Degradation of gonococcal peptidoglycan by granule extract from human neutrophils: demonstration of N-acetylglucosaminidase activity that utilizes peptidoglycan substrates

Abstract

The degradation of purified Neisseria gonorrhoeae peptidoglycan (PG) by granule extract derived from normal human polymorphonuclear leukocytes was examined. Hen egg lysozyme-resistant, extensively 0acetylated [3H]PG (O-PG) from strain FA19 and lysozyme-sensitive, non-O-acetylated ['4CJPG (non-O-PG) from strain RD5 (each containing label in both glucosamine and muramic acid) were mixed and incubated with granule extract at pHs 4.5, 5.5, and 6.5. The rate of degradation of O-PG was uniformly slower than that of non-O-PG in the same tube, but ultimately, even the O-PG was rendered completely soluble. Molecular-sieve high-performance liquid chronmatography revealed that both PGs were degraded by granule extract at the pH values tested to disaccharide peptide monomers and peptide-cross-linked oligomers, reflecting the action of human lysozyme. Of particular interest was the appearance of a peak containing free N-acetylglucosamine which was quite prominent in reaction mixtures at pH 4.5, less prominent at pH 5.5, and not detectable at pH 6.5. Free N-acetylglucosamine was not released from control PG samples at any pH in the absence of granule extract. Treatment of purified gonococcal PG monomers with granule extract at pH 4.5 yielded exclusively free N-acetylglucosamine and muramyl peptides with no N-acetylglucosamine. These data suggest that granule extract contains a previously undescribed pH-dependent N-acetylglucosaminidase with specificity for PG as well as an N-acetylmuramidase activity that degrades O-PG less efficiently than it does non-O-PG.on August 5, 2020 by guest http://iai.asm.org/ Downloaded from 2580 STRIKER ET AL.

Purification and characterization of N -acetylmuramyl- l -Alanine amidase from human plasma using monoclonal antibodies

“…It was shown that NAMLAA is a glycoprotein because the molecular mass decreased after incubating the enzyme with N-glycosidase F. This was confirmed by experiments where to some extent NAMLAA activity bound to Sambuccus Nigra Agglutinin (SNA) coupled to sepharose beads (results not shown). SNA has a specific affinity for oz-NeuNAc [2][3][4][5][6] GalNAc [17] which is a charged group.…”

“…An important factor in the induction of the biological effects is the ability of the peptidoglycan products to persist in human tissues. Bacterial cell wall products can be degraded by 3 different human enzymes: lysozyme [1], /3-N-acetylglucosaminidase [2] and the not well characterised N-acetylmuramyl-L-alanine amidase (NAMLAA). This NAMLAA hydrolyses peptidoglycan by cleaving the lactamide bond between N-acetyl muramic acid and L-alanine in the peptide side chain of the peptidoglycan molecule.…”

The role of bacteriolysis in the pathophysiology of inflammation, infection and post‐infectious sequelae

“…Granule extracts from human neutrophils containing a pH‐dependent N‐acetylglucosaminidase, degraded non‐O‐acetylated peptidoglycan (PPG) from gonococci, at acidic pH. This was much faster than the hydrolysis of O‐acetylated PPG shown to be highly resistant to lysozyme action (42, 94). The role of O‐acetylation in the pathogenicity of bacterial peptidoglycan has been discussed in detail (29–32, 52–55, 94).…”

Interactions of Neisseria gonorrhoeae with human neutrophils.

“…Certainly lysozyme (present in both specific and azurophil granules [104]) may have a role in degrading gonococcal PG in phagolysosomes. An unrelated lysosomal enzyme has been described by Striker et al (108) that is activated by acidic conditions and appears to remove terminal glucosamine residues in PG. Since this enzyme is activated by acidic conditions, it probably performs a PGmodifying activity in the terminal stages of phagolysosome development when the environment is acidic.…”