanna, P.E. 1986. N-Acetyltransferase multiplicity and the bioactivation of N-arylhydroxamic acids by hamster hepatic and intestinal enzymes. Carcinogenesis, 7: 697-702. Snowden, M.A., and Perkins, H.R. 1990. Peptidoglycan crosslinking in Staphylococcus aureus. An apparent random polymerization process. Eur. J. Biochem. 191: 373-377. Snowden, M.A., Perkins, H.R., Wyke, A.W., et al. 1989. Crosslinking and 0-acetylation of newly synthesized peptidoglycan in Staphylococcus aureus H. J. Gen. Microbiol. 135: 301 5-3022. Stimpson, S.A., Brown, R.R., Anderle, S.K., et al. 1986. Arthropathic properties of cell wall polymers from normal flora bacteria. Infect. Immun. 51: 240-249. Swim, S.G., Gfell, M.A., Wilde, C.E., 111, and Rosenthal, R.S. 1983. Strain distribution in extents of lysozyme resistance and 0-acetylation of gonococcal peptidoglycan determined by highperformance liquid chromatography. Infect. Immun. 42: 446-452. Verbrugh, H.A., van Dijk, W.C., Peters, R., et al. 1980. Opsonic recognition of staphylococci mediated by cell wall peptidoglycan: antibody-independent activation of human complement and opsonic activity of peptidoglycan antibodies. J . Immunol. 124: 1167-1 173. Waxman, D. J., and Strominger, J .L. 1983. Penicillin-binding proteins and the mechanism of action of /3-lactam antibiotics. Annu. Rev. Biochem. 52: 825-869. White, T. 1938. Amino sugars. I. A case of acyl migration. J. Chem. Soc. 1938: 1498-1500.
0-Acetylated peptidoglycan: its occurrence, pathobiological significance, and biosynthesis
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by Université Laval Bibliotheque on 07/15/14 For personal use only.
ANTHONY J.
CLARKE' AND
CLAUDED U P O N T ~
Guelph- Waterloo Centre for Graduate Work in Chemistry, Department of Microbiology, University of Guelph, Guelph, Ont., Canada N I G 2 WI Received July 22, 1991 Revision received October 9, 1991 Accepted October 15, 1991 CLARKE, A. J., and DUPONT,C. 1992. 0-Acetylated peptidoglycan: its occurrence, pathobiological significance, and biosynthesis. Can. J . Microbiol. 38: 85-91. Bacterial cell walls and their structural units, particularly peptidoglycan, induce a vast variety of biological effects in host organisms. The pathobiological effects of peptidoglycan are greatly enhanced by various modifications and substitutions to its basic composition and structure. One such modification is the presence of acetyl moieties at the C-6 hydroxyl group of N-acetylmuramyl residues, and to date, 11 species of eubacteria, including some important human pathogens, such as Neisseria gonorrhoeae, Proteus mirabilis, and Staphylococcus aureus, are known to possess 0-acetylated peptidoglycan. This review addresses the influence of 0-acetylation of peptidoglycan on its resistance to degradation both in vitro and in vivo, the clinical importance of the modification, and the currently held views on the pathway for its biosynthesis. Key words: peptidoglycan, murein, 0-acetylation, lysozyme, arthritis. CLARKE,A. J., et DUPONT,C. 1992. 0-Acetylated peptidoglycan: its occurrence, pathobiological significance, and biosynthesis. Can. J . Microbiol. 38 : 85-91. Les parois cellulaires bacteriennes et leurs unites structurales, notamment le peptidoglycan, induisent une grande variete d'effets biologiques sur les organismes hates. Les effets pathobiologiques du peptidoglycan sont accentues grandement par les modifications et les substitutions variees de sa composition de base et de sa structure. La presence de radicaux acetyles a la position du groupe hydroxyl en C-6 des residus N-acetylmuramyles constitue l'une de ces modifications. Jusqu'a present, 11 espkces d'eubacteries, incluant quelques pathogknes humains importants comme Neisseria gonorrhoeae, Proteus mirabilis et Staphylococcus aureus, posskdent un peptidoglycan 0-acetyle. Cette revue traite de l'influence de ce type d'acetylation du peptidoglycan sur sa resistance a la degradation in vitro et in vivo, de l'importance clinique de la modification ainsi que des opinions qui ont cours actuellement concernant le sentier de sa biosynthkse. Mots elks : peptidoglycan, mureine, 0-acetylation, lysozyme, arthrite. [Traduit par la redaction]
Introduction Peptidoglycan in a heteropolymer of distinctive composition and structure and is uniquely associated with prokaryotic cell walls. Its chemistry and biosynthesis have been reviewed (Park 1987; Shockman and Barrett 1983). Basically, it is composed of 0-1,4-linked N-acetylglucosamine and N-acetylmuramic acid residues, the latter being modified by a tetrapeptide composed of both L and D amino acids. In most cases, these peptide side chains are involved in the direct cross-linking between adjacent glycan strands, but in some instances more complicated arrangements exist. For example, a pentaglycine peptide bridges the neighboring peptide side chains within the peptidoglycan sacculus of Staphylococcus aureus. In Gram-positive bacteria, the highly cross-linked peptidoglycan strands are arranged in a thick .three-dimensionalconcentric array (Snowden and Perkins 1990), whereas those of Gram-negative bacteria are probably no more than three layers deep (Glauner et al. 1988; Labischinski et al. 1991). The continuous peptidoglycan layer that covers the inner (cytoplasmic) membrane provides shape and rigidity to ' ~ u t h o rto whom all correspondence should be addressed. 2 ~ r e s e n taddress: Department of Chemistry, Carlsberg Laboratorium, Gamle Carlsbergvej 10 Valby, Copenhagen, DK2500, Denmark. Printed in Canada / Irnprirne au Canada
bacterial cells and, more importantly, contains the turgor pressure exerted by the cytoplasm. Peptidoglycan composition and structure are now known to be complex, as exemplified in a recent study by Glauner et al. (1988). From a digest of Escherichia coli peptidoglycan, 30 different types of muropeptides were isolated by HPLC and characterized. The diversity of structures is due to the free combination of seven different types of side chains with two types of cross-bridges between the peptides of adjacent strands. In this review, we discuss the effect and biosynthesis of one clinically significant modification to peptidoglycan, that of the 6-0-acetylation of muramyl residues.
Biological properties of peptidoglycan Following the lysis of invading microorganisms, mammalian tissues clear cellular debris, including peptidoglycan, very rapidly through the action of the hydrolytic enzymes of either phagocytic cells or serum. However, in some instances, high molecular weight fragments of peptidoglycan have been observed to persist and circulate in the host organism. These large peptidoglycan fragments have been shown to induce diverse pathobiological and pathophysiological effects, many of which have been recently reviewed (Seidl and Schleifer 1985). Some of these include the induction of slow-wave sleep (Johannsen et al. 1989, 1990), com-
CAN. J. MICROBIOL. VOL. 38, 1992
TABLE1 . Bacteria reported to possess peptidoglycan containing 6-0-acetylmuramyl residues Species
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Gram positive Streptococcus faecalis Micrococcus luteus Staphylococcus aureus Lactobacillus acidophilus Lactobacillus fermentum Gram negative Moraxella glucidolytica Pseudomonas alcaligenes Neisseria perflava Neisseria gonorrhoeae
Proteus vulgaris Proteus mirabilis
Strain
% 0-acetyl
Reference
ATCC 9790 nr Copenhagen SG5 11Berlin H(NC1B 6571) 63 AM Gasser ATCC 9338
Abrams 1958 Brumfitt et al. 1958 Ghuysen and Strominger 1963 Burghaus et al. 1983 Snowden et al. 1989 Coyette and Ghuysen 1970 Logardt and Neujahr 1975
nr nr nr I 1260 FA19 FA 140 FA171 FA136 F62 FA 102 CL1 BR87 1342 7502 2686 7405 CS7 609 624 8035 8038 129 1 JW3 1 P18 19 19 Perkins Perkins GB8 ATCC 7002 ATCC 29245 ATCC 33583 ATCC 4307 1 ATCC 25933 ATCC 33659 ATCC 12453 ATCC 14273 TGH 9041 TGH 7341 F 16 F49 1
Martin et al. 1973 Martin et al. 1973 Martin et al. 1973 Blundell et al. 1980 Blundell et al. 1980 Swim et al. 1983 Swim et al. 1983 Swim et al. 1983 Swim et al. 1983 Swim et al. 1983 Swim et al. 1983 Swim et al. 1983 Swim et al. 1983 Swim et al. 1983 Swim et al. 1983 Swim et al. 1983 Swim et al. 1983 Swim et al. 1983 Swim et al. 1983 Swim et al. 1983 Swim et al. 1983 Swim et al. 1983 Swim et al. 1983 Fleck et al. 197 1 Martin and Gmeiner 1979 Dupont and Clarke 1991a Blundell and Perkins 198 1 Dupont and Clarke 1991a Dupont and Clarke 1991a Dupont and Clarke 1991a Dupont and Clarke 1991a Dupont and Clarke 1991a Dupont and Clarke 1991a Dupont and Clarke 1991a Dupont and Clarke 1991a Dupont and Clarke 1991a Dupont and Clarke 1991a Dupont and Clarke 1991a Dupont and Clarke 1991a Dupont and Clarke 1991a Dupont and Clarke 1991a
NOTE:nr, not recorded.
plement activation (Kawasaki et al. 1987; Verbrugh et al. 1980), pyrogenicity (Johannsen et al. 1990; Masek et al. 1968), and arthritogenicity (Cromartie 1981). As early as 1966, Hamerman proposed that oligosaccharide or glycopeptide fragments derived from bacterial cell walls or capsules may be the inciting agents for rheumatoid arthritis in mankind (Hamerman 1966). Although extrapolation of recent findings to mankind is still premature, their is a plethora of data in the literature to indicate that such occurs in animal models (Chedid et al. 1978; Cromartie 1981; Esser et al. 1985; Fleming et al. 1986; Fox et al. 1982;
Koga et al. 1985; Koga and Pearson 1976; Ginsburg and Sela 1976; Rosenthal and Fleming 1985; Stimpson et al. 1986). That high molecular weight fragments of peptidoglycan are required to induce arthritic conditions was clearly demonstrated in studies by Fox et al. (1982) and Janusz et al. (1984, 1986). Purified peptidoglycan digested with mutanolysin (a muramidase isolated from Streptomyces globisporus) was shown to lose its arthropathic potential. In a separate experiment, a single intravenous injection of mutanolysin into peptidoglycan-induced arthritic rats prevented the development of recurrent, chronic erosive arthritis. In vivo studies
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0 II
o \*
Stage 1
l o
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NHCCH, II 0
0 NHCCH, CH3CH II 0 C=O beptide
Stage 2
FIG. 1. Structure of the glycan portion of the repeating subunit of 0-acetylated peptidoglycan. with the pathogens Staphylococcus aureus and Neisseria gonorrhoeae have indicated that the phenomenon of peptidoglycan persistence in a host is directly attributable, in-these instances, to the presence of numerous 0-acetyl substituents on the glycan backbone (Blundell et al. 1980; Fleming et al. 1986; Rosenthal et al. 1982; Rosenthal and Fleming 1985; Swim et al. 1983).
6-0-Acetylated peptidoglycan 0-Acetylated peptidoglycan was first observed independently in Steptococcus faecalis by Abrams (1958) and in Micrococcus luteus by Brumfitt and co-workers (Brumfitt et al. 1958) 30 years ago. Since then, 0-acetylated peptidoglycan has been observed in a total of 47 strains of 11 species of both Gram-positive and Gram-negative bacteria, including some important pathogens (Table 1). It is quite conceivable that many other bacteria possess 0-acetylated peptidoglycan but have escaped detection as a result of the base lability of the modification. 0-Acetylation of peptidoglycan occurs at the C-6 hydroxyl group of N-acetylmuramyl residues producing the corresponding 2,6-diacetylmuramyl derivative (Fig. 1). The extent of naturally occurring 0-acetylation in microorganisms ranges between < 10 and 70%, while a spontaneous mutant of Micrococcus luteus cultured in the presence of hen egg-white lysozyme was reported to have a molar ratio of N-acetylmuramic acid : 0acetyl of 1:l (Brumfitt et al. 1958). Despite intensive investigations by a number of groups, the physiological function of this modification to peptidoglycan as it pertains to .the bacterium remains unclear. However, the role of 0-acetylation in conferring resistance to the hydrolytic activity of hen egg-white lysozyme was discerned soon after the initial discovery of the modification (Brumfitt 1959). Subsequent studies have confirmed the resistance of most muramidases to 0-acetylated peptidoglycan, but it should be noted that the efficacy of others, such as the N,Odiacetylmuramidases of Chalaropsis (Hash and Rothlauf 1967) and Streptomyces globisporus (mutanolysin) (Hamada et al. 1978), is not affected by 0-acetyl groups. As with other types of modifications to peptidoglycan, such as the presence of teichoic acids at the C-6 atom of murarnyl residues of Gram-positive bacteria, the 0-acetylation of the peptidoglycan of a pathogenic microorganism thus provides protection from the hydrolytic action of host muramidases. Correlations have been made between the strain-related differences in muramidase sensitivity and the extent of 0-acetylation of gonococcal peptidoglycan for both the hen egg-white (Blundell et al. 1980; Rosenthal et al.
-0l , -0 -0 -0 - q - f, A
-~-o-o-o-o~o Stage 3
-0-0-0-0 A
-fi-o-o-fio - ~ - ~ stage~ 4 -
FIG. 2. Proposed scheme of peptidoglycan biosynthesis in P. mirabilis. , pre-existing ("old") 0-acetylated subunits; 0, newly incorporated 0-acetylated subunits; , newly incorporated non-0-acetylated subunits; -, 0-1,4 glycosidic linkages between subunits; A , peptide cross-links between glycan strands. (Adapted from Gmeiner and Kroll, 1981.) 1982; Swim et al. 1983) and human sera and polymorphonuclear (Rosenthal et al. 1983) enzymes. The extent of muramidase resistance to the solubilization of peptidoglycan by both human and hen egg-white enzymes was also shown to be dependent upon the degree of 0-acetylation in 14 strains of Proteus mirabilis (Dupont and Clarke 1991a). In the latter case, an increase in the overall change in the standard Gibbs free energy of activation (A(AG)) as a consequence of increasing 0-acetylation was observed. This was shown to result from the weaker affinity of the enzyme for the modified substrate, presumably through steric hinderance since the C-6 hydroxyl moieties of the substrate directly participate in its binding to the active-site cleft of the enzyme (Blake et al. 1967; Imoto et al. 1972). The consequences of this phenomenon are thus considerable in view of the detrimental effects exerted by circulating peptidoglycan fragments in a mammalian host organism.
Biosynthesis of 6-0-acetyl peptidoglycan Whereas its role in resistance to muramidases and the consequences of such have been well documented, very little is known concerning the biosynthetic sequence of reactions leading to the assembly and 0-acetylation of peptidoglycan. There is substantial evidence to suggest that 0-acetylation occurs after nascent peptidoglycan strands have been both attached and cross-linked to the pre-existing sacculus (Dougherty 1983a; Gmeiner and Kroll 1981; Gmeiner and Sarnow 1987; Lear and Perkins 1983, 1986, 1987; Snowden
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CAN. J. MICROBIOL. VOL. 38, 1992
et al. 1989). By pulse-chasing radiolabelled N-acetylglucosamine into the peptidoglycan of P. mirabilis, Gmeiner and co-workers (Gmeiner and Kroll 1981; Gmeiner and Sarnow 1987) showed that only non-0-acetylated peptidoglycan subunits are incorporated into the growing polymer, and they are subsequently 0-acetylated following the crosslinking of peptide chains by transpeptidases. In their model (Fig. 2), newly inserted subunits are attached to "old" 0-acetylated subunits by peptide cross-linkage, and the new chain is extended by non-0-acetylated subunits. A second non-0-acetylated chain is assembled and linked to the previously incorporated chain. This implies that both 0-acetylated and non-0-acetylated subunits may serve as acceptors in the transpeptidation reactions. The new peptidoglycan region subsequently becomes 0-acetylated chain by chain. Employing similar experimental strategies, analogous results have been obtained by others with N. gonorrhoeae (Lear and Perkins 1983, 1986, 1987, Dougherty 1983a). The 0-acetylation of gonococcal peptidoglycan was also observed to be a slower process than crosslinking, indicating that subunits already incorporated into the pre-existing peptidoglycan must then undergo O-acetylation (Lear and Perkins 1983). However, rather than observing a time lag between incorporation and 0-acetylation of new peptidoglycan subunits as reported for P. mirabilis, the acetylation appeared to proceed immediately after their cross-linkage to the growing polymer. The presence of a significant degree of 0-acetylation after a very short time of labelling raised the possibility of a cytoplasmic or pretranslocated 0-acetylation of the subunits. Searches for lipid (bactoprenvl) - linked N-acetylglucosaminyl-N,O-diacetylmuramyl pentapeptide precursors in either the cytoplasm or the cytoplasmic membrane, however, proved futile (Lear and Perkins 1986). The short-term 0-acetylation characteristic of the gonococcal species was also observed in the Gram-positive S. aureus, and again no 0-acetylated peptidoglycan bactoprenyl intermediates could be detected (Snowden et al. 1989). Taken together, these observations imply that O-acetylation of peptidoglycan takes place outside the cytoplasmic membrane and that an acetyl transferase, in addition to an acetyl donor, must be present external to the cytoplasm. Indeed, 0-acetylation of peptidoglycan has been observed to continue in a cell-free biosynthetic system of P. mirabilis peptidoglycan (Martin 1984). The fact that the required enzyme(s) and cosubstrate are not lost in the preparation of the permeabilized cells suggests that the components remain firmly cell bound, presumably to the cytoplasmic membrane. Further details on both the biosynthesis and physiological role of peptidoglycan 0-acetylation have been obtained from experiments with various antibiotics. The bacteriostatic antibiotic chloramphenicol has been shown to enhance the degree of 6-0-acetylation of the peptidoglycan of both N. gonorrhoeae (Rosenthal et al. 1985) and S. aureus (Johannsen et al. 1983). Increases in 6-0-acetyl content from 46 to 70 and from 35 to above 85% were reported for N. gonorrhoeae and S. aureus, respectively. The chloramphenicol-induced increase in the degree of 6-0acetylation of the N. gonorrhoeae peptidoglycan was observed to immediately follow the cessation of protein synthesis and that subsequent acetylation was limited to newly incorporated peptidoglycan. Therefore, while the process of
0-acetylation occurs outside the cytoplasm, it would appear that it is regulated by agents that act directly on cytoplasmic targets. Of therapeutic concern, the increased 6-0-acetyl content of cell walls in bacteria treated with this antibiotic serves to increase its resistance to the lysosomal muramidases of the phagolysosomes (Ginsburg and Sela 1976) and thereby enhances the potential for the induction of rheumatic arthritis in mammalian joints. A sharp decline in the degree of peptidoglycan O-acetylation has been observed in N. gonorrhoeae (Blundell and Perkins 1981; Dougherty 1983b, 1985), S. aureus (Burghaus et al. 1983), and P. mirabilis (Martin and Gmeiner 1979) upon treatment with penicillin G. Comparison of the penicillin-binding protein (PBP) profiles of both wild-type (FA 19) and penicillin G sensitive (FA 19101 ( penA)) strains of N. gonorrhoeae with the extent of peptidoglycan 0-acetylation led Dougherty (1983b, 1985) to implicate PBP-2 as having a specific and possibly direct role in 0-acetylation. At levels below minimal inhibitory concentration (MIC), penicillin G binds PBP-2 and PBP-3 of the wild-type strain and causes a substantial decrease in the 0-acetyl modification of the peptidoglycan. With the strain PA 19101, however, penicillin G only binds PBP-3, and there is no apparent change in 0-acetyl content. High-affinity binding to PBP-2 was subsequently found to inhibit D,Dcarboxypeptidase activity, leading to increased pentapeptidecontaining subunits in the wall (Garcia-Bustos and Dougherty 1987). Since 0-acetylation is a postsynthetic modification of the peptidoglycan, it was proposed that pentapeptide subunits are a poor substrate for this reaction and, thus, the failure to process incorporated disaccharide pentapeptide to tetrapeptide via D,D-carboxypeptidase(presumably PBP-2) would indirectly inhibit further O-acetylation of the peptidoglycan. An analogous mechanism for the penicillin G inhibition of 0-acetylation in P. mirabilis was previously postulated by Martin and Gmeiner (1979). The P. mirabilis peptidoglycan sacculus was viewed as comprising areas of the polymer that differ in their composition and thought that penicillin G selectively inhibits the synthesis of only those areas that accommodate the 0-acetyl modification. In a more recent study, Labischinski and co-workers correlated the decrease in 0-acetylation of S. aureus peptidoglycan with the onset of penicillin G induced autolysis (Sidow et al. 1990). Changes in the extent of 0-acetylation were detected before the onset of autolysis, suggesting that for S. aureus, decreased 0-acetylation may be a prerequisite for bacteriolysis. Their observations on cross-linking and 0-acetylation of peptidoglycan under the influence of penicillin G also provided some insight into possible alterations of the portion of the peptidoglycan sacculus synthesized before addition of the antibiotic. Material synthesized before the addition of penicillin G was only marginally affected regarding its degree of cross-linking but strongly affected with respect to 0-acetylation. This is consistent with the interpretation of others regarding the peptidoglycan chemistry of P. mirabilis (Gmeiner and Kroll 1981) and N. gonorrhoeae (Lear and Perkins 1987) in that the 0-acetyl groups are attached after assembly of the peptidoglycan sacculus. Until recently, nothing specific was known about the pathway of peptidoglycan 0-acetylation other than it must be a postsynthetic or maturation event. A putative acetyl transferase(s) was assumed to exist, but even the source of
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acetate was unknown. Through both in vivo and in vitro labelling experiments with P. mirabilis, we have recently provided evidence to suggest that acetate is transferred from the N-2 position of either N-acetylglucosaminyl or N-acetylmuramyl residues to the C-6 hydroxyl group of the latter via an N--0 acetyl migration during peptidoglycan turnover (Dupont and Clarke 1991b, 1991c). Radioactive 0-linked acetate was recovered from the purified pe tidoglycan of cells grown in the presence of [acetyl- H]Nacetylglucosamine, whereas the inclusion of other potential sources of transferable acetate, such as pyruvate, acetylphosphate, or acetate, did not interfere with radiolabel transfer. No such transfer of radiolabel was observed in peptidoglycan isolated from either Escherichia coli incubated with the same compound or P. mirabilis grown with [l , 6 - 3 ~N-acetylglucosamine ] or [1- 1 4 ~ ] g l u c o s a m i n e (Dupont and Clarke 1991b). The specific transfer of radioactive acetate was also observed with a cell-free biosynthetic system of peptidoglycan when supplemented with UDP[acetyl-1- 14c]N-acetylglucosamine (Dupont and Clarke 1991c), providing further evidence for a N-- 0 transacetylation as the pathway for the 0-acetylation of peptidoglycan. The activity of the putative enzyme involved is thus thought to be similar to N-arylhydroxarnic acid N,O-acetyltransferase (AHAT) (EC 2.3.1.56), which transfers the N-acetyl group of some aromatic acethydroxamates to the 0-position of some aromatic hydroxylamines. AHAT has recently been proven to exist as a unique and independent enzyme in a variety of mammalian tissues (Smi,th and Hanna 1986). In view of the absence of readily available activated acetate (e.g., acetyl CoA or acetyl phosphate) in the milieu external to the cytoplasm, it is conceivable that P. mirabilis has developed a method for utilizing the conserved bond energies stored within the peptidoglycan sacculus to achieve 0-acetylation in a manner analogous to transpeptidation. The fact that free amino sugars are not detected in the peptidoglycan sacculus but have been observed in the spent culture medium as turnover products (Dupont and Clarke 1991b) indicates that the process of peptidoglycan turnover and 0-acetylation could be associated. Although there is a dearth of information concerning the details of peptidoglycan turnover in P. mirabilis, extensive studies have been pursued with E. coli and members of the genus Bacillus (for a recent review see Doyle et al., 1989). With E. coli, components of -the peptidoglycan polymer, including oligopeptides which comprise -thepeptide moiety of ,the muramyl side chain, are recovered by the cell for reutilization after their hydrolysis from the sacculus (Goodell and Higgins 1987). This observation of recycling further lends support to the hypothesis of N - 0 acetyl transfer during peptidoglycan turnover. That both peptidoglycan autolytic (Blundell and Perkins 1985; Chapman and Perkins 1983) and N - 0 transacetylation activities are retained in a cell-free system led to the speculation that, by analogy with some of the penicillin-binding proteins of E. coli (for a review see Waxman and Strominger, 1983), the two activities maybe catalyzed by a single bifunctional enzyme (Dupont 1991). However, much further intensive investigations are required to bear out ,this postulate. The phenomenon of N- 0 acyl transfer has been widely investigated since 1924, when Bergman and Mickeley demonstrated the migration of acyl groups from N-acylamino alcohol to form an 0-acyl analog under the influence of
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anhydrous acidic reagents (Bergman and Mickeley 1924). It was shown by White (White 1938) and later by Fodor and 0tvos (Fodor and 0tvos 1956) that the N-acetyl group of N-acetylglucosamine derivatives easily migrates under the influence of acid to the oxygen atom at C-3. Hence, it appears feasible that N--0 acetyl transfer may occur in peptidoglycan, especially if it is intimately coupled to the release of glucosamine during the process of turnover. Acknowledgements These studies were supported by operating grants to A.J.C. from both the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Medical Research Council. C.D. was a recipient of an NSERC postgraduate scholarship. Abrams, A. 1958.0-Acetyl groups in the cell wall of Streptococcus faecalis. J. Biol. Chem. 230: 949-959. Bergman, M., and Mickeley, A. 1924. Rearrangements of peptidelike substances. 111. Derivatives of dl-serine. Z. Physiol. Chem. 140: 128-145. Blake, C.C.F., Johnson, L.N., Mair, G.A., et al. 1967. Crystallographic studies of the activity of hen egg-white lysozyme. Proc. R. Soc. London, Ser. B, 167: 378-388. Blundell, J.K., and Perkins, H.R. 1981. Effects of P-lactam antibiotics on peptidoglycan synthesis in growing Neisseria gonorrhoeae, including changes in the degree of 0-acetylation. J. Bacteriol. 147: 633-641. Blundell, J.K., and Perkins, H.R. 1985. Selectivity for 0-acetylated peptidoglycan during endopeptidase action by permeabilized Neisseria gonorrhoeae. FEMS Microbiol. Lett. 30: 67-69. Blundell, J.K., Smith, G.J., and Perkins, H.R. 1980. The peptidoglycan of Neisseria gonorrhoeae: 0-acetyl groups and lysozyme sensitivity. FEMS Microbiol. Lett. 9: 259-261. Brumfitt, W. 1959. The mechanism of development of resistance to lysozyme by some Gram-positive bacteria and its results. Br. J . Exp. Pathol. 40: 441-451. Brumfitt, W., Wardlaw, A.C., and Park, J.T. 1958. Development of lysozyme resistance in Micrococcus lysodeikticus and its association with an increased 0-acetyl content of the cell wall. Nature (London), 181: 1783-1784. Burghaus, P., Johannsen, L., Naumann, D., et al. 1983. The influence of different antibiotics on the degree of 0-acetylation of staphylococcal cell walls. In The target of penicillin. The murein sacculus of bacterial cell walls, architecture and growth. Edited by R. Hakenbeck, J. Holtje, and H. Labischinski. Walter de Gruyter and Co., New York. pp. 317-322. Chapman, S.J., and Perkins, H.R. 1983. Peptidoglycan degrading enzymes in ether-treated cells of Neisseria gonorrhoeae. J . Gen. Microbiol. 129: 877-883. Chedid, L., Audibert, F., and Johnson, A.G. 1978. Biological activities of muramyl dipeptide, a synthetic glycopeptide analogous to bacterial immunoregulating agents. Prog. Allergy, 25: 63-105. Coyette, J., and Ghuysen, J.M. 1970. Structure of the walls of Lactobacillus acidophilus strain 63 AM Gasser. Biochemistry, 9: 2935-2943. Cromartie, W. J . 198 1. Arthropathic properties of peptidoglycanpolysaccharide complexes of microbial origin. In Arthritis models and mechanisms. Edited by H. Deicker and L.C. Schultz. Sprinter-Verlag, New York. pp. 24-38. Dougherty, T. J. 1983a. Synthesis and modification of the peptidoglycan in Neisseria gonorrhoeae. FEMS Microbiol. Lett. 17: 51-53.
Dougherty, T. J . 1983b. Peptidoglycan synthesis in Neisseria gonorrhoeae strains sensitive and intrinsically resistant to P-lactam antibiotics. J. Bacteriol. 153: 429-435.
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rheumatoid arthritis. Am. J. Med. 40: 1-9. Hash, J.H., and Rothlauf, M.V. 1%7. The N,O-diacetylrnuramidase of Chalaropsis species. 1. Purification and crystallization. J. Biol. Chem. 242: 5586-5590. Imoto, T., Johnson, L.N., North, A.T.C., et al. 1972. Vertebrate lysozymes. In The enzymes. Vol. 7. 3rd ed. Edited by P.D. Boyer. pp. 666-868. Janusz, M.J., Chetty, C., Eisenberg, R.A., et al. 1984. Treatment of experimental erosive arthritis in rats by injection of the muralytic enzyme mutanolysin. J. Exp. Med. 160: 1360-1 374. Janusz, M.J., Esser, R.E., and Schwab, J.H. 1986. In vivo degradation of bacterial cell wall by the muralytic enzyme mutanolysin. Infect. Immun. 52: 459-467. Johannsen, L., Labischinski, H., Burghaus, P., and Giesbrecht, P . 1983. Acetylation in different phases of growth of staphylococci and their relation to cell wall degradability by lysozyme. In The target of penicillin. International FEMS Symposium on the Murein Sacculus of Bacterial Cell Walls. Edited by R. Hakenbeck, J . Holtje, and H . Labischinski. Walter de Gruyter and Co., Berlin. pp. 261-266. Johannsen, L., Rosenthal, R.S., Martin, S.A., et al. 1989. Somnogenic activity of 0-acetylated and dimeric muramyl peptides. Infect. Immun. 57: 2726-2732. Johannsen, L., Toth, L.A., Rosenthal, R.S., et al. 1990. Sommnogenic, pyrogenic and hematologic effects of bacterial peptidoglycan. Am. J. Physiol. 259: R182-R186. Kawasaki, A., Takada, H., Kotani, S., et al. 1987. Activation of the human complement cascade by bacterial cell walls, peptidoglycans, water-soluble peptidoglycan components, and synthetic muramylpeptides. Studies on active components and structural requirements. Microbiol. Immunol. 31: 554-569. Koga, T., and Pearson, C.M. 1976. Induction of polyarthritis in the rat with a water-soluble component of Mycobacterium tuberculosis wax D and with various bacterial cell walls or their watersoluble components. In Rheumatic fever and rheumatoid arthritis. Edited by Y. Otaka. Igaku Shoin Ltd., Tokyo. pp. 242-259. Koga, T., Hirofuji, T., Kotani, S., et a/. 1985. Acute joint inflammation in mice after systemic injections of the cell wall, its peptidoglycan, and chemically defined peptidoglycan subunits from various bacteria. Infect. Immun. 50: 27-34. Labischinski, H., Goodell, E. W., Goodell, A., and Hochberg, M.L. 1991. Direct proof of a "more-than-single-layered" peptidoglycan architecture of Escherichia coli W7: a neutron small-angle scattering study. J . Bacteriol. 173: 75 1-756. Lear, A.L., and Perkins, H.R. 1983. Degrees of 0-acetylation and cross-linking of the peptidoglycan of Neisseria gonorrhoeae during growth. J. Gen. Microbiol. 129: 885-888. Lear, A.L., and Perkins, H.R. 1986. 0-Acetylation of peptidoglycan in Neisseria gonorrhoeae. Investigation of lipid-linked intermediates and glycan chains newly incorporated into the cell wall. J. Gen. Microbiol. 132: 2413-2420. Lear, A.L., and Perkins, H.R. 1987. Progress of 0-acetylation and cross-linking of peptidoglycan in Neisseria gonorrhoeae grown in the presence of penicillin. J . Gen. Microbiol. 133: 1743-1750. Logardt, I.M., and Neujahr, H.Y. 1975. Lysis of modified walls from Lactobacillus fermentum. J. Bacteriol. 124: 73-77. Martin, H.H. 1984. In vitro synthesis of peptidoglycan by spheroplasts of Proteus mirabilis grown in the presence of penicillin. Arch. Microbiol. 139: 371 -375. Martin, H.H., and Gmeiner, J. 1979. Modification of peptidoglycan structure by penicillin-action in cell walls of Proteus mirabilis. Eur. J. Biochem. 95: 487-495. Martin, J.P., Fleck, J., Mock, M., and Ghuysen, J.M. 1973. The wall peptidoglycans of Neisseria perflava, Maraxella glucidolytica, Pseudomonas alcaligenes and Proteus vulgaris strain P18. Eur. J . Biochem. 38: 301-306. Masek, K., Raskova, H., and Rotta, J. 1968. The mechanism of
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Smith, T. J., and Hanna, P.E. 1986. N-Acetyltransferase multiplicity and the bioactivation of N-arylhydroxamic acids by hamster hepatic and intestinal enzymes. Carcinogenesis, 7: 697-702. Snowden, M.A., and Perkins, H.R. 1990. Peptidoglycan crosslinking in Staphylococcus aureus. An apparent random polymerization process. Eur. J. Biochem. 191: 373-377. Snowden, M.A., Perkins, H.R., Wyke, A.W., et al. 1989. Crosslinking and 0-acetylation of newly synthesized peptidoglycan in Staphylococcus aureus H. J. Gen. Microbiol. 135: 301 5-3022. Stimpson, S.A., Brown, R.R., Anderle, S.K., et al. 1986. Arthropathic properties of cell wall polymers from normal flora bacteria. Infect. Immun. 51: 240-249. Swim, S.G., Gfell, M.A., Wilde, C.E., 111, and Rosenthal, R.S. 1983. Strain distribution in extents of lysozyme resistance and 0-acetylation of gonococcal peptidoglycan determined by highperformance liquid chromatography. Infect. Immun. 42: 446-452. Verbrugh, H.A., van Dijk, W.C., Peters, R., et al. 1980. Opsonic recognition of staphylococci mediated by cell wall peptidoglycan: antibody-independent activation of human complement and opsonic activity of peptidoglycan antibodies. J . Immunol. 124: 1167-1 173. Waxman, D. J., and Strominger, J .L. 1983. Penicillin-binding proteins and the mechanism of action of /3-lactam antibiotics. Annu. Rev. Biochem. 52: 825-869. White, T. 1938. Amino sugars. I. A case of acyl migration. J. Chem. Soc. 1938: 1498-1500.
