A Single Dual-Function Enzyme Controls the Production of Inflammatory NOD Agonist Peptidoglycan Fragments by Neisseria gonorrhoeae

Abstract

Neisseria gonorrhoeae gonococcus (GC) is a Gram-negative betaproteobacterium and causative agent of the sexually transmitted infection gonorrhea. During growth, GC releases lipooligosaccharide (LOS) and peptidoglycan (PG) fragments, which contribute significantly to the inflammatory damage observed during human infection. In ascending infection of human Fallopian tubes, inflammation leads to increased risk of ectopic pregnancy, pelvic inflammatory disease, and sterility. Of the PG fragments released by GC, most are disaccharide peptide monomers, and of those, 80% have tripeptide stems despite the observation that tetrapeptide stems make up 80% of the assembled cell wall. We identified a serine-protease l,d-carboxypeptidase, NGO1274 (LdcA), as the enzyme responsible for converting cell wall tetrapeptide-stem PG to released tripeptide-stem PG. Unlike characterized cytoplasmic LdcA homologs in gammaproteobacteria, LdcA in GC is exported to the periplasm, and its localization is critical for its activity in modifying PG fragments for release. Distinct among other characterized l,d-carboxypeptidases, LdcA from GC is also capable of catalyzing the cleavage of specific peptide cross-bridges (endopeptidase activity). To define the role of ldcA in pathogenesis, we demonstrate that ldcA disruption results in both loss of NOD1-dependent NF-κB activation and decreased NOD2-dependent NF-κB activation while not affecting Toll-like receptor (TLR) agonist release. Since the human intracellular peptidoglycan receptor NOD1 (hNOD1) specifically recognizes PG fragments with a terminal meso-DAP rather than d-alanine, we conclude that LdcA is required for GC to provoke NOD1-dependent responses in cells of the human host.

A Francisella tularensis L,D-carboxypeptidase plays important roles in cell morphology, envelope integrity, and virulence

“…At this time, we can only speculate on why most Gram-negative bacteria encode cytosolic LdcAs but F. tularensis encodes a periplasmic LdcA, but in the context of PG repair and recycling, periplasmic LdcA certainly offers a fitness advantage. This is not the first report of a periplasmic LdcA, as C. jejuni Pgp2 is predicted to be periplasmic and N. gonorrhoeae LdcA is periplasmic (31, 32).…”

“…1, P. aeruginosa LdcA contains a Ser-His-Glu catalytic triad which is essential for function and is characteristic of Ldc in the Peptidase_S66 family (30). The Ser-His-Glu catalytic triad also has been confirmed in Ldc from E. coli (47), Novosphingobium aromaticivorans (48), and N. gonorrhoeae (31). Given the relatively conserved spacing of Ser-His-Glu residues in FTL1678 (Fig.…”

“…S1). Ldc proteins have been studied in a number of Gram-negative bacteria, including E. coli (26–29), Pseudomonas aeruginosa (30), N. gonorrhoeae (31), and Campylobacter jejuni (32). To further explore this conserved domain, amino acid sequences of FTL1678 ( F. tularensis subsp.…”

“…In addition, we demonstrated that F. tularensis LdcA cleaves PG pentapeptides without a prior cleavage event by a D,D-carboxypeptidase/Penicillin Binding Protein (PBP), such as DacD. It should be noted that although N. gonorrhoeae LdcA has been reported to have L,D-endopeptidase activity, this activity is specific to tetrapeptide cross links (4-3 and 3-3 links) (31). Interestingly, only two previous studies have examined putative F. tularensis PG modifying enzymes.…”

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

“…An N-acetylmuramyl-L-alanine amidase cleaves the peptide chains from the glycan strands (10,11). PG peptidases, including endopeptidases and carboxypeptidases, cut the peptide cross-links and shorten the peptide stems (12,13). The PG fragments produced by these enzymes are released in significant amounts by growing N. gonorrhoeae cells (8,14).…”