The Immunochemistry of Peptidoglycan

Abstract

An albumin-peptide conjugate was synthesized, which carries pentaglycine peptides with Cterminal glycine residues as found in the interpeptide bridges of the peptidoglycan of many staphylococci. Immunization of rabbits with this synthetic immunogen yielded antisera containing predominantly antibodies against the peptide moiety of the conjugate. The quantitative precipitin and the Ouchterlony agar gel reaction with several synthetic protein-peptide-conjugates, immunoaffinity chromatography of the antisera on Sepharose-(Gly-Gly-Gly-Gly-Gly), and hapten inhibition studies with several synthetic peptides and peptide derivatives demonstrated that the antibodies were highly specific for oligoglycine peptides with C-terminal glycine. These antibodies also reacted strongly with staphylococcal peptidoglycans with an interpeptide bridge composed of pentaglycine peptides or of pentaglycine peptides in which part of the glycine residues were replaced by L-serine. In contrast, all the peptidoglycans lacking interpeptide bridges composed of glycine residues gave no precipitin reaction at all. The final proof for identical determinant groups of albumin-(CHZCO-Gly-Gly-GlyGly-Gly)31 and the staphylococcal peptidoglycans applied in the precipitin reaction was furnished by double gel diffusion studies and by hapten inhibition of the precipitin reaction between antisera to albumin-(CHKO-Gly-Gly-Gly-Gly-Gly)3 I and the corresponding peptidoglycans.For rapid screening of the different peptidoglycans, a latex agglutination test was elaborated. Purified antibodies were adsorbed to latex particles, and the titers with the particular peptidoglycans were then determined. The test was highly sensitive, in that 10 nanogr,ams of peptidoglycan could still be detected.Peptidoglycan (murein, mucopeptide) is a typical cell wall polymer of prokaryotes. It is composed of glycan strands which are cross-linked through short peptides. The glycan portion of peptidbglycan consists of alternating N-acetylglucosamine and N-acetylniuramic acid residues. The peptide moiety usually is made up of a tetrapeptide or pentapeptide of the sequence L-Ala-D-Glu[L-diamino acid-~-Ala-(~-Ala)], whereby the L-alanine residue is linked to muramic acid by a peptide bond. These peptide side chains (peptide subunits) are cross-linked, generally between the distal amino group of the diamino acid at position 3 and the cc-carboxyl group of D-alanine at position 4' from an adjacent chain. In many cases, the peptide subunits are connected by an interpeptide bridge consisting of a single amino acid or a short peptide. A review on the different primary structures of the peptidoglycans of bacteria is given in reference [l]. The immunogenicity of peptidoglycan is well established. Antibodies to the glycan moiety 12-5,9] and to the peptide subunit [5-111 were detected. Investigations of Helgeland et al. [12] indicated the presence of antibodies to the pentaglycine interpeptide bridge in antisera to Staphylococcus aureus. In recent papers [lo, 111 synthetic antigens were reported ca...

Cloning, sequence, and expression of the lysostaphin gene from Staphylococcus simulans.

“…Hybridization analyses show that the 13-lactamase determinants from S. aureus and S. simulans are homologous, and it seems likely that the lysostaphin plasmid will show other similarities to the S. aureus 13-lactamase plasmids. Lysostaphin itself may be related to autolytic enzymes, which appear to be widely distributed in staphylococci (45). Although little is known about their properties, these enzymes are apparently associated with the cell wall of the producing organism and may play an essential role in reshaping the wall during cell growth and division.…”

Immunoglobulin E antibodies against Staphylococcus aureus cell walls in the sera of patients with hyperimmunoglobulinemia E and recurrent staphylococcal infection

“…The glycan backbone and the peptide subunit attached to muramic acid are believed to be essentially identical in all staphylococcal strains. Some variation occurs in the interpeptide bridge with L-alanine or L-serine present in this structure, in addition to glycine, in some species of staphylococci (23).…”

“…The interpeptide bridge shows some species variation, and small structural differences have been detected immunologically in the interpeptide bridge of certain staphylococcal species. For example, Seidl and Schleifer (23) have demonstrated that antibodies raised in rabbits against a synthetic protein-peptide conjugate carrying pentaglycine peptides with free carboxyl groups can differentiate staphylococcal peptidoglycans without Cterminal glycine residues. Karakawa et al (11) have recently found evidence for strain-specific immunological difference in sonically treated S. aureus peptidoglycan preparations.…”

Antibodies Reactive with Streptococcal Peptidoglycan-Polysaccharide Complexes in Rheumatic Fever, Subacute Bacterial Endocarditis and Tuberculosis

“…The humoral immune response to bacterial PG is complex, with at least five well-defined antigenic de terminants expressed by the molecule [11], namely: (1) the glycan strand [12]; (2) the N-terminal moiety of the non-cross-linked interpeptide bridge [13]; (3) C-terminal sequences of the interpeptide bridge [14]; (4) the non-cross-linked tetrapeptide of the peptide subunits, with C-terminal L-lysyl-D-alanine as the immunodominant group [15], and (5) the non cross-linked pentapeptide of the peptide subunits, with C-terminal D-alanyl-D-alanine as the immuno dominant group [16].…”