Progress of O-acetylation and cross-linking of peptidoglycan in Neisseria gonorrhoeae grown in the presence of penicillin

The synthesis, cross-linking and O-acetylation of gonococcal peptidoglycan in growing cells were studied by following incorporation of radioactive glucosamine and separation of the SDS-insoluble peptidoglycan into uncross-linked (monomer) and cross-linked (dimer and oligomer) fragments. Cultures to which penicillin or piperacillin at concentrations near the minimum growth inhibitory concentration (MIC) had been added 20 min before the radioactive label were compared with controls. The beta-lactams affected the early stage of cross-linking (up to 3 min) but had no effect thereafter. The deficit of cross-linking, however, did not recover. The O-acetylation, particularly of the monomer fraction, was decreased by beta-lactams even at concentrations that had no effect on culture optical density, viable counts or overall peptidoglycan synthesis. These effects on O-acetylation occurred mainly after the first 3 min of incorporation, rather than before. O-Acetylation of the oligomer fraction was also followed. Here penicillin led to increased levels of O-acetylation during the early stages of incorporation but the final value was never exceeded; indeed at higher drug concentrations the later stages of O-acetylation of oligomers were inhibited (e.g. almost completely at 2.5 X MIC).     

Cross-linking and O-acetylation of newly synthesized peptidoglycan in Staphylococcus aureus H

Staphylococcus aureus H growing exponentially was labelled with N-acetyl[14C]glucosamine, which became incorporated into the peptidoglycan. The portion of peptidoglycan not linked to teichoic acid (60-75% of the whole) was degraded with Chalaropsis muramidase to yield disaccharide-peptide monomers and dimers, trimers and oligomers formed by biosynthetic cross-linking of the monomers. The degree of O-acetylation of these fragments was also examined. Pulse-chase experiments showed that the proportion of label initially in the monomer fraction immediately after the 1 min pulse declined rapidly during a 3 min chase, while the oligomer fraction (fragments greater than trimer) gained the radioactivity proportionately. The radioactivity of the dimer and trimer fractions remained virtually unchanged. At 4 min after the commencement of labelling (i.e. approx. one-tenth of a generation time) final values had been reached. The O-acetylation of all fragments had achieved final values even at 1 min, except for the monomer fraction, which showed an increase from 40% to 60% during the first 3 min of chase. Although O-acetylation was clearly a very rapid process, no O-acetylated peptidoglycan lipid-intermediates could be detected.     

Effects of beta-lactam antibiotics on peptidoglycan synthesis in growing Neisseria gonorrhoeae, including changes in the degree of O-acetylation

Low concentrations of beta-lactam antibiotics caused an increased uptake of radioactive glucosamine into the sodium dodecyl sulfate-insoluble peptidoglycan of growing Neisseria gonorrhoeae. There was no appreciable change in the (small) amount of sodium dodecyl sulfate-soluble polymer present in the cultures. The sodium dodecyl sulfate-insoluble product in control cells was only partially dissolved by egg-white lysozyme (about 40%), but could all be released by the Chalaropsis B muramidase. In cells exposed to beta-lactams the proportion of labeled peptidoglycan susceptible to lysozyme increased to 60%. Examination of the Chalaropsis B digests by thin-layer chromatography showed that they contained disaccharide-peptide monomers with and without O-acetylation and bis-disaccharide-peptide dimers with one or two O-acetyl groups, or with none. beta-Lactam antibiotics caused a decrease in the degree of O-acetylation but did not greatly affect the amount of peptidoglycan cross-linking. They also had the effect of enlarging the bacteria and conserving and thickening the septa that could be observed in thin sections under the electron microscope. The relationship between these results and the effects of beta-lactams on in vitro synthesis of peptidoglycan by ether-treated N. gonorrhoeae is discussed.     

O-acetylation of peptidoglycan in Neisseria gonorrhoeae. Investigation of lipid-linked intermediates and glycan chains newly incorporated into the cell wall

Radioactive labelling of the amino sugars in gonococcal peptidoglycan was followed by treatment with Chalaropsis muramidase and TLC separation of the products. Even after very brief periods of labelling (0.5 min) the peptidoglycan was already cross-linked to some 80% of the final value and little change occurred within 2 min. The remaining cross-linking was achieved only over a period of about one generation time. Streptomycete endopeptidase was used to show the extent to which new chains were cross-linked to old. Even at the earliest times many cross-linked units contained new material in both moieties and by 3 min there was little distinction in relative labelling, indicating that in Neisseria gonorrhoeae most newly synthesized glycan chains are cross-linked to other new chains rather than to pre-existing peptidoglycan. A model is proposed in which newly polymerized monomer units are predestined either towards dimer formation with other new chains, which are then rapidly O-acetylated and not further cross-linked, or towards the formation of trimers and higher oligomers, the latter being a slower process. Although significant O-acetylation of peptidoglycan was detectable even at the earliest times, efforts to detect O-acetylated lipid intermediates were unsuccessful. The chief lipid intermediate found was apparently the disaccharide-peptide unit linked to undecaprenol.     

Peptidoglycan biosynthesis in Neisseria gonorrhoeae strains sensitive and intrinsically resistant to beta-lactam antibiotics.

Treatment of penicillin-sensitive and intrinsically resistant Neisseria gonorrhoeae strains with their respective inhibitory concentrations of penicillin caused rapid cell death. When the peptidoglycan syntheses of these two strains were examined in the presence of penicillin, the sensitive strain continued to make this cell wall polymer for an extended time, whereas the resistant strain underwent a rapid and marked depression in synthesis. Examination of the labeled sodium dodecyl sulfate-insoluble peptidoglycan made in the presence of inhibitory concentrations of penicillin revealed further differences. The primary effect on the penicillin-sensitive gonococcus was a slight change in peptide cross-linking and a sharp decline in the degree of O-acetylation. In contrast, the resistant strain exhibited a substantial decline in cross-linking, with a very moderate change in O-acetylation. The degree of saturation of the individual penicillin-binding proteins (PBPs) was assessed under these conditions. PBP 2, which exhibits a reduced affinity for penicillin in the resistant strain, appeared to be related to O-acetylation, whereas PBP 1 was implicated in the transpeptidation reaction.     

Dependence of lysozyme-catalysed solubilization of Proteus mirabilis peptidoglycan on the extent of O-acetylation

The degree of peptidoglycan O-acetylation in 14 strains of Proteus mirabilis has been accurately determined by a procedure which employs the quantitation of mild-base-released acetic acid by HPLC, and the estimation of peptidoglycan concentration by cation-exchange amino acid analysis. The beta-D-N,6-O-diacetylmuramyl content of all isolated and purified peptidoglycans was ranged 20-52.8%, relative to the total muramic acid concentration. Each of the O-acetylated peptidoglycans was found to be resistant to solubilization by both human and hen egg-white lysozymes and for hen egg-white lysozyme, the extent of this resistance was dependent upon the degree of O-acetylation. The steady-state parameters, Km and V, for the hen-egg-white-lysozyme-catalysed solubilization of various peptidoglycan preparations were determined at pH 6.61 and 25 degrees C. Values of Km for the different peptidoglycan samples were found to increase with increasing O-acetylation, whereas with V no such relationship appeared to exist. An increase in the overall change in the standard Gibbs free energy of activation [delta(delta G#)], a consequence of increasing O-acetylation, was observed, and is shown to result from the weaker affinity of the enzyme for the modified substrates.     

Detailed structural analysis of the peptidoglycan of the human pathogen Neisseria meningitidis

We used reverse-phase high pressure liquid chromatography (HPLC), matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and post source decay analysis (MALDI-PSD) to determine the muropeptide composition of the human pathogen Neisseria meningitidis. Structural assignment was determined for 28 muropeptide species isolated after HPLC separation and purification. Fourteen of these muropeptides were O-acetylated to different degrees. We identified the entire O-acetylation spectrum of dimers and trimers both in muropeptides and 1,6-anhydromuropeptides. On average, one of three disaccharides was O-acetylated. Furthermore, the degree of cross-linking of the N. meningitidis peptidoglycan was around 39% in all the strains analyzed. MALDI-PSD analysis of several muropeptide species indicated that meningococci only synthesize D-alanyl-meso-diaminopimelate cross-bridges. No muropeptides representative of covalent linkages of lipoproteins to the peptidoglycan could be identified, unlike in Escherichia coli. Finally, comparison of the muropeptide composition of penicillin-susceptible and penicillin-intermediate clinical strains of meningococci showed a positive correlation between the minimum inhibitory concentration (MIC) of penicillin G and the amount of muropeptides carrying an intact pentapeptide chain in the peptidoglycan. This suggests that reduced susceptibility to penicillin G in N. meningitidis is associated with a decrease in d,d-carboxypeptidase activity and/or D,D-transpeptidase activity.     

O-Acetylation of peptidoglycan is required for proper cell separation and S-layer anchoring in Bacillus anthracis

O-Acetylation of the MurNAc moiety of peptidoglycan is typically associated with bacterial resistance to lysozyme, a muramidase that serves as a central component of innate immunity. Here, we report that the peptidoglycan of Bacillus anthracis, the etiological agent of anthrax, is O-acetylated and that, unusually, this modification is produced by two unrelated families of O-acetyltransferases. Also, in contrast to other bacteria, O-acetylation of B. anthracis peptidoglycan is combined with N-deacetylation to confer resistance of cells to lysozyme. Activity of the Pat O-acetyltransferases is required for the separation of the daughter cells following bacterial division and for anchoring of one of the major S-layer proteins. Our results indicate that peptidoglycan O-acetylation modulates endogenous muramidase activity affecting the cell-surface properties and morphology of this important pathogen.     

The peptidoglycan of Neisseria gonorrhoeae, with or without O-acetyl groups, contains anhydro-muramyl residues

Muramidase digests of alkali-treated SDS-insoluble peptidoglycan from two strains of Neisseria gonorrhoeae were examined. Both strains contained disaccharide peptide monomers that had intramolecular 1,6-anhydro-muramyl ends. In contrast to strain 1L260, in which 50% of the monomer fraction is O-acetylated, the monomer fraction from strain RD5 was completely devoid of O-acetyl groups, as shown by HPLC. Penicillin decreased the O-acetylation of peptidoglycan but did not affect the proportion of anhydro-muramyl residues.     

O-Acetylated peptidoglycan: controlling the activity of bacterial autolysins and lytic enzymes of innate immune systems

The O-acetylation of peptidoglycan is now known to occur in 50 different bacterial species, both Gram positive and Gram negative, including a number of important human pathogens. This modification to the essential cell wall component of bacteria provides both a level of control over endogenous autolysins and protection from the lysozymes of innate immune systems. In this review, we describe the details of the pathways for peptidoglycan O-acetylation that are now beginning to emerge and we explore the possibility that the associated enzymes may present new candidates for antibacterial targets.     

Penicillin-induced changes in the cell wall composition of Staphylococcus aureus before the onset of bacteriolysis

To analyze if chemical cell wall alterations contribute to penicillin-induced bacteriolysis, changes in the amount, stability, and chemical composition of staphylococcal cell walls were investigated. All analyses were performed before onset of bacteriolysis i.e. during the first 60 min following addition of different penicillin G doses. Only a slight reduction of the amount of cell wall material incorporated after penicillin addition at the optimal lytic concentration was observed as compared to control cells. However, the presence of higher penicillin G concentrations reduced the incorporation of wall material progressively without bacteriolysis. Losses of wall material during isolation of dodecylsulfate insoluble cell walls were monitored to assess the stability of the wall material following penicillin addition. Wall material grown at the lytic penicillin concentration was least stable but about 30% of the newly incorporated wall material withstood even the harsh conditions of mechanical breakage and dodecylsulfate treatment. Dodecylsulfate insoluble cell walls were used for chemical analyses. While peptidoglycan chain length was unaffected in the presence of penicillin, other wall parameters were considerably altered: peptide cross-linking was reduced in the wall material synthesized after addition of penicillin; reductions from approx. 85% in controls to about 60% were similar for lytic and also for very high penicillin concentrations leading to nonlytic death. O-acetylation was also reduced after treatment with penicillin; this effect paralleled the occurence of subsequent bacteriolysis at different drug concentrations. The results are not consistent with hypotheses explaining penicillin-induced lysis as a result of an overall weakened cell wall structure or an overall activation of autolytic wall enzymes but not conflicting with the model that ascribes penicillin-induced bacteriolysis as the result of a very restricted, local perforation of the peripheral cell wall (murosome-induced bacteriolysis).Abbreviations CL Cross-linking - DNFB 2,4-dinitro-1-fluorobenzole - MIC Minimal inhibitory concentration - OD Optical density at 578 nm - PEN Penicillin G     

O-acetylated peptidoglycan: its occurrence, pathobiological significance, and biosynthesis.

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 O-acetylated peptidoglycan. This review addresses the influence of O-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.     

Identification and characterization of O-acetylpeptidoglycan esterase: a novel enzyme discovered in Neisseria gonorrhoeae

Modification of the bacterial cell wall heteropolymer peptidoglycan by addition of an acetyl group to the C-6 hydroxyl group of N-acetylmuramoyl residues is known to inhibit the activity of muramidases (lysozymes) of innate immune systems. The O-acetylation of peptidoglycan also precludes the action of intrinsic lytic transglycosylases, enzymes that require a free C-6 hydroxyl group to generate their 1,6-anhydromuropeptide products. This class of autolysins is ubiquitous in peptidoglycan-synthesizing bacteria as they are responsible for insertion of pores and flagella, spore formation, and the general metabolism of peptidoglycan. We recently discovered a cluster of genes in the Neisseria gonorrhoeae chromosome that are proposed to participate in peptidoglycan O-acetylation (Weadge, J. T., Pfeffer, J. M., and Clarke, A. J. (2005) BMC Microb. 5, 49). In the current study, we demonstrate that one of these genes, ape1 functions as an O-acetylpeptidoglycan esterase. The ape1 gene was cloned and overexpressed in Escherichia coli as a fusion protein with a hexa-histidine tag. The expressed protein was purified to apparent homogeneity and assayed for activity as an esterase using three different assays involving high-performance liquid chromatography and chromogenic detection methods which measured the release of ester-linked acetate from a variety of polymer and soluble substrates. These assays demonstrated that Ape1 has a higher specific activity on O-acetylated peptidoglycan compared to O-acetylated xylan. Consequently, Ape1 represents the first enzyme characterized as an O-acetylpeptidoglycan esterase. The physicochemical and kinetic parameters of Ape1 were determined using soluble chromogenic substrates for convenience. Thus, its pH optima for stability and activity were observed to be 6.0 and 6.2, respectively, while its optimum temperature for activity was 55 degrees C. Two forms of truncated Ape1 are generated in E. coli, one lacked the complete predicted N-terminal signal sequence, while the second involved a proteolytic cleavage within this signal sequence. The smaller truncated form was localized predominantly to the periplasm, whereas the larger form was mainly associated with the outer membrane, and to a lesser extent, the cytoplasmic membrane, sites expected for the maintenance of peptidoglycan.     

The prevalence of gentamicin 2'-N-acetyltransferase in the Proteeae and its role in the O-acetylation of peptidoglycan

Abstract The prevalence of aac(2')-Ia , a gene coding for gentamicin 2'-JV-acetyltransferase in Providenda stuartii , among species of the Proteeae was investigated to determine if it is a common resistance factor and whether the correlation observed in P. stuartii between its expression and the levels of peptidoglycan O -acetylation represents a general feature of bacteria producing this form of modified peptidoglycan. An evaluation of the MICs of gentamicin for each of the species of the Proteeae did not reveal any apparent relationship between resistance and the degree of O-acetylation of peptidoglycan. The entire aac(2')-Ia gene was used as a probe in Southern hybridization experiments against genomic DNA from each species of the Proteeae. A sequence with strong homology to aac(2')-Ia was present only in Proteus penneri while weak hybridization was also observed to the restriction digested DNA from Providenda rettgeri . Other bacteria that O -acetylate peptidoglycan were also screened with this probe and a homologous DNA sequence was only found in Neisseria subflava . These data suggest that AAC(2')-Ia may contribute to the rO -acetylation of peptidoglycan in P. stuartii , but a more specific enzyme must also be produced for this function.     

Peptidoglycan synthetic activities in membranes of Escherichia coli caused by overproduction of penicillin-binding protein 2 and rodA protein

Penicillin-binding protein (PBP)-2 and the RodA protein are known to function in determining the rod shape of Escherichia coli cells. Peptidoglycan biosynthetic reactions that required these two proteins were demonstrated in the membrane fraction prepared from an E. coli strain that overproduced both of these two proteins and which lacked PBP-1B activity (the major peptidoglycan synthetase activity in the normal E. coli membranes). The cross-linked peptidoglycan was synthesized from UDP-N-acetylmuramylpentapeptide and UDP-N-acetylglucosamine in the presence of a high concentration of cefmetazole that inhibited all of PBPs except PBP-2. The peptidoglycan was synthesized via a lipid intermediate and showed up to 30% cross-linking. The cross-linking reaction was strongly inhibited by the amidinopenicillin, mecillinam, and by other beta-lactam antibiotics that have a high affinity for PBP-2, but not by beta-lactams that had very low affinity for PBP-2. The formation of peptidoglycan required the presence of high levels of both PBP-2 and the RodA protein in the membranes, but it is unclear which of the two proteins was primarily responsible for the extension of the glycan chains (transglycosylation). However, the sensitivity of the cross-linking reaction to specific beta-lactam antibiotics strongly suggested that it was catalyzed by PBP-2. The transglycosylase activity of the membranes was sensitive to enramycin and vancomycin and was unusual in being stimulated greatly by a high concentration of a chelating agent.     

Structural variation in the glycan strands of bacterial peptidoglycan

The normal, unmodified glycan strands of bacterial peptidoglycan consist of alternating residues of beta-1,4-linked N-acetylmuramic acid and N-acetylglucosamine. In many species the glycan strands become modified after their insertion into the cell wall. This review describes the structure of secondary modifications and of attachment sites of surface polymers in the glycan strands of peptidoglycan. It also provides an overview of the occurrence of these modifications in various bacterial species. Recently, enzymes responsible for the N-deacetylation, N-glycolylation and O-acetylation of the glycan strands were identified. The presence of these modifications affects the hydrolysis of peptidoglycan and its enlargement during cell growth. Glycan strands are frequently deacetylated and/or O-acetylated in pathogenic species. These alterations affect the recognition of bacteria by host factors, and contribute to the resistance of bacteria to host defence factors such as lysozyme.     

Structure of Bordetella pertussis peptidoglycan.

Bordetella pertussis Tohama phases I and III were grown to the late-exponential phase in liquid medium containing [3H]diaminopimelic acid and treated by a hot (96 degrees C) sodium dodecyl sulfate extraction procedure. Washed sodium dodecyl sulfate-insoluble residue from phases I and III consisted of complexes containing protein (ca. 40%) and peptidoglycan (60%). Subsequent treatment with proteinase K yielded purified peptidoglycan which contained N-acetylglucosamine, N-acetylmuramic acid, alanine, glutamic acid, and diaminopimelic acid in molar ratios of 1:1:2:1:1 and less than 2% protein. Radiochemical analyses indicated that 3H added in diaminopimelic acid was present in peptidoglycan-protein complexes and purified peptidoglycan as diaminopimelic acid exclusively and that pertussis peptidoglycan was not O acetylated, consistent with it being degraded completely by hen egg white lysozyme. Muramidase-derived disaccharide peptide monomers and peptide-cross-linked dimers and higher oligomers were isolated by molecular-sieve chromatography; from the distribution of these peptidoglycan fragments, the extent of peptide cross-linking of both phase I and III peptidoglycan was calculated to be ca. 48%. Unambiguous determination of the structure of muramidase-derived peptidoglycan fragments by fast atom bombardment-mass spectrometry and tandem mass spectrometry indicated that the pertussis peptidoglycan monomer fraction was surprisingly homogeneous, consisting of greater than 95% N-acetylglucosaminyl-N-acetylmuramyl-alanyl-glutamyl-diaminopimelyl++ +-alanine.     

Cross-linkage and cross-linking of peptidoglycan in Escherichia coli: definition, determination, and implications.

The glycan chains in peptidoglycan or murein are cross-linked by transpeptidation of the peptide side chains. To assess the fraction of side chains involved in cross-bridges, distinction has been made between cross-linkage and cross-linking. The first expression refers to the situation in unlabeled (or fully labeled) peptidoglycan, and the second refers to pulse-labeled peptidoglycan. It is argued that for the determination of the cross-linking value, the mode of insertion as denoted by the so-called acceptor/donor radioactivity ratio should be taken into account.     

Penicillin-resistant temperature-sensitive mutants of Escherichia coli which synthesize hypo- or hyper-cross-linked peptidoglycan

A group of Escherichia coli mutants which are ampicillin resistant at 32 C and which either are unable to grow or lyse at 42 C has been selected. These mutants have been classified by a number of characteristics: total peptidoglycan synthesis measured by [(14)C]diaminopimelic acid incorporation, extent of cross-linking of the peptidoglycan which is synthesized, growth characteristics at the two temperatures, and morphology. Two especially interesting groups of mutants have been described. In one of these, a hypo-cross-linked peptidoglycan was synthesized at the nonpermissive temperature. Most of these organisms lysed at 42 C. In another group, the peptidoglycan synthesized at 42 C was hyper-cross-linked. Many of these organisms were spherical. Studies of revertants indicated that ampicillin resistance, temperature sensitivity, cross-linking, growth characteristics, and morphological changes may be related to a single mutational event in both of these groups.