Neisseria gonorrhoeae PBP3 and PBP4 Facilitate NOD1 Agonist Peptidoglycan Fragment Release and Survival in Stationary Phase

Abstract

Neisseria gonorrhoeae releases peptidoglycan fragments during growth, and these molecules induce an inflammatory response in the human host. The proinflammatory molecules include peptidoglycan monomers, peptidoglycan dimers, and free peptides. These molecules can be released by the actions of lytic transglycosylases or an amidase. However, Ͼ40% of the gonococcal cell wall is cross-linked, where the peptide stem on one peptidoglycan strand is linked to the peptide stem on a neighboring strand, suggesting that endopeptidases may be required for the release of many peptidoglycan fragments. Therefore, we characterized mutants with individual or combined mutations in genes for the low-molecular-mass penicillin-binding proteins PBP3 and PBP4. Mutations in either dacB, encoding PBP3, or pbpG, encoding PBP4, did not significantly reduce the release of peptidoglycan monomers or free peptides. A mutation in dacB caused the appearance of a larger-sized peptidoglycan monomer, the pentapeptide monomer, and an increased release of peptidoglycan dimers, suggesting the involvement of this enzyme in both the removal of C-terminal D-Ala residues from stem peptides and the cleavage of cross-linked peptidoglycan. Mutation of both dacB and pbpG eliminated the release of tripeptide-containing peptidoglycan fragments concomitantly with the appearance of pentapeptide and dipeptide peptidoglycan fragments and higher-molecular-weight peptidoglycan dimers. In accord with the loss of tripeptide peptidoglycan fragments, the level of human NOD1 activation by the dacB pbpG mutants was significantly lower than that by the wild type. We conclude that PBP3 and PBP4 overlap in function for cross-link cleavage and that these endopeptidases act in the normal release of peptidoglycan fragments during growth. KEYWORDS NOD1, NOD1 agonist, carboxypeptidase, endopeptidase, penicillinbinding proteins, peptidoglycan Citation Schaub RE, Perez-Medina KM, Hackett KT, Garcia DL, Dillard JP. 2019. Neisseria gonorrhoeae PBP3 and PBP4 facilitate NOD1 agonist peptidoglycan fragment release and survival in stationary phase. Infect Immun

Structural Modeling of Cell Wall Peptidase CwpFM (EntFM) Reveals Distinct Intrinsically Disordered Extensions Specific to Pathogenic Bacillus cereus Strains

“…A recent work has demonstrated that the PG hydrolase Cwp19 contributes to Clostridium difficile autolysis, which induces the release of bacterial toxins [72]. Markedly, PG fragments released by CWPs can also act as signaling molecules to promote the presence of antimicrobial agents or to interact with the component of the host [73,74].…”

The Pathogenic Neisseria Use a Streamlined Set of Peptidoglycan Degradation Proteins for Peptidoglycan Remodeling, Recycling, and Toxic Fragment Release

“…Strain FA19 was found to cause a large NOD2 response in epithelial cells both to soluble PG fragments released by the bacteria during growth and to purified sacculi. Compositional analyses demonstrated that both the sacculus and the released fragments contained much larger amounts of dipeptide chains compared to other gonococcal strains (Schaub et al, 2019). This observation suggests that Neisseria have another, yet uncharacterized, endopeptidase and that this enzyme may differ between strains or may be regulated to give larger or smaller NOD2 responses.…”

“…However, the requirement for peptidases for NOD1 agonist production was not quite so obvious. Mutations affecting dacB (encoding PBP3) and pbpG (encoding PBP4) were also demonstrated to decimate NOD1 activation by N. gonorrhoeae (Schaub et al, 2019). These enzymes both cleave the common peptide crosslinks (Ala-DAP) and remove the fifth amino acid ( D -Ala) (Stefanova et al, 2003; Schaub et al, 2019).…”

“…Mutations affecting dacB (encoding PBP3) and pbpG (encoding PBP4) were also demonstrated to decimate NOD1 activation by N. gonorrhoeae (Schaub et al, 2019). These enzymes both cleave the common peptide crosslinks (Ala-DAP) and remove the fifth amino acid ( D -Ala) (Stefanova et al, 2003; Schaub et al, 2019). When the fifth amino acid is not removed, LdcA cannot cleave the fourth amino acid to leave a strand terminating in DAP (Schaub et al, 2019).…”

“…These enzymes both cleave the common peptide crosslinks (Ala-DAP) and remove the fifth amino acid ( D -Ala) (Stefanova et al, 2003; Schaub et al, 2019). When the fifth amino acid is not removed, LdcA cannot cleave the fourth amino acid to leave a strand terminating in DAP (Schaub et al, 2019). Also, the L,D -transpeptidase cannot act to make DAP–DAP crosslinks and in the process cleave an Ala-DAP bond.…”