Synthesis and immunoadjuvant activities of the repeating,disaccharide-dipeptide unit of the bacterial,cell-wall peptidoglycan and of some carbohydrate analogs

The repeating disaccharide-dipeptide units of the bacterial, cell-wall peptidoglycan, one being O-(N-acetyl-β-muramoyl-l-alanyl-d-isoglutamine)-(1→4)-2-acetamido-2-deoxy-d-glucose, and the other, O-(2-acetamido-2-deoxy-β-d-glucosyl)-(1→4)-N-acetyl-muramoyl-l-alanyl-d-isoglutamine, have been synthesized. Some carbohydrate analogs, such as O-(N-acetyl-β-muramoyl-l-alanyl-d-isoglutamine)- (1→4)-N-acetylmuramoyl-l-alanyl-d-isoglutamine, O-β-d-glucosyl-(1→4)-N-acetylmuramoyl-l-alanyl-d-isoglutamine, and O-(6-acetamido-6-deoxy-β-d-glucosyl)-(1→4)-N-acetylmuramoyl-l-alanyl-d-isoglutamine, were also synthesized. Their immunoadjuvant activities were examined in guinea-pigs.     

Peptidoglycan composition and taxonomy of group D, E, and H streptococci, and Streptococcus mutans

The qualitative and quantitative composition of the peptidoglycan from the cell wall of groups D, E, and H streptococci, and Streptococcus mutans, was determined. In group D, S. faecalis and the closely related species S. liquefaciens and S. zymogenes were separated from S. faecium and the closely related species S. durans on the basis of their peptidoglycan composition. A relationship among S. bovis, S. equinus, and some strains of S. mutans was indicated by the presence in each of a similar peptidoglycan containing threonine. Threonine was released from the S. mutans polymer as a threonine-lysine dipeptide. Hydrolysis of the dipeptide at 100 C for 24 hr in 6 n HC1 was required to break the peptide bond. Motile group D streptococci possessed a peptidoglycan of the same composition as S. faecium. Group E and H strains were also similar in the composition of their peptidoglycan. The results demonstrate that peptidoglycan composition can be used to (i) aid in the division of members of an immunological group into subgroups, and (ii) indicate a relationship between members of the same genus which are not related on an immunological basis.     

Isolation, Characterization, and Ultrastructure of the Peptidoglycan Layer of a Marine Pseudomonad

The peptidoglycan layer of a marine pseudomonad was observed by electron microscopy in thin sections of plasmolyzed intact cells and mureinoplasts but not in untreated intact cells. Only fragments of this layer could be isolated by sodium lauryl sulfate (SLS) treatment of mureinoplast envelopes. Sacculus-like peptidoglycan structures were obtained from growing cells by immediate heat inactivation of cellular autolytic enzymes and subsequent SLS, trypsin, and nuclease treatments. Recently, similar peptidoglycan sacculus-like structures have been obtained by adding SLS to the growing culture and treating the isolated particulate material with nucleases. Thin-sectioned and negatively stained preparations of whole cell peptidoglycan showed compressed profiles of cell-shaped sacculi. Peptidoglycan prepared by SLS treatment of mureinoplast envelopes had a similar composition to that prepared from whole cells. The major amino sugars and amino acids in the peptidoglycan component were glucosamine, muramic acid, alanine, glutamic acid and diaminopimelic acid in the molar ratios 1.18:1.24:1.77:1.00:0.79. Forty-five per cent of the epsilon-amino groups of diaminopimelic acid were cross-linked. The peptidoglycan was estimated to account for about 1% of the cell dry weight.     

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

Cell walls isolated from 29 strains of 24 gram-positive bacterial species, whose peptidoglycans belong to the group A type of Schleifer and Kandler's classification, with one exception (Arthrobacter sp.), were shown to activate the complement cascade in pooled fresh human serum mainly through the alternative pathway and partly through the classical one. The complement-activating effect of cell walls (5 species) possessing group B type peptidoglycan, except those of Corynebacterium insidiosum, was weaker than that of the walls with group A type peptidoglycan. Preparations of peptidoglycan isolated from cell walls of Staphylococcus aureus, Streptococcus pyogenes, and Lactobacillus plantarum also activated the alternative pathway of the complement cascade, but less effectively than the respective parent cell walls. A water-soluble "polymer" of peptidoglycan subunits (SEPS), which was prepared from Staphylococcus epidermidis peptidoglycans by treatment with a cross-bridge degrading endopeptidase, retained most of the complement-activating ability of the parent cell walls. A peptidoglycan "monomer," SEPS-M, which was obtained by hydrolysis of the glycan chain of SEPS with endo-N-acetylmuramidase to disaccharide units did not activate complement. In conformity with this finding, neither synthetic N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) nor MDP-L-Lys-D-Ala activated the complement cascade. Among several lipophilic derivatives of MDP, 6-O-(3-hydroxy-3-docosylhexacosanoyl)-MDP-L-Lys-D-Ala (BH48-MDP-L-Lys-D-Ala) and 6-O-(2-tetradecylhexadecanoyl)-MDP (B30-MDP) were shown to activate complement through the alternative as well as the classical pathway and exclusively through the classical pathway, respectively. The finding that a D-isoasparagine analog of B30-MDP caused the same effect as the parent molecule strongly suggests that the activation of complement by B30-MDP is different from that caused by cell wall peptidoglycans and a water-soluble "polymer" of peptidoglycan subunits.     

Peptidoglycan isolated from Lactobacillus bulgaricus: its effect, mediated by the complement system, on pre-T-cell maturation

Peptidoglycan from Lactobacillus bulgaricus converts mouse pre-T-cells into theta-positive cells and activates the complement. It has been observed that the effect depends on serum peptidoglycan concentration and the time of the interaction of the activated complement with mouse pre-T-cells. The action of peptidoglycan was replaced by C3a complement component.     

Biological characteristics of peptidoglycans of group A streptococcus and some other bacterial species. I. Tolerance and effect of antibody in fever response, and heart damaging effect in rabbits.

Induced tolerance to the pyrogenic action of group A streptococcus peptidoglycan decreased after one week and was no longer detectable after the second week. However, one or two further doses of peptidoglycan rapidly restored the tolerance. The passive transfer of plasma from rabbits tolerant to streptococcus peptidoglycan to nontolerant animals failed to transfer tolerance. Antiserum to streptococcus peptidoglycan neutralized the pyrogenic effect of not only streptococcus but also staphylococcus and pneumococcus peptidoglycan; it did not influence the febrile response to endotoxin. Histopathologic changes in the rabbit heart produced by the intravenous injection of staphylococcus or pneumococcus peptidoglycans were similar and were characterized by various stages of degeneration and necrosis. The changes were less pronounced than after streptococcus peptidoglycan. Antiserum to streptococcus peptidoglycan had modest or no counteracting effect on the development of heart alterations after staphylococcus or pneumococcus peptidoglycan.     

Seminalplasmin, an antimicrobial protein from bovine seminal plasma, inhibits peptidoglycan synthesis in Escherichia coli

Seminalplasmin, an antimicrobial protein from bovine seminal plasma, inhibited peptidoglycan synthesis in Escherichia coli in a concentration-dependent manner. The inhibition of peptidoglycan synthesis appears to be a cause rather than a consequence of growth inhibition as it was observed soon after the addition of the antibiotic even in E. coli cells whose growth was totally inhibited by chloramphenicol.     

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.     

Induction of antibacterial protein synthesis by soluble peptidoglycan in isolated fat body from larvae of the silkworm, Bombyx mori

Synthesis and secretion of bactericidal protein (cecropin) and lysozyme were induced by soluble peptidoglycan fragments (SPG) from Escherichia coli in a culture of fat body from Bombyx mori larvae. The rate of the secretion by fat body increased as a function of SPG concentration added to the culture medium. The induction of bactericidal activity was specific for peptidoglycan of a particular structure. Thus, SPG from Micrococcus luteus was 500-times less potent than E. coli SPG, and various glucans and peptides structurally related to peptidoglycan were all ineffective as elicitor. These results support the hypothesis that bacteria invading the haemocoel have to be partially degraded to generate peptidoglycan fragments as a signal molecule, which subsequently acts on a receptor on fat body cells and induces antibacterial protein synthesis.     

Peptidoglycan of a Chemolithotrophic Bacterium, Ferrobacillus ferrooxidans

The rigid layer (peptidoglycan) of the wall of the chemolithotroph Ferrobacillus ferrooxidans was isolated after various chemical treatments. The removal of specific components was followed by noting in an electron microscope changes in the appearance of the cell surface. The final peptidoglycan was virtually free from proteins and was sensitive to the action of lysozyme. Results of chemical analyses of acidhydrolyzed peptidoglycan revealed three major amino acids and two amino sugars: glutamic acid, α,ε-diaminopimelic acid, alanine, glucosamine, and muramic acid in a ratio of 1:1:2.33:062:088.     

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.     

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.     

Minimum structure of peptidoglycan required for induction of antibacterial protein synthesis in the silkworm, Bombyx mori

Various peptidoglycan fragments, different in mode of cross-linking and molecular size, were isolated, and the elicitor activity was tested for induction of antibacterial protein synthesis in larvae of Bombyx mori. Linear uncross-linked peptidoglycans from Bacillus licheniformis and Micrococcus luteus were effective elicitors, similar to the directly cross-linked peptidoglycan from B. licheniformis cell wall. The fragments of uncross-linked peptidoglycan with a sugar chain length of four or more were active elicitors, but the disaccharide unit had no elicitor activity. The minimum structure of peptidoglycan required for induction of antibacterial protein synthesis was determined to be two repeating N-acetylglucosamine-N-acetylmuramic acid units with peptide side chains.     

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.     

Peptidoglycan N-acetylglucosamine deacetylases from Bacillus cereus, highly conserved proteins in Bacillus anthracis

The genomes of Bacillus cereus and its closest relative Bacillus anthracis contain 10 polysaccharide deacetylase homologues. Six of these homologues have been proposed to be peptidoglycan N-acetylglucosamine deacetylases. Two of these genes, namely bc1960 and bc3618, have been cloned and expressed in Escherichia coli, and the recombinant enzymes have been purified to homogeneity and further characterized. Both enzymes were effective in deacetylating cell wall peptidoglycan from the Gram(+) Bacillus cereus and Bacillus subtilis and the Gram(-) Helicobacter pylori as well as soluble chitin substrates and N-acetylchitooligomers. However, the enzymes were not active on acetylated xylan. These results provide insight into the substrate specificity of carbohydrate esterase family 4 enzymes. It was revealed that both enzymes deacetylated only the GlcNAc residue of the synthetic muropeptide N-acetyl-D-glucosamine-(beta-1,4)-N-acetylmuramyl-L-alanine-D-isoglutamine. Analysis of the constituent muropeptides of peptidoglycan from B. subtilis and H. pylori resulting from incubation of the enzymes BC1960 and BC3618 with these polymers and subsequent hydrolysis by Cellosyl and mutanolysin, respectively, similarly revealed that both enzymes deacetylate GlcNAc residues of peptidoglycan. Kinetic analysis toward GlcNAc(2-6) revealed that GlcNAc4 was the favorable substrate for both enzymes. Identification of the sequence of N-acetychitooligosaccharides (GlcNAc(2-4)) following enzymatic deacetylation by using 1H NMR revealed that both enzymes deacetylate all GlcNAc residues of the oligomers except the reducing end ones. Enzymatic deacetylation of chemically acetylated vegetative peptidoglycan from B. cereus by BC1960 and BC3618 resulted in increased resistance to lysozyme digestion. This is the first biochemical study of bacterial peptidoglycan N-acetylglucosamine deacetylases.     

Different binding specificities of S-layer homology modules from Clostridium thermocellum AncA, Slp1, and Slp2

S-layer homology (SLH) module polypeptides were derived from Clostridium thermocellum S-layer proteins Slp1 and Slp2 and cellulosome anchoring protein AncA as rSlp1-SLH, rSlp2-SLH, and rAncA-SLH respectively. Their binding specificities were investigated using C. thermocellum cell-wall preparations. rAncA-SLH associated with native peptidoglycan-containing sacculi from C. thermocellum, including both peptidoglycan and secondary cell wall polymers (SCWP), but not to hydrofluoric acid-extracted peptidoglycan-containing sacculi (HF-EPCS) lacking SCWPs, suggesting that SCWPs are responsible for binding with SLH modules of AncA. On the other hand, rSlp1-SLH and rSlp2-SLH associated with HF-EPCS, suggesting that these polypeptides had an affinity for peptidoglycan. A binding assay using a peptidoglycan fraction prepared from Escherichia coli cells definitely confirmed that rSlp1-SLH and rSlp2-SLH specifically interacted with peptidoglycan but not with SCWP.     

Structural analysis of the fundamental polymer of the sheath constructed by Sphaerotilus natans

The sheath of Sphaerotilus natans is composed of cysteine-rich peptide and polysaccharide moieties. The polysaccharide was prepared by treating the sheath with hydrazine, and was determined to be a mucopolysaccharide containing beta-D-GlcA, beta-D-Glc, alpha-D-GalN, and beta-D-GalN. To elucidate the structure of the peptide, the sheath was labeled with a thiol-selective fluorogenic reagent, 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole. Enantiomeric determination of the S-derivatized Cys in the fluorescent sheath suggested that it contained L-Cys mainly. Fluorescent cysteinylglycine was detected in the partial acid hydrolysate of the fluorescent sheath. The sheath-degrading enzyme secreted by Paenibacillus koleovorans produced a fluorescent disaccharide-dipeptide composed of GalN, Gly, and N-acetylated Cys from the fluorescent sheath. The disaccharide and dipeptide moieties were found to be connected by an amide bond. Based on these results, the sheath was deduced to be formed by association of a mucopolysaccharide modified with N-acetyl-L-cysteinylglycine.     

PapA,a peptidoglycan-associated protein,interacts with OmpC and maintains cell envelope integrity

The bacterial cell envelope is critical to support and maintain cellular life. In Gram-negative bacterial cells, the outer membrane and the peptidoglycan layer are two important parts of the cell envelope and they harbour abundant proteins. Here, we report the identification and characterization of a previously unknown p eptidoglycan-a ssociated p rotein, PapA, from the Gram-negative Comamonas testosteroni. PapA bound peptidoglycan with its C-terminal domain and interacted with the outer-membrane porin OmpC. The PapA-OmpC complex riveted the outer membrane and the peptidoglycan layer, and played a role in maintaining cell envelope integrity. When papA was disrupted, the mutant CNB-1ΔpapA apparently had an outer membrane partly separated from the peptidoglycan layer. Phenotypically, the mutant CNB-1ΔpapA lost chemotactic responses and had longer lag-phase of growth, less flagellation and higher sensitivity to harsh environments. Totally, 1093 functionally unknown PapA homologues were identified from the public NR protein database and they were mainly distributed in Burkholderiales of Betaproteobacteria. Our finding provides a clue that the PapA homologous proteins might function as a rivet to maintain cell envelope integrity in those Gram-negative bacteria.     

murH, a new genetic locus in Escherichia coli involved in cell wall peptidoglycan biosynthesis.

A temperature-sensitive mutant of Escherichia coli defective in peptidoglycan synthesis was characterized. The incorporation of radiolabeled meso-diaminopimelate into peptidoglycan by the mutant was inhibited at the restrictive growth temperature, resulting in autolysis. The defective step appeared to be part of the terminal stage in peptidoglycan synthesis involving the incorporation of disaccharide peptide units into the wall peptidoglycan. The mutation was assigned to a new locus, designated murH, at 99.2 min on the E. coli linkage map.