The low‐molecular‐mass, penicillin‐binding proteins DacB and DacC combine to modify peptidoglycan cross‐linking and allow stable Type IV pilus expression in Neisseria gonorrhoeae

Abstract

Neisseria gonorrhoeae is the causative agent of the sexually transmitted infection gonorrhea and is adapted to survive in humans, its only host. The N. gonorrhoeae cell wall is critical for maintaining envelope integrity, resisting immune cell killing and production of cytotoxic peptidoglycan (PG) fragments. Deletion of the N. gonorrhoeae strain FA1090 genes encoding two predicted low-molecular-mass, penicillin-binding proteins (LMM PBPs), DacB and DacC, substantially altered the PG cross-linking. Loss of the DacB peptidase resulted in global alterations to the PG composition, while loss of the DacC protein affected a much narrower subset of PG peptide components. A double ΔdacB/ΔdacC mutant resembled the ΔdacB single mutant, but had an even greater level of cross-linked PG. While single ΔdacB or ΔdacC mutants did not show any major phenotypes, the ΔdacB/ΔdacC mutant displayed an altered cellular morphology, decreased resistance to antibiotics and increased sensitivity to detergent-mediated death. Loss of the two proteins also drastically reduced the number of Type IV pili (Tfp), a critical virulence factor. The decreased piliation reduced transformation efficiency and correlated with increased growth rate. While these two LMM PBPs differentially alter the PG composition, their overlapping effects are essential to proper envelope function and expression of factors critical for pathogenesis.

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

“…As reported previously for a dacB mutant of strain FA1090, mutation of dacB in FA19 resulted in a substantial decrease in the amount of tetrapeptides, a finding consistent with the idea that PBP3 acts as a carboxypeptidase to remove the fifth amino acid of the peptide ( Fig. 3) (26). In agreement with this conclusion, there was a corresponding increase in the amount of pentapeptide fragments.…”

“…Sacculi were prepared from each strain and were digested with mutanolysin to degrade the glycan strands. PG composition analysis for WT N. gonorrhoeae has been reported before, including the commonly studied strains MS11 and FA1090 (8,26). Those studies showed a sacculus containing mostly tetrapeptide side chains, a substantial amount of tripeptide side chains, and a minority of pentapeptides and dipeptides.…”

“…Peptidoglycan purification and composition analysis. PG sacculi were purified from N. gonorrhoeae strains grown in liquid culture as described previously (26). Purified sacculi were digested with mutanolysin and reduced with sodium borohydride, and PG fragments were separated by reversedphase HPLC.…”

RNA polymerase mutations cause cephalosporin resistance in clinicalNeisseria gonorrhoeaeisolates

“…Transcription of the putative L,D-transpeptidase gene ynhG was not altered in 159 the CRO RS strains ( Supplementary Figure 3). However, PBP1 (ponA) is upregulated in each of 160 the CRO RS strains compared to GCGS0457, while expression of PBP3, pbpG, and dacC is 161 decreased in these strains ( Figure 3A Table 2), in agreement with reported structural 166 changes of cell walls from strains lacking the DacB carboxypeptidase 18 . The increased 167 expression of PBP1 in these RNAP mutants does not appear to increase the abundance of 168 crosslinked peptidoglycan (Supplementary Figure 5 and Supplementary Table 2).…”

“…Since ESCs, like other β-lactams, block cell wall biosynthesis by 148 covalently inhibiting penicillin-binding proteins (PBPs), we examined how CRO RS -associated 149 RNAP mutations affect expression of enzymes in this target pathway. N. gonorrhoeae encodes 150 four known PBPs 17,18 : the essential high-molecular weight bifunctional 151 transpeptidase/transglycosylase PBP1 (encoded by ponA); the essential high-molecular weight 152 transpeptidase PBP2 (encoded by penA), which is the primary target of ESCs 8,11 ; and the 153 nonessential carboxypeptidases PBP3 (encoded by dacB) and PBP4 (encoded by pbpG), as well 154 as DacC (encoded by dacC), a third putative low-molecular weight carboxypeptidase that lacks 155 conserved active site motifs 18 . A homologue of the L,D-transpeptidase YnhG is also present in 156 the genome, although it has not been reported to be functional in N. gonorrhoeae.…”