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.     

Analysis of peptidoglycan structure from vegetative cells of Bacillus subtilis 168 and role of PBP 5 in peptidoglycan maturation.

The composition and fine structure of the vegetative cell wall peptidoglycan from Bacillus subtilis were determined by analysis of its constituent muropeptides. The structures of 39 muropeptides, representing 97% of the total peptidoglycan, were elucidated. About 99% analyzed muropeptides in B. subtilis vegetative cell peptidoglycan have the free carboxylic group of diaminopimelic acid amidated. Anhydromuropeptides and products missing a glucosamine at the nonreducing terminus account for 0.4 and 1.5%, respectively, of the total muropeptides. These two types of muropeptides are suggested to end glycan strands. An unexpected feature of B. subtilis muropeptides was the occurrence of a glycine residue in position 5 of the peptide side chain on monomers or oligomers, which account for 2.7% of the total muropeptides. This amount is, however, dependent on the composition of the growth media. Potential attachment sites for anionic polymers to peptidoglycan occur on dominant muropeptides and account for 2.1% of the total. B. subtilis peptidoglycan is incompletely digested by lysozyme due to de-N-acetylation of glucosamine, which occurs on 17.3% of muropeptides. The cross-linking index of the polymer changes with the growth phase. It is highest in late stationary phase, with a value of 33.2 or 44% per muramic acid residue, as determined by reverse-phase high-pressure liquid chromatography or gel filtration, respectively. Analysis of the muropeptide composition of a dacA (PBP 5) mutant shows a dramatic decrease of muropeptides with tripeptide side chains and an increase or appearance of muropeptides with pentapeptide side chains in monomers or oligomers. The total muropeptides with pentapeptide side chains accounts for almost 82% in the dacA mutant. This major low-molecular-weight PBP (DD-carboxypeptidase) is suggested to play a role in peptidoglycan maturation.     

Primary structure of cyanelle peptidoglycan of Cyanophora paradoxa: a prokaryotic cell wall as part of an organelle envelope.

The peptidoglycan layer surrounding the photosynthetic organelles (cyanelles) of the protist Cyanophora paradoxa is thought to be a relic of their cyanobacterial ancestors. The separation of muropeptides by gel filtration and reverse-phase high-performance liquid chromatography revealed four different muropeptide monomers. A number of muropeptides were identical in retention behavior to muropeptides of Escherichia coli, while others had remarkably long retention times with respect to their sizes, as indicated by gel filtration. Molecular mass determination by plasma desorption and matrix-assisted laser desorption ionization mass spectrometry showed that these unusual muropeptides had molecular masses greater by 112 Da or a multiple thereof than those of ones common to both species. Fast atom bombardment-tandem mass spectrometry of these reduced muropeptide monomers allowed the localization of the modification to D-glutamic acid. High-resolution fast atom bombardment-mass spectrometry and amino acid analysis revealed N-acetylputrescine to be the substituent (E. Pittenauer, E. R. Schmid, G. Allmaier, B. Pfanzagl, W. Löffelhardt, C. Quintela, M. A. de Pedro, and W. Stanek, Biol. Mass Spectrom. 22:524-536, 1993). In addition to the 4 monomers already known, 8 dimers, 11 trimers, and 6 tetramers were characterized. An average glycan chain length of 51 disaccharide units was determined by the transfer of [U-14C]galactose to the terminal N-acetylglucosamine residues of cyanelle peptidoglycan. The muropeptide pattern is discussed with respect to peptidoglycan biosynthesis and processing.     

Distribution of muropeptides in walls of Bacillus subtilis and a temperature-sensitive mutant

Attempts to correlate differences in cell shape with aspects of peptidoglycan structure were investigated. The parent strain, Bacillus subtilis 168, and its temperature-sensitive tagB mutant were grown in the chemostat under different growth conditions. The composition of the peptidoglycan was similar in all samples, regardless of cellular shape and anionic polymer content. Muropeptides, released by digestion with muramidase, comprised mainly dimers and monomers with only small amounts of trimer and traces of tetramer muropeptide. Overall, cross-linking did not vary greatly and the cross-linking index was less than 38%. Reverse-phase HPLC separation showed a complex fine structure. The principal muropeptides in all samples appeared to be the tetra monomer, tetra-tetra dimer and tetra-tetra-tetra trimer. While the major components looked the same in all samples, two specific components, a monomer and a dimer, were seen exclusively in the samples that had coccal morphology.     

The Listeria monocytogenes serotype 4b autolysin IspC has N-acetylglucosaminidase activity

IspC is a novel peptidoglycan (PG) hydrolase that is conserved in Listeria monocytogenes serotype 4b strains and is involved in virulence. The aim of this study was to establish the hydrolytic bond specificity of IspC. Purified L. monocytogenes peptidoglycan was digested by recombinant IspC and the resulting muropeptides were separated by reverse phase high-performance liquid chromatography. The structure of each muropeptide was determined using matrix-assisted laser desorption ionization (MALDI)-time-of-flight mass spectrometry in combination with MALDI-post-source decay mass spectrometry. The structure of muropeptides resulting from IspC-mediated hydrolysis indicated that IspC has N-acetylglucosaminidase activity. These muropeptides also had a high proportion of N-acetylated glucosamine residues. To determine whether IspC is more effective at hydrolysing N-acetylated peptidoglycan than de-N-acetylated peptidoglycan, a peptidoglycan deacetylase (PgdA) in-frame deletion mutant was created. This mutant was shown to have fully N-acetylated peptidoglycan and was more susceptible to hydrolysis by IspC when compared with the partially de-N-acetylated wild-type peptidoglycan. This indicates that IspC is more efficient when hydrolysing a fully N-acetylated peptidoglycan substrate. The finding that IspC acts as an N-acetylglucosaminidase is consistent with its categorization, based on amino acid sequence, as a member of the GH73 family. As with other members of this family, de-N-acetylation seems to be an important mechanism for regulating the activity of IspC.     

N-acetylputrescine as a characteristic constituent of cyanelle peptidoglycan in glaucocystophyte algae.

Cyanelle peptidoglycan from the glaucocystophyte algae Glaucocystis nostochinearum and Cyanoptyche gloeocystis was investigated by high-performance liquid chromatography of muropeptides, supported by matrix-assisted laser desorption-ionization mass spectrometry. The peptidoglycans of both species are modified with N-acetylputrescine, as has been demonstrated for cyanelle peptidoglycan of Cyanophora paradoxa.     

Peptidoglycan structure analysis of Lactococcus lactis reveals the presence of an L,D-carboxypeptidase involved in peptidoglycan maturation

Detailed structural analysis of Lactococcus lactis peptidoglycan was achieved by identification of its constituent muropeptides separated by reverse phase high-performance liquid chromatography. Modification of the classical elution buffer allowed direct and sensitive analysis of the purified muropeptides by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The structures of 45 muropeptides were assigned for L. lactis strain MG1363. Analysis of the muropeptide composition of an MG1363 dacB mutant showed that the dacB-encoded protein has l,d-carboxypeptidase activity and is involved in peptidoglycan maturation.     

Characterization of the murMN operon involved in the synthesis of branched peptidoglycan peptides in Streptococcus pneumoniae

The murMN operon, recently identified in the genome of Streptococcus pneumoniae, encodes for enzymes involved in the synthesis of branched structured muropeptides in the pneumococcal peptidoglycan; inactivation of murMN causes production of a peptidoglycan composed exclusively of linear muropeptides and a virtually complete loss of resistance in penicillin-resistant strains (Filipe, S. R., and Tomasz, A. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 4891-4896). The experiments described in this paper follow up these observations. Primer extension analysis was used to identify the putative promoter region of the murMN operon in penicillin-susceptible and -resistant strains. Selective inactivation of the murN gene in the penicillin-resistant strain Pen6 caused production of an unusual peptidoglycan that contained only single amino acid residues in the muropeptide branches, indicating that the product of murN was involved with the addition of the second amino acid and the product of murM was involved with the addition of the first amino acid (alanine or serine) to the peptidoglycan cross-bridge. Allelic replacement of the mosaic murM gene of strain Pen6 with murM of the penicillin-susceptible laboratory strain caused enrichment of the peptidoglycan in linear muropeptides. The findings suggest that the genetic determinant primarily controlling the synthesis of branched muropeptides in the pneumococcal peptidoglycan is murM.     

Cell wall assembly in Bacillus megaterium: incorporation of new peptidoglycan by a monomer addition process.

The pattern of cross-linking in the peptidoglycan of Bacillus megaterium has been studied by the pulsed addition of radiolabeled diaminopimelic acid. The distribution of label in muropeptides, generated by digestion with Chalaropsis muramidase and separated by high-performance liquid chromatography, stabilized after 0.15 of a generation time. The proportion of label in the acceptor and donor positions of isolated muropeptide dimers stabilized over the same period of time. The results have led to the formulation a new model for the assembly of peptidoglycan into the cylindrical wall of B. megaterium by a monomer addition process. Single nascent glycan peptide strands form cross-linkages only with material at the inner surface of the wall. Maturation is a direct consequence of subsequent incorporation of further new glycan peptide strands, and there is no secondary cross-linking process. The initial distribution of muropeptides is constant. It follows that the final pattern of cross-linking in the wall is determined solely by, and can be forecast from, this repetitive pattern of incorporation. In a modified form, this model can also be applied to assembly of cell walls in rod-shaped gram-negative bacteria.     

Cloning of a DNA fragment encoding part of a 70-kDa heat shock protein of Candida albicans

Abstract Muropeptide composition of peptidoglycan from the Gram-negative bacteria Aeromonas sp., Acinetobacter acetoaceticus, Agrobacterium tumefaciens, Enterobacter cloacae, Proteus morganii, Pseudomonas aeruginosa, Pseudomonas putida, Vibrio parahaemolyticus Yersinia enterocolitica and Escherichia coli , was analyzed by HPLC In all instances peptidoglycan was built up from the same subunits. A wide disparity in the relative abundance of muropeptides and all structural parameters was observed. The contribution of LD-A2pm-A2pm cross-linked muropeptides was extremely variable; from 1 to 45% of cross-linked muropeptides. Muropeptides with the dipeptides Lys-Lys or Arg-Lys, indicative of murein-bound (lipo)proteins, were detected in all instances although abundance was very variable.     

Effect of D-amino acids on structure and synthesis of peptidoglycan in Escherichia coli.

Growth of Escherichia coli in the presence of certain D-amino acids, such as D-methionine, results in the incorporation of the D-amino acid into macromolecular peptidoglycan and can be lethal at high concentrations. Previous studies suggested that incorporation was independent of the normal biosynthetic pathway. An enzymatic reaction between the D-amino acid and macromolecular peptidoglycan was proposed as the mechanism of incorporation. The application of more advanced analytical techniques, notably high-pressure liquid chromatography, revealed that the presence of a D-amino acid susceptible to incorporation induced a multiplicity of alterations in peptidoglycan metabolism. Results derived basically from the study of samples treated with D-Met, D-Trp, and D-Phe indicated that the incorporation of a D-amino acid results in the accumulation of two major new muropeptides whose general structures most likely are GlucNAc-MurNAc-L-Ala-D-Glu-m-diaminopimelic acid-D-aa and GlucNAc-MurNAc-L-Ala-D-Glu-m-diaminopimelic acid-D-Ala-GlucNAc-MurNAc-L-Ala-D-Glu-m-diaminopimelic acid-D-aa, where D-aa represents a residue of the added D-amino acid. Resting cells are proficient in the incorporation of D-amino acids and can reach peptidoglycan modification levels comparable to those in growing cells. Under our conditions, D-amino acids had no apparent effect on growth or morphology but caused a severe inhibition of peptidoglycan synthesis and cross-linking, possibly leading to a reduction in the amount of peptidoglycan per cell. The properties of the reaction support the involvement of a penicillin-insensitive LD-transpeptidase enzyme in the synthesis of modified muropeptides and a possible inhibitory action of D-amino acids on high-molecular-weight penicillin-binding proteins.     

Analysis of Neisseria gonorrhoeae peptidoglycan by reverse-phase, high-pressure liquid chromatography

The muramidase digest of peptidoglycan from Neisseria gonorrhoeae was isolated and analyzed by the use of a reverse-phase, high-pressure liquid chromatography system. As was found previously in the case of Escherichia coli, gonococci peptidoglycan is also composed of a greater number of muropeptides than can be resolved with thin-layer chromatography systems. Preliminary classification of the muropeptide components into subclasses based on O-acetyl modification and degree of cross-linkage was achieved. Examination of a penicillin-susceptible strain and a highly resistant strain with two penicillin-binding protein alterations synthesized distinctly different peptidoglycan structures, as revealed by this technique.     

N Glycolylation of the nucleotide precursors of peptidoglycan biosynthesis of Mycobacterium spp. is altered by drug treatment

The peptidoglycan of Mycobacterium spp. reportedly has some unique features, including the occurrence of N-glycolylmuramic rather than N-acetylmuramic acid. However, very little is known of the actual biosynthesis of mycobacterial peptidoglycan, including the extent and origin of N glycolylation. In the present work, we have isolated and analyzed muramic acid residues located in peptidoglycan and UDP-linked precursors of peptidoglycan from Mycobacterium tuberculosis and Mycobacterium smegmatis. The muramic acid residues isolated from the mature peptidoglycan of both species were shown to be a mixture of the N-acetyl and N-glycolyl derivatives, not solely the N-glycolylated product as generally reported. The isolated UDP-linked N-acylmuramyl-pentapeptide precursor molecules also contain a mixture of N-acetyl and N-glycolyl muramyl residues in apparent contrast to previous observations in which the precursors isolated after treatment with d-cycloserine consisted entirely of N-glycolyl muropeptides. However, nucleotide-linked peptidoglycan precursors isolated from M. tuberculosis treated with d-cycloserine contained only N-glycolylmuramyl-tripeptide precursors, whereas those from similarly treated M. smegmatis consisted of a mixture of N-glycolylated and N-acetylated residues. The full pentapeptide intermediate, isolated following vancomycin treatment of M. smegmatis, consisted of the N-glycolyl derivative only, whereas the corresponding M. tuberculosis intermediate was a mixture of both the N-glycolyl and N-acetyl products. Thus, treatment with vancomycin and d-cylcoserine not only caused an accumulation of nucleotide-linked intermediate compounds but also altered their glycolylation status, possibly by altering the normal equilibrium maintained by de novo biosynthesis and peptidoglycan recycling.     

Improved high-performance liquid chromatographic separation of peptidoglycan isolated from various Staphylococcus aureus strains for mass spectrometric characterization

Reversed-phase high-performance liquid chromatography (RP-HPLC) of muropeptides, obtained by muramidase digestion of peptidoglycan in combination with amino acid analysis and plasma desorption time-of-flight mass spectrometry is today by far the best tool to analyze the fine structure of the peptidoglycans. Here we report further improvements of the RP-HPLC separation of muropeptides for analyzing the peptidoglycans of various methicillin-resistant strains of Staphylococcus aureus, with emphasis on a more detailed characterization of the interpeptide bridge of the peptidoglycans of this species.     

The role of peptidoglycan in pathogenesis

Bacterial pathogens rely on a variety of virulence factors to establish the colonization of a new niche. Although peptidoglycan and its muropeptide derivatives have been known to possess potent biological properties, until recently the molecular bases were poorly understood. With the identification of the cytosolic surveillance mechanism mediated by the nucleotide-binding oligomerization domain (Nod)1 and Nod2 proteins, which detect unique peptidoglycan-derived muropeptides, these muropeptides should be considered as potential virulence factors. Recent research highlights the role of peptidoglycan in the pathogenesis of different human pathogens such as Streptococcus pneumoniae, Listeria monocytogenes or Helicobacter pylori.     

Use of 4-sulfophenyl isothiocyanate labeling and mass spectrometry to determine the site of action of the streptococcolytic peptidoglycan hydrolase zoocin A

Zoocin A is a streptococcolytic peptidoglycan hydrolase with an unknown site of action that is produced by Streptococcus equi subsp. zooepidemicus 4881. Zoocin A has now been determined to be a d-alanyl-l-alanine endopeptidase by digesting susceptible peptidoglycan with a combination of mutanolysin and zoocin A, separating the resulting muropeptides by reverse-phase high-pressure liquid chromatography, and analyzing them by mass spectrometry (MS) in both the positive- and negative-ion modes to determine their compositions. In order to distinguish among possible structures for these muropeptides, they were N-terminally labeled with 4-sulfophenyl isothiocyanate (SPITC) and analyzed by tandem MS in the negative-ion mode. This novel application of SPITC labeling and MS/MS analysis can be used to analyze the structure of peptidoglycans and to determine the sites of action of other peptidoglycan hydrolases.     

Clostridium difficile has an original peptidoglycan structure with a high level of N-acetylglucosamine deacetylation and mainly 3-3 cross-links

The structure of the vegetative cell wall peptidoglycan of Clostridium difficile was determined by analysis of its constituent muropeptides with a combination of reverse-phase high pressure liquid chromatography separation of muropeptides, amino acid analysis, mass spectrometry and tandem mass spectrometry. The structures assigned to 36 muropeptides evidenced several original features in C. difficile vegetative cell peptidoglycan. First, it is characterized by a strikingly high level of N-acetylglucosamine deacetylation. In addition, the majority of dimers (around 75%) contains A(2)pm(3) → A(2)pm(3) (A(2)pm, 2,6-diaminopimelic acid) cross-links and only a minority of the more classical Ala(4) → A(2)pm(3) cross-links. Moreover, a significant amount of muropeptides contains a modified tetrapeptide stem ending in Gly instead of D-Ala(4). Two L,D-transpeptidases homologues encoding genes present in the genome of C. difficile 630 and named ldt(cd1) and ldt(cd2), were inactivated. The inactivation of either ldt(cd1) or ldt(cd2) significantly decreased the abundance of 3-3 cross-links, leading to a marked decrease of peptidoglycan reticulation and demonstrating that both ldt(cd1)-and ldt(cd2)-encoded proteins have a redundant L,D-transpeptidase activity. The contribution of 3-3 cross-links to peptidoglycan synthesis increased in the presence of ampicillin, indicating that this drug does not inhibit the L,D-transpeptidation pathway in C. difficile.     

Mode of action of glycine on the biosynthesis of peptidoglycan

The mechanism of glycine action in growth inhibition was studied on eight different species of bacteria of various genera representing the four most common peptidoglycan types. To inhibit the growth of the different organisms to 80%, glycine concentrations from 0.05 to 1.33 M had to be applied. The inhibited cells showed morphological aberrations. It has been demonstrated that glycine is incorporated into the nucleotide-activated peptidoglycan precursors. The amount of incorporated glycine was equivalent to the decrease in the amount of alanine. With one exception glycine is also incorporated into the peptidoglycan. Studies on the primary structure of both the peptidoglycan precursors and the corresponding peptidoglycan have revealed that glycine can replace l-alanine in position 1 and d-alanine residues in positions 4 and 5 of the peptide subunit. Replacement of l-alanine in position 1 of the peptide subunit together with an accumulation of uridine diphosphate-muramic acid (UDP-MurNAc), indicating an inhibition of the UDP-MurNAc:l-Ala ligase, has been found in three bacteria (Staphylococcus aureus, Lactobacillus cellobiosus and L. plantarum). However, discrimination against precursors with glycine in position 1 in peptidoglycan synthesis has been observed only in S. aureus. Replacement of d-alanine residues was most common. It occurred in the peptidoglycan with one exception in all strains studied. In Corynebacterium sp., C. callunae, L. plantarum, and L. cellobiosus most of the d-alanine replacing glycine occurs C-terminal in position 4, and in C. insidiosum and S. aureus glycine is found C-terminal in position 5. It is suggested that the modified peptidoglycan precursors are accumulated by being poor substrates for some of the enzymes involved in peptidoglycan synthesis. Two mechanisms leading to a more loosely cross-linked peptidoglycan and to morphological changes of the cells are considered. First, the accumulation of glycine-containing precursors may lead to a disrupture of the normal balance between peptidoglycan synthesis and controlled enzymatic hydrolysis during growth. Second, the modified glycine-containing precursors may be incorporated. Since these are poor substrates in the transpeptidation reaction, a high percentage of muropeptides remains uncross-linked. The second mechanism may be the more significant in most cases.     

The peptidoglycan sacculus of Myxococcus xanthus has unusual structural features and is degraded during glycerol-induced myxospore development

Upon nutrient limitation cells of the swarming soil bacterium Myxococcus xanthus form a multicellular fruiting body in which a fraction of the cells develop into myxospores. Spore development includes the transition from a rod-shaped vegetative cell to a spherical myxospore and so is expected to be accompanied by changes in the bacterial cell envelope. Peptidoglycan is the shape-determining structure in the cell envelope of most bacteria, including myxobacteria. We analyzed the composition of peptidoglycan isolated from M. xanthus. While the basic structural elements of peptidoglycan in myxobacteria were identical to those in other gram-negative bacteria, the peptidoglycan of M. xanthus had unique structural features. meso- or LL-diaminopimelic acid was present in the stem peptides, and a new modification of N-acetylmuramic acid was detected in a fraction of the muropeptides. Peptidoglycan formed a continuous, bag-shaped sacculus in vegetative cells. The sacculus was degraded during the transition from vegetative cells to glycerol-induced myxospores. The spherical, bag-shaped coats isolated from glycerol-induced spores contained no detectable muropeptides, but they contained small amounts of N-acetylmuramic acid and meso-diaminopimelic acid.     

Requirements of peptidoglycan structure that allow detection by the Drosophila Toll pathway

The Drosophila immune system is able to discriminate between classes of bacteria. Detection of Gram-positive bacteria involves a complex of two pattern recognition receptors: peptidoglycan recognition protein SA (PGRP-SA) and Gram-negative binding protein 1 (GNBP1). These activate the Toll signalling pathway. To define the cell wall components sensed by the host, we used highly purified peptidoglycan fragments of two principal Gram-positive bacterial pathogens Staphylococcus aureus and Streptococcus pneumoniae. We report that in both peptidoglycans, the minimal structure needed to activate the Toll pathway is a muropeptide dimer and that the free reducing end of the N-acetyl muramic acid residues of the muropeptides is essential for activity. Monomeric muropeptides were inactive and inhibitory in combination with dimers. Finally, peptidoglycan was degraded by the haemolymph of wild-type but not GNBP1 mutant flies. We suggest a model whereby GNBP1 is involved in the hydrolysis of Gram-positive peptidoglycan producing new glycan reducing ends, which are subsequently detected by PGRP-SA.