Peptidoglycan fragment release and NOD activation by commensal Neisseria species from humans and other animals.

Neisseria gonorrhoeae, a human restricted pathogen, releases inflammatory peptidoglycan (PG) fragments that contribute to the pathophysiology of pelvic inflammatory disease. The genus Neisseria is also home to multiple species of human- or animal-associated Neisseria that form part of the normal microbiota. Here we characterized PG release from the human-associated nonpathogenic species Neisseria lactamica and Neisseria mucosa and animal-associated Neisseria from macaques and wild mice. An N. mucosa strain and an N. lactamica strain were found to release limited amounts of the proinflammatory monomeric PG fragments. However, a single amino acid difference in the PG fragment permease AmpG resulted in increased PG fragment release in a second N. lactamica strain examined. Neisseria isolated from macaques also showed substantial release of PG monomers. The mouse colonizer Neisseria musculi exhibited PG fragment release similar to that seen in N. gonorrhoeae with PG monomers being the predominant fragments released. All the human-associated species were able to stimulate NOD1 and NOD2 responses. N. musculi was a poor inducer of mouse NOD1, but ldcA mutation increased this response. The ability to genetically manipulate N. musculi and examine effects of different PG fragments or differing amounts of PG fragments during mouse colonization will lead to a better understanding of the roles of PG in Neisseria infections. Overall, we found that only some nonpathogenic Neisseria have diminished release of proinflammatory PG fragments, and there are differences even within a species as to types and amounts of PG fragments released.

Mutation of mltG increases peptidoglycan fragment release, cell size, and antibiotic susceptibility in Neisseria gonorrhoeae.

IMPORTANCE: Neisseria gonorrhoeae is unusual in that the bacteria release larger amounts of cell wall material as they grow as compared to related bacteria, and the released cell wall fragments induce inflammation that leads to tissue damage in infected people. The study of MltG revealed the importance of this enzyme for controlling cell wall growth, cell wall fragment production, and bacterial cell size and suggests a role for MltG in a cell wall synthesis and degradation complex. The increased antibiotic sensitivities of mltG mutants suggest that an antimicrobial drug inhibiting MltG would be useful in combination therapy to restore the sensitivity of the bacteria to cell wall targeting antibiotics to which the bacteria are currently resistant.

The AmiC/NlpD Pathway Dominates Peptidoglycan Breakdown in Neisseria meningitidis and Affects Cell Separation, NOD1 Agonist Production, and Infection.

The human-restricted pathogen Neisseria meningitidis, which is best known for causing invasive meningococcal disease, has a nonpathogenic lifestyle as an asymptomatic colonizer of the human naso- and oropharyngeal space. N. meningitidis releases small peptidoglycan (PG) fragments during growth. It was demonstrated previously that N. meningitidis releases low levels of tripeptide PG monomer, which is an inflammatory molecule recognized by the human intracellular innate immune receptor NOD1. In the present study, we demonstrated that N. meningitidis released more PG-derived peptides than PG monomers. Using a reporter cell line overexpressing human NOD1, we showed that N. meningitidis activates NOD1 using PG-derived peptides. The generation of such peptides required the presence of the periplasmic N-acetylmuramyl-l-alanine amidase AmiC and the outer membrane lipoprotein NlpD. AmiC and NlpD were found to function in cell separation, and mutation of either amiC or nlpD resulted in large clumps of unseparated N. meningitidis cells instead of the characteristic diplococci. Using stochastic optical reconstruction microscopy, we demonstrated that FLAG epitope-tagged NlpD localized to the septum, while similarly tagged AmiC was found at the septum in some diplococci but was distributed around the cell in most cases. In a human whole-blood infection assay, an nlpD mutant was severely attenuated and showed particular sensitivity to complement. Thus, in N. meningitidis, the cell separation proteins AmiC and NlpD are necessary for NOD1 stimulation and survival during infection of human blood.

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

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, >40% 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.

Two lytic transglycosylases in Neisseria gonorrhoeae impart resistance to killing by lysozyme and human neutrophils.

Symptomatic infection by Neisseria gonorrhoeae (Gc) produces a potent inflammatory response, resulting in a neutrophil-rich exudate. A population of Gc can survive the killing activities of neutrophils for reasons not completely understood. Unlike other Gram-negative bacteria, Gc releases monomeric peptidoglycan (PG) extracellularly, dependent on two nonessential, nonredundant lytic transglycosylases (LTs), LtgA and LtgD. PG released by LtgA and LtgD can stimulate host immune responses. We report that ΔltgAΔltgD Gc were decreased in survival in the presence of primary human neutrophils but otherwise grew equally to wild-type Gc. Adding PG monomer failed to alter ΔltgAΔltgD Gc survival. Thus, LTs protect Gc from neutrophils independently of monomer release. We found two reasons to explain decreased survival of the double LT mutant. First, ΔltgAΔltgD Gc was more sensitive to the neutrophil antimicrobial proteins lysozyme and neutrophil elastase, but not others. Sensitivity to lysozyme correlated with decreased Gc envelope integrity. Second, exposure of neutrophils to ΔltgAΔltgD Gc increased the release of neutrophil granule contents extracellularly and into Gc phagosomes. We conclude that LtgA and LtgD protect Gc from neutrophils by contributing to envelope integrity and limiting bacterial exposure to select granule-localized antimicrobial proteins. These observations are the first to link bacterial degradation by lysozyme to increased neutrophil activation.

Amidase Activity of AmiC Controls Cell Separation and Stem Peptide Release and Is Enhanced by NlpD in Neisseria gonorrhoeae.

The human-restricted pathogen Neisseria gonorrhoeae encodes a single N-acetylmuramyl-l-alanine amidase involved in cell separation (AmiC), as compared with three largely redundant cell separation amidases found in Escherichia coli (AmiA, AmiB, and AmiC). Deletion of amiC from N. gonorrhoeae results in severely impaired cell separation and altered peptidoglycan (PG) fragment release, but little else is known about how AmiC functions in gonococci. Here, we demonstrated that gonococcal AmiC can act on macromolecular PG to liberate cross-linked and non-cross-linked peptides indicative of amidase activity, and we provided the first evidence that a cell separation amidase can utilize a small synthetic PG fragment as substrate (GlcNAc-MurNAc(pentapeptide)-GlcNAc-MurNAc(pentapeptide)). An investigation of two residues in the active site of AmiC revealed that Glu-229 is critical for both normal cell separation and the release of PG fragments by gonococci during growth. In contrast, Gln-316 has an autoinhibitory role, and its mutation to lysine resulted in an AmiC with increased enzymatic activity on macromolecular PG and on the synthetic PG derivative. Curiously, the same Q316K mutation that increased AmiC activity also resulted in cell separation and PG fragment release defects, indicating that activation state is not the only factor determining normal AmiC activity. In addition to displaying high basal activity on PG, gonococcal AmiC can utilize metal ions other than the zinc cofactor typically used by cell separation amidases, potentially protecting its ability to function in zinc-limiting environments. Thus gonococcal AmiC has distinct differences from related enzymes, and these studies revealed parameters for how AmiC functions in cell separation and PG fragment release.

Targeted mutagenesis of intergenic regions in the Neisseria gonorrhoeae gonococcal genetic island reveals multiple regulatory mechanisms controlling type IV secretion.

Gonococci secrete chromosomal DNA into the extracellular environment using a type IV secretion system (T4SS). The secreted DNA acts in natural transformation and initiates biofilm development. Although the DNA and its effects are detectable, structural components of the T4SS are present at very low levels, suggestive of uncharacterized regulatory control. We sought to better characterize the expression and regulation of T4SS genes and found that the four operons containing T4SS genes are transcribed at very different levels. Increasing transcription of two of the operons through targeted promoter mutagenesis did not increase DNA secretion. The stability and steady-state levels of two T4SS structural proteins were affected by a homolog of tail-specific protease. An RNA switch was also identified that regulates translation of a third T4SS operon. The switch mechanism relies on two putative stem-loop structures contained within the 5' untranslated region of the transcript, one of which occludes the ribosome binding site and start codon. Mutational analysis of these stem loops supports a model in which induction of an alternative structure relieves repression. Taken together, these results identify multiple layers of regulation, including transcriptional, translational and post-translational mechanisms controlling T4SS gene expression and DNA secretion.

TraK and TraB are conserved outer membrane proteins of the Neisseria gonorrhoeae Type IV secretion system and are expressed at low levels in wild-type cells.

Neisseria gonorrhoeae uses a type IV secretion system (T4SS) to secrete chromosomal DNA into the medium, and this DNA is effective in transforming other gonococci via natural transformation. In addition, the T4SS is important in the initial stages of biofilm development and mediates intracellular iron uptake in the absence of TonB. To better understand the mechanism of type IV secretion in N. gonorrhoeae, we examined the expression levels and localization of two predicted T4SS outer membrane proteins, TraK and TraB, in the wild-type strain as well as in overexpression strains and in a strain lacking all of the T4SS proteins. Despite very low sequence similarity to known homologues, TraB (VirB10 homolog) and TraK (VirB9 homolog) localized similarly to related proteins in other systems. Additionally, we found that TraV (a VirB7 homolog) interacts with TraK, as in other T4SSs. However, unlike in other systems, neither TraK nor TraB required the presence of other T4SS components for proper localization. Unlike other gonococcal T4SS proteins we have investigated, protein levels of the outer membrane proteins TraK and TraB were extremely low in wild-type cells and were undetectable by Western blotting unless overexpressed or tagged with a FLAG3 triple-epitope tag. Localization of TraK-FLAG3 in otherwise wild-type cells using immunogold electron microscopy of thin sections revealed a single gold particle on some cells. These results suggest that the gonococcal T4SS may be present in single copy per cell and that small amounts of T4SS proteins TraK and TraB are sufficient for DNA secretion.

IL-17C is a driver of damaging inflammation during Neisseria gonorrhoeae infection of human Fallopian tube.

The human pathogen Neisseria gonorrhoeae ascends into the upper female reproductive tract to cause damaging inflammation within the Fallopian tubes and pelvic inflammatory disease (PID), increasing the risk of infertility and ectopic pregnancy. The loss of ciliated cells from the epithelium is thought to be both a consequence of inflammation and a cause of adverse sequelae. However, the links between infection, inflammation, and ciliated cell extrusion remain unresolved. With the use of ex vivo cultures of human Fallopian tube paired with RNA sequencing we defined the tissue response to gonococcal challenge, identifying cytokine, chemokine, cell adhesion, and apoptosis related transcripts not previously recognized as potentiators of gonococcal PID. Unexpectedly, IL-17C was one of the most highly induced genes. Yet, this cytokine has no previous association with gonococcal infection nor pelvic inflammatory disease and thus it was selected for further characterization. We show that human Fallopian tubes express the IL-17C receptor on the epithelial surface and that treatment with purified IL-17C induces pro-inflammatory cytokine secretion in addition to sloughing of the epithelium and generalized tissue damage. These results demonstrate a previously unrecognized but critical role of IL-17C in the damaging inflammation induced by gonococci in a human explant model of PID.

Mating pair formation homologue TraG is a variable membrane protein essential for contact-independent type IV secretion of chromosomal DNA by Neisseria gonorrhoeae.

Neisseria gonorrhoeae uses a type IV secretion system (T4SS) to secrete chromosomal DNA into the surrounding milieu. The DNA is effective in transforming gonococci in the population, and this mechanism of DNA donation may contribute to the high degree of genetic diversity in this species. Similar to other F-like T4SSs, the gonococcal T4SS requires a putative membrane protein, TraG, for DNA transfer. In F-plasmid and related systems, the homologous protein acts in pilus production, mating pair stabilization, and entry exclusion. We characterized the localization, membrane topology, and variation of TraG in N. gonorrhoeae. TraG was found to be an inner-membrane protein with one large periplasmic region and one large cytoplasmic region. Each gonococcal strain carried one of three different alleles of traG. Strains that carried the smallest allele of traG were found to lack the peptidoglycanase gene atlA but carried a peptidoglycan endopeptidase gene in place of atlA. The purified endopeptidase degraded gonococcal peptidoglycan in vitro, cutting the peptide cross-links. Although the other two traG alleles functioned for DNA secretion in strain MS11, the smallest traG did not support DNA secretion. Despite the requirement for a mating pair stabilization homologue, static coculture transformation experiments demonstrated that DNA transfer was nuclease sensitive and required active uptake by the recipient, thus demonstrating that transfer occurred by transformation and not conjugation. Together, these results demonstrate the TraG acts in a process of DNA export not specific to conjugation and that different forms of TraG affect what substrates can be transported.

Neisseria gonorrhoeae virulence factor NG1686 is a bifunctional M23B family metallopeptidase that influences resistance to hydrogen peroxide and colony morphology.

Symptomatic gonococcal infection, caused exclusively by the human-specific pathogen Neisseria gonorrhoeae (the gonococcus), is characterized by the influx of polymorphonuclear leukocytes (PMNs) to the site of infection. Although PMNs possess a potent antimicrobial arsenal comprising both oxidative and non-oxidative killing mechanisms, gonococci survive this interaction, suggesting that the gonococcus has evolved many defenses against PMN killing. We previously identified the NG1686 protein as a gonococcal virulence factor that protects against both non-oxidative PMN-mediated killing and oxidative killing by hydrogen peroxide. In this work, we show that deletion of ng1686 affects gonococcal colony morphology but not cell morphology and that overexpression of ng1686 does not confer enhanced survival to hydrogen peroxide on gonococci. NG1686 contains M23B endopeptidase active sites found in proteins that cleave bacterial cell wall peptidoglycan. Strains of N. gonorrhoeae expressing mutant NG1686 proteins with substitutions in many, but not all, conserved metallopeptidase active sites recapitulated the hydrogen peroxide sensitivity and altered colony morphology of the Δng1686 mutant strain. We showed that purified NG1686 protein degrades peptidoglycan in vitro and that mutations in many conserved active site residues abolished its degradative activity. Finally, we demonstrated that NG1686 possesses both dd-carboxypeptidase and endopeptidase activities. We conclude that the NG1686 protein is a M23B peptidase with dual activities that targets the cell wall to affect colony morphology and resistance to hydrogen peroxide and PMN-mediated killing.

Peptidoglycan fragment release from Neisseria meningitidis.

Neisseria meningitidis (meningococcus) is a symbiont of the human nasopharynx. On occasion, meningococci disseminate from the nasopharynx to cause invasive disease. Previous work showed that purified meningococcal peptidoglycan (PG) stimulates human Nod1, which leads to activation of NF-κB and production of inflammatory cytokines. No studies have determined if meningococci release PG or activate Nod1 during infection. The closely related pathogen Neisseria gonorrhoeae releases PG fragments during normal growth. These fragments induce inflammatory cytokine production and ciliated cell death in human fallopian tubes. We determined that meningococci also release PG fragments during growth, including fragments known to induce inflammation. We found that N. meningitidis recycles PG fragments via the selective permease AmpG and that meningococcal PG recycling is more efficient than gonococcal PG recycling. Comparison of PG fragment release from N. meningitidis and N. gonorrhoeae showed that meningococci release less of the proinflammatory PG monomers than gonococci and degrade PG to smaller fragments. The decreased release of PG monomers by N. meningitidis relative to N. gonorrhoeae is partly due to ampG, since replacement of gonococcal ampG with the meningococcal allele reduced PG monomer release. Released PG fragments in meningococcal supernatants induced significantly less Nod1-dependent NF-κB activity than released fragments in gonococcal supernatants and tended to induce less interleukin-8 (IL-8) secretion in primary human fallopian tube explants. These results support a model in which efficient PG recycling and extensive degradation of PG fragments lessen inflammatory responses and may be advantageous for maintaining meningococcal carriage in the nasopharynx.

Mutation of mltG increases peptidoglycan fragment release, cell size, and antibiotic susceptibility in Neisseria gonorrhoeae.

Infection with the Gram-negative species Neisseria gonorrhoeae leads to inflammation that is responsible for the disease symptoms of gonococcal urethritis, cervicitis, and pelvic inflammatory disease. During growth these bacteria release significant amounts of peptidoglycan (PG) fragments which elicit inflammatory responses in the human host. To better understand the mechanisms involved in PG synthesis and breakdown in N. gonorrhoeae, we characterized the effects of mutation of mltG. MltG has been identified in other bacterial species as a terminase that stops PG strand growth by cleaving the growing glycan. Mutation of mltG in N. gonorrhoeae did not affect bacterial growth rate but resulted in increased PG turnover, more cells of large size, decreased autolysis under non-growth conditions, and increased sensitivity to antibiotics that affect PG crosslinking. An mltG mutant released greatly increased amounts of PG monomers, PG dimers, and larger oligomers. In the mltG background, mutation of either ltgA or ltgD, encoding the lytic transglycosylases responsible for PG monomer liberation, resulted in wild-type levels of PG monomer release. Bacterial two-hybrid assays identified positive interactions of MltG with synthetic penicillin-binding proteins PBP1 and PBP2 and the PG-degrading endopeptidase PBP4 (PbpG). These data are consistent with MltG acting as a terminase in N. gonorrhoeae and suggest that absence of MltG activity results in excessive PG growth and extra PG in the sacculus that must be degraded by lytic transglycosylases including LtgA and LtgD. Furthermore, absence of MltG causes a cell wall defect that is manifested as large cell size and antibiotic sensitivity.

Prevalence and detailed mapping of the gonococcal genetic island in Neisseria meningitidis.

The 57-kb gonococcal genetic island (GGI) encodes a type IV secretion system (T4SS) that is found in most strains of N. gonorrhoeae. This T4SS functions to secrete single-stranded DNA that is active in natural transformation. The GGI has also been found in some strains of N. meningitidis. We screened 126 isolates of N. meningitidis and found the GGI in 17.5% of strains, with the prevalence varying widely among serogroups. The GGI is found in a significant number of serogroup C, W-135, and X strains but was not found in strains of serogroup A, B, or Y. Through detailed PCR mapping and DNA sequencing, we identified five distinct GGI types in meningococci. DNA sequencing and a genetic assay revealed that the GGI was likely integrated into the meningococcal chromosome by the site-specific recombinase XerCD and that the GGI can be excised and lost from the genome. Functional studies showed that in contrast with the gonococcal T4SS, the meningococcal T4SS does not secrete DNA, nor does it confer Ton-independent intracellular survival. Deletion of T4SS genes did not affect association with or invasion of host cells. These results demonstrate that the GGI is found in a significant proportion of meningococcal strains and that while some strains carry multiple insertions and deletions in the GGI, other strains carry intact T4SS genes and may produce functional secretion systems.

New complementation constructs for inducible and constitutive gene expression in Neisseria gonorrhoeae and Neisseria meningitidis.

We have created new complementation constructs for use in Neisseria gonorrhoeae and Neisseria meningitidis. The constructs contain regions of homology with the chromosome and direct the insertion of a gene of interest into the intergenic region between the genes iga and trpB. In order to increase the available options for gene expression in Neisseria, we designed the constructs to contain one of three different promoters. One of the constructs contains the isopropyl-ß-d-thiogalactopyranoside-inducible lac promoter, which has been widely used in Neisseria. We also designed a construct that contains the strong, constitutive promoter from the gonococcal opaB gene. The third construct contains a tetracycline-inducible promoter, a novel use of this promoter in Neisseria. We demonstrate that anhydrotetracycline can be used to induce gene expression in the pathogenic Neisseria at very low concentrations and without negatively affecting the growth of the organisms. We use these constructs to complement an arginine auxotrophy in N. gonorrhoeae as well as to express a translational fusion of alkaline phosphatase with TraW. TraW is a component of the gonococcal type IV secretion system, and we demonstrate that TraW localizes to the periplasm.

XerCD-mediated site-specific recombination leads to loss of the 57-kilobase gonococcal genetic island.

Most strains of Neisseria gonorrhoeae carry the 57-kb gonococcal genetic island (GGI), as do a few strains of Neisseria meningitidis. The GGI is inserted into the chromosome at the dif site (difA) and is flanked by a partial repeat of the dif site (difB). Since dif is a sequence recognized by the site-specific recombinases XerC and XerD and the GGI shows evidence of horizontal acquisition, we hypothesized that the GGI may be acquired or lost by XerCD-mediated site-specific recombination. We show that while the GGI flanked by wild-type dif sites, difA and difB, is not readily lost from the gonococcal chromosome, the substitution of difB with another copy of difA allows the frequent excision and loss of the GGI. In mutants carrying two difA sites (difA(+) difA(+)), the GGI can be detected as an extrachromosomal circle that exists transiently. A mutation of xerD diminished GGI excision from the chromosome of a difA(+) difA(+) strain, while mutations in recA or type IV secretion genes had no effect on the loss of the GGI. These data indicate that the GGI is maintained by the replication of the chromosome and that GGI excision and loss are dependent upon the dif sequence and xerD. The detection of a circular form of the GGI in a wild-type strain suggests that GGI excision may occur naturally and could function to facilitate GGI transfer. These data suggest a model of GGI excision and loss explaining the absence of the GGI from some gonococcal strains and the maintenance of variant GGIs in some gonococcal and meningococcal isolates.

Expression, Localization, and Protein Interactions of the Partitioning Proteins in the Gonococcal Type IV Secretion System.

Partitioning proteins are well studied as molecular organizers of chromosome and plasmid segregation during division, however little is known about the roles partitioning proteins can play within type IV secretion systems. The single-stranded DNA (ssDNA)-secreting gonococcal T4SS has two partitioning proteins, ParA and ParB. These proteins work in collaboration with the relaxase TraI as essential facilitators of type IV secretion. Bacterial two-hybrid experiments identified interactions between each partitioning protein and the relaxase. Subcellular fractionation demonstrated that ParA is found in the cellular membrane, whereas ParB is primarily in the membrane, but some of the protein is in the soluble fraction. Since TraI is known to be membrane-associated, these data suggest that the gonococcal relaxosome is a membrane-associated complex. In addition, we found that translation of ParA and ParB is controlled by an RNA switch. Different mutations within the stem-loop sequence predicted to alter folding of this RNA structure greatly increased or decreased levels of the partitioning proteins.

Increased expression of the type IV secretion system in piliated Neisseria gonorrhoeae variants.

Neisseria gonorrhoeae produces a type IV secretion system that secretes chromosomal DNA. The secreted DNA is active in the transformation of other gonococci in the population and may act to transfer antibiotic resistance genes and variant alleles for surface antigens, as well as other genes. We observed that gonococcal variants that produced type IV pili secreted more DNA than variants that were nonpiliated, suggesting that the process may be regulated. Using microarray analysis, we found that a piliated strain showed increased expression of the gene for the putative type IV secretion coupling protein TraD, whereas a nonpiliated variant showed increased expression of genes for transcriptional and translational machinery, consistent with its higher growth rate compared to that of the piliated strain. These results suggested that type IV secretion might be controlled by either traD expression or growth rate. A mutant with a deletion in traD was found to be deficient in DNA secretion. Further mutation and complementation analysis indicated that traD is transcriptionally and translationally coupled to traI, which encodes the type IV secretion relaxase. We were able to increase DNA secretion in a nonpiliated strain by inserting a gene cassette with a strong promoter to drive the expression of the putative operon containing traI and traD. Together, these data suggest a model in which the type IV secretion system apparatus is made constitutively, while its activity is controlled through regulation of traD and traI.

Neisseria gonorrhoeae uses two lytic transglycosylases to produce cytotoxic peptidoglycan monomers.

Peptidoglycan fragments released by Neisseria gonorrhoeae contribute to the inflammation and ciliated cell death associated with gonorrhea and pelvic inflammatory disease. However, little is known about the production and release of these fragments during bacterial growth. Previous studies demonstrated that one lytic transglycosylase, LtgA, was responsible for the production of approximately half of the released peptidoglycan monomers. Systematic mutational analysis of other putative lytic transglycosylase genes identified lytic transglycosylase D (LtgD) as responsible for release of peptidoglycan monomers from gonococci. An ltgA ltgD double mutant was found not to release peptidoglycan monomers and instead released large, soluble peptidoglycan fragments. In pulse-chase experiments, recycled peptidoglycan was not found in cytoplasmic extracts from the ltgA ltgD mutant as it was for the wild-type strain, indicating that generation of anhydro peptidoglycan monomers by lytic transglycosylases facilitates peptidoglycan recycling. The ltgA ltgD double mutant showed no growth abnormalities or cell separation defects, suggesting that these enzymes are involved in pathogenesis but not necessary for normal growth.

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

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.IMPORTANCE The macromolecular meshwork of peptidoglycan serves essential functions in determining bacterial cell shape, protecting against osmotic lysis, and defending cells from external assaults. The conserved peptidoglycan structure, however, is also recognized by eukaryotic pattern recognition receptors, which can trigger immune responses against bacteria. Many bacteria can induce an inflammatory response through the intracellular peptidoglycan receptor NOD1, but Neisseria gonorrhoeae serves as an extreme example, releasing fragments of peptidoglycan into the environment during growth that specifically antagonize human NOD1. Understanding the peptidoglycan breakdown mechanisms that allow Neisseria to promote NOD1 activation, rather than avoiding or suppressing immune detection, is critical to understanding the pathogenesis of this increasingly drug-resistant organism. We identify a peptidoglycan l,d-carboxypeptidase responsible for converting liberated peptidoglycan fragments into the human NOD1 agonist and find that the same enzyme has endopeptidase activity on certain peptidoglycan cross-links, the first described combination of those two activities in a single enzyme.