Range of the antigenic specificity of peptidoglycan in Staphylococcus aureus compared to other representatives of the Staphylococcus genus

In investigations based on the use of a highly sensitive test system permitting the detection of normal human antibodies to S. aureus peptidoglycan, the antigenic relationships between the peptidoglycans of S. aureus and other representatives of the genus Staphylococcus have been studied. Among other staphylococcal species, S. simulans, S. xylosus, S. hyicus, S. cohnii, S. hyicus s. s. chromogenes have been found to possess peptidoglycans most closely related to S. aureus peptidoglycans, while S. warneri and S. epidermidis peptidoglycans have proved to be least closely related to it.     

Biological characteristics of peptidoglycans of group A streptococcus and some other bacterial species. II. Immunological mechanisms involved in thrombocytolysis.

Immunological mechanisms are involved in the thrombocytolytic activity of peptidoglycan of Group A streptococcus, Streptococcus pneumoniae and Staphylococcus aureus. Inactivation of particular components of complement (heating of blood serum to 56 degrees C,incubation with zymosan or NH4OH) inhibited the thrombocytolytic activity of group A streptococcus peptidoglycan. So did preincubation of Group A streptococcus peptidoglycan with homologous antipeptidoglycan antibody. On the other hand, antibody to Group A streptococcus peptidoglycan did not inhibit the thrombocytolytic effect of Streptococcus pneumoniae or Staphylococcus aureus peptidoglycan. Human platelets are resistant to peptidoglycans. They remain resistant in the presence of rabbit serum although rabbit platelets are highly sensitive to peptidoglycans. This suggests that, for the expression of the thrombocytolytic activity of bacterial peptidoglycan, specific receptors on the surface of platelets must be present in addition to serum factors.     

The characteristics of the polyclonal action and immunomodulating activity of bacterial peptidoglycans in oppositely reacting mouse strains]

The data on the specific features of the polyclonal action of gram-negative bacteria on primary immune response to sheep red blood cells in oppositely reacting mouse strains are presented. Staphylococcus aureus and Bacterium flavum peptidoglycans have been found to increase the amount of antibody producing cells to thymus-dependent antigen only in the spleen of low-responsive mice. At the same time differences in the amino acid composition of these heteropolymers do not affect their stimulating activity. Differences between the capacity of B. flavum peptidoglycans to induce the activation of B cells in mice of different strains have been established.     

Chemiluminescence of human lymphocytes in reactions with Staphylococcus aureus peptidoglycan

The capacity of S. aureus peptidoglycan (PG) for inducing the luminol-dependent chemiluminescence of human lymphocytes has been studied. Lymphocytes taken from adult donors have been found to give dose-dependent reaction to S. aureus PG, while lymphocytes from newborn infants have been inert under the same conditions. Essential differences in the kinetics of response to PG (the maximum intensity of chemiluminescence occurs in 25-30 minutes) and to phytohemagglutinin (the maximum intensity is reached in 1 minute) were observed. These results are considered as the manifestation of specific sensitization to bacterial peptidoglycans, which may be rapidly detected by reactive chemiluminescence.     

Quantitative characteristics of human antibodies to the peptidoglycans of different Staphylococcus species

The content of antibodies to peptidoglycans of 13 staphylococcal species was studied in the ELISA with the IgG fraction isolated from commercial human immunoglobulin. The content of antibodies to S. aureus peptidoglycan (calculated per mg of protein) was 3.0 micrograms and that of antibodies to peptidoglycans of other species amounted to 1.8-4.1 micrograms. The maximal value was obtained for S. xylosus and the minimal value, for S. lentus. No relationship between the content of antibodies to peptidoglycans and the degree of their antigenic affinity to S. aureus peptidoglycan was noted: the scatter of the values in species with relatively low and even minimal affinity did not exceed the range of variance in the group with a high degree of homology (2.2-2.4 micrograms). The production of antibodies to staphylococcal peptidoglycans was supposed to result from the action of a complex of antigenic determinants, both common and specific for each staphylococcal species.     

The characteristics of Staphylococcus aureus peptidoglycan in the spectrum of the reactivity of human lymphocytes to bacterial peptidoglycans]

The sensitivity of lymphocytes of healthy persons to S. aureus peptidoglycan as compared with that to the polyclonal stimulator zymosan C3b and peptidoglycans of other bacteria (Streptococcus faecalis, Escherichia coli, Bacterium bifidum) was analyzed with a test system permitting the determination of specific reactivity to peptidoglycans. The analysis showed that at the peak of luminol-dependent chemiluminescence (25-30 minutes) individual reactivity to S. aureus peptidoglycan varied within wide limits (the coefficient of lymphocyte stimulation was 1.4-9.6, 3.5 +/- 0.6), exceeding sensitivity to other bacteria, as well as the values obtained in the negative control. The conclusion of the wide spread of sensitization to S. aureus peptidoglycan and the possibility of using this preparation for the study of cell-mediated immunity reactions was made.     

Peptidoglycans synthesized by a membrane preparation of Micrococcus luteus.

By incubation of cell-free particulate preparations from Micrococcus luteus with nucleotidic precursors uridine 5'-diphosphate-N-acetylglucosamine and uridine 5'-diphosphate-N-acetylmuramic acid-L-Ala-D-iso-Glu-L-Lys-D-Ala-D-Ala, several types of peptidoglycans were obtained: soluble peptidoglycan, insoluble peptidoglycan bound to the membrane and solubilized by trypsin, and peptidoglycan, which remained insoluble after the action of trypsin. The structure of each type of peptidoglycan was studied by action of lytic enzymes and separation of the fragments on Sephadex. Soluble peptidoglycans consist of a mixture of un-cross-linked polymers of various molecular weights. Trypsin-solubilized peptidoglycans are also a mixture of polymers of various sizes. They contain a preponderance of un-cross-linked material and some bridges with dimer peptides. Insoluble peptidoglycans, after the action of trypsin, contain about 50% of un-cross-linked peptide residues; in the other moiety, peptide units are cross-linked by D-Ala leads to L-Lys and D-Ala leads to L-Ala bonds which characterize the natural peptidoglycan. Therefore, the cell-free particulate preparation possesses the whole enzymatic system necessary for synthesis of cross-linked peptidoglycan.     

The lysostaphin endopeptidase resistance gene (epr) specifies modification of peptidoglycan cross bridges in Staphylococcus simulans and Staphylococcus aureus.

Staphylococcus simulans biovar staphylolyticus produces an extracellular glycylglycine endopeptidase (lysostaphin) that lyses other staphylococci by hydrolyzing the cross bridges in their cell wall peptidoglycans. The genes for endopeptidase (end) and endopeptidase resistance (epr) reside on plasmid pACK1. An 8.4-kb fragment containing end was cloned into shuttle vector pL150 and was then introduced into Staphylococcus aureus RN4220. The recombinant S. aureus cells produced endopeptidase and were resistant to lysis by the enzyme, which indicated that the cloned fragment also contained epr. Treatments to remove accessory wall polymers (proteins, teichoic acids, and lipoteichoic acids) did not change the endopeptidase sensitivity of walls from strains of S. simulans biovar staphylolyticus or of S. aureus with and without epr. Immunological analyses of various wall fractions showed that there were epitopes associated with endopeptidase resistance and that these epitopes were found only on the peptidoglycans of epr+ strains of both species. Treatment of purified peptidoglycans with endopeptidase confirmed that resistance or susceptibility of both species was a property of the peptidoglycan itself. A comparison of the chemical compositions of these peptidoglycans revealed that cross bridges in the epr+ cells contained more serine and fewer glycine residues than those of cells without epr. The presence of the 8.4-kb fragment from pACK1 also increased the susceptibility of both species to methicillin.     

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.     

Electromechanical Interactions in Cell Walls of Gram-Positive Cocci

Isolated cell walls of Staphylococcus aureus and Micrococcus lysodeikticus were found to expand and contract in response to changes in environmental pH and ionic strength. These volume changes, which could amount to as much as a doubling of wall dextran-impermeable volume, were related to changes in electrostatic interactions among fixed, ionized groups in wall polymers, including peptidoglycans. S. aureus walls were structurally more compact in the hydrated state and had a higher maximum charge density than M. lysodeikticus walls. However, they were less responsive to changes in electrostatic interactions, apparently because of less mechanical compliance. In media of nearly neutral pH, S. aureus walls had a net positive charge whereas M. lysodeikticus walls had a net negative charge. These charge differences were reflected in Donnan distributions of mobile ions between wall phases and bulk medium phases. Cell walls of unfractionated cocci also could be made to swell and contract, and wall tonus in intact cells appeared to be set partly by electrostatic interactions and partly by mechanical tension in the elastic structures due to cell turgor pressure. The experimental results led to the conclusions that bacterial cell walls have many of the properties of polyelectrolyte gels and that peptidoglycans are flexible polymers. A reasonable mechanical model for peptidoglycan structure might be a sort of three-dimensional rope ladder with relatively rigid, polysaccharide rungs and relatively flexible polypeptide ropes. Thus, the peptidoglycan network surrounding cocci appeared to be predominantly an elastic restraining structure rather than a rigid shell.     

Reactivity of neutrophils in systems with peptidoglycans of different species of Staphylococcus

The work presents the results of the comparative study of the luminol-dependent chemiluminescence of human neutrophils stimulated with peptidoglycans of 14 staphylococcal species. The intensity of chemiluminescence greatly varied, which was not linked with specific antigenic and structural features of peptidoglycans. The maximal chemiluminescence was induced by S. aureus and S. cohnii peptidoglycans and the minimal chemiluminescence, by S. lentus and S. epidermidis peptidoglycans. The data obtained in experiments on cross restimulation with homologous and heterologous peptidoglycans suggest that the mechanisms of neutrophil activation are similar in character, differing from the mechanism of latex-induced chemiluminescence. These data were analyzed from the viewpoint of the heterogeneity of bacterial peptidoglycans and its effect on their relationship with neutrophils.     

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.     

The range of antigenic specificity of Bifidobacterium peptidoglycan]

The antigenic relationships of Bifidobacterium bifidum 1 peptidoglycans with different strains of this species (LVA-3, 791, GO-4), bifidobacteria of other species (B. adolescentis GO-13, B. breve 79-38, B. lactentis 79-41, B. longum GO-3) and bacteria of remote taxonomic groups (Streptococcus faecalis 6-3. Staphylococcus aureus COM 885, S. epidermidis COM 2124. Lactobacillus plantarum 1, Escherichia coli M-17) were studied on the basis of a highly sensitive test system permitting the registration of normal human antibodies to peptidoglycans. The level of cross reactions with staphylococci and streptococci correspond to intraspecific and antigenic affinity to L. plantarum and E. coli was considerably less pronounced. Copying a number of epitopes of bifidobacteria, S. aureus peptidoglycan seems to possess additional antigenic determinants which participate in the formation of immunological responsiveness in man.     

Structural requirements for arthritogenicity of peptidoglycans from Staphylococcus aureus and Lactobacillus plant arum and analogous synthetic compounds.

The comparative studies on the arthritogenicity of chemically well defined peptidoglycans (PG)2 from the cell walls of Staphylococcus aureus (FDA 209P) and Lactobacillus plantarum (ATCC 8014) showed that 1) a polymer of disaccharide-peptide with or without the presence of N-acetylglucosaminyl-ribitol-teichoic acid produced severe arthritis; 2) oligosaccharide-peptides with or without the special structure (N-acetylglucosaminyl-ribitol-teichoic acid in S. aureus, a polymer of rhamnose and glucose in L. plantarum) produced severe disease; 3) disaccharide-heptapeptide-disaccharide with or without the presence of either glucose-ribitol-teichoic acid or a polymer of rhamnose and glucose appeared to be arthritogenic but much less effective for disease production; 4) N-acetylmuramyl-heptapeptide-N-acetylmuramic acid and disaccharide-hexapeptide were non-arthritogenic; 5) none of the synthetic N-acetylmuramyl-peptides, including tetrapeptide, produced the disease. Thus it is concluded that arthritis-inducing activity is related to the peptidoglycan moiety but not to the special structure, and the most important moiety responsible for disease production may be located in a chain length of two or more disaccharide units on PG subunits. However, it is discussed that non-arthritogenicity of peptidoglycans, including synthetic compounds, may be due to a lack of adjuvanticity in rats rather than a lack of antigenic determinant(s) responsible for production of arthritis.     

Cell wall-targeting domain of glycylglycine endopeptidase distinguishes among peptidoglycan cross-bridges

ALE-1, a homologue of lysostaphin, is a peptidoglycan hydrolase that specifically lyses Staphylococcus aureus cell walls by cleaving the pentaglycine linkage between the peptidoglycan chains. Binding of ALE-1 to S. aureus cells through its C-terminal 92 residues, known as the targeting domain, is functionally important for staphylolytic activity. The ALE-1-targeting domain belongs to the SH3b domain family, the prokaryotic counterpart of the eukaryotic SH3 domains. The 1.75 angstroms crystal structure of the targeting domain shows an all-beta fold similar to typical SH3s but with unique features. The structure reveals patches of conserved residues among orthologous targeting domains, forming surface regions that can potentially interact with some common features of the Gram-positive cell wall. ALE-1-targeting domain binding studies employing various bacterial peptidoglycans demonstrate that the length of the interpeptide bridge, as well as the amino acid composition of the peptide, confers the maximum binding of the targeting domain to the staphylococcal peptidoglycan. Truncation of the highly conserved first 9 N-terminal residues results in loss of specificity to S. aureus cell wall-targeting, suggesting that these residues confer specificity to S. aureus cell wall.     

Expression of the Staphylococcus aureus UDP-N-acetylmuramoyl- L-alanyl-D-glutamate:L-lysine ligase in Escherichia coli and effects on peptidoglycan biosynthesis and cell growth.

The monomer units in the Escherichia coli and Staphylococcus aureus cell wall peptidoglycans differ in the nature of the third amino acid in the L-alanyl-gamma-D-glutamyl-X-D-alanyl-D-alanine side chain, where X is meso-diaminopimelic acid or L-lysine, respectively. The murE gene from S. aureus encoding the UDP-N-acetylmuramoyl-L-alanyl-D-glutamate: L-lysine ligase was identified and cloned into plasmid vectors. Induction of its overexpression in E. coli rapidly results in abnormal morphological changes and subsequent cell lysis. A reduction of 28% in the peptidoglycan content was observed in induced cells, and analysis of the peptidoglycan composition and structure showed that ca. 50% of the meso-diaminopimelic acid residues were replaced by L-lysine. Lysine was detected in both monomer and dimer fragments, but the acceptor units from the latter contained exclusively meso-diaminopimelic acid, suggesting that no transpeptidation could occur between the epsilon-amino group of L-lysine and the alpha-carboxyl group of D-alanine. The overall cross-linking of the macromolecule was only slightly decreased. Detection and analysis of meso-diaminopimelic acid- and L-lysine-containing peptidoglycan precursors confirmed the presence of L-lysine in precursors containing amino acids added after the reaction catalyzed by the MurE ligase and provided additional information about the specificity of the enzymes involved in these latter processes.     

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.     

Investigation of cell wall components in actinomycin D resistant Staphylococcus aureus]

Cell walls in 2 strains of Staphylococcus aureus 209P, i.e. actinomycin D susceptible and resistant ones were comparatively investigated. The resistant cells contained much more wall material per a unit of the biomass weight vs the susceptible strain cells, that conformed to thickening of the resistant cell walls detected by electron microscopy and a sharp increase of their electron density. Investigation of peptidoglycans and teichoic acids did not reveal any significant alterations in the structure of the wall components in the actinomycin D resistant cells. Only some increase of glucosamine in the peptidoglycan fraction of the resistant cells vs the susceptible ones was observed. It was shown that preparations of the resistant cell walls and peptidoglycan isolated from the resistant cells were able to bind somewhat lower quantities of actinomycin D vs the analogous preparations of the susceptible cells. The significant decrease of the antibiotic binding by live cells of the resistant strain probably slightly depended on the structure characteristics of the main wall components. The barrier properties of the walls in resistant staphylococci are most likely defined by the wall thickening and consolidation while adapting to actinomycin D.