Characterization of Staphylococcus aureus cell wall glycan strands, evidence for a new beta-N-acetylglucosaminidase activity

Using sequential digestion with the glycyl-glycine endopeptidase lysostaphi n followed by the pneumococcal N-acetylmuramyl-L-alanine amidase (amidase), the glycan strands of the peptidoglycan of Staphylococcus aureus were puri fied and analyzed by a combination of reverse-phase-high pressure Liquid ch romatography (HPLC) and mass spectrometry. Reverse-phase-RPLC resolved the glycan strands to a family of major peaks, which represented oligosaccharid es composed of repeating disaccharide units (N-acetylglucosamine-[beta-1,4] -N-acetylmuramic acid) with different degrees of polymerization and termina ting with N-acetylmuramic acid residues at the reducing ends. The method al lowed separation of strands up to 23-26 disaccharide units with a predomina nt length between 3 and 10 and an average degree of polymerization of simil ar to 6. Glycan strands with a higher degree of polymerization (>26 disacch aride units) represented 10-15% of the total UV absorbing glycan material. A unique feature of the staphylococcal glycan strands was the presence of m inor satellite peaks that were present throughout the HPLC elution profile eluting either just prior or shortly after the major oligosaccharide peaks. A number of observations including mass spectrometric analysis suggest tha t the satellites are the products of an N-acetylglucosaminidase activity th at differs from the atl gene product and that appears to be involved with m odification of the glycan strand structure.