The Neisseria gonorrhoeae type IV pilus promotes resistance to hydrogen peroxide- and LL-37-mediated killing by modulating the availability of intracellular, labile iron.

The Neisseria gonorrhoeae Type IV pilus is a multifunctional, dynamic fiber involved in host cell attachment, DNA transformation, and twitching motility. We previously reported that the N. gonorrhoeae pilus is also required for resistance against hydrogen peroxide-, antimicrobial peptide LL-37-, and non-oxidative, neutrophil-mediated killing. We tested whether the hydrogen peroxide, LL-37, and neutrophil hypersensitivity phenotypes in non-piliated N. gonorrhoeae could be due to elevated iron levels. Iron chelation in the growth medium rescued a nonpiliated pilE mutant from both hydrogen peroxide- and antimicrobial peptide LL-37-mediated killing, suggesting these phenotypes are related to iron availability. We used the antibiotic streptonigrin, which depends on free cytoplasmic iron and oxidation to kill bacteria, to determine whether piliation affected intracellular iron levels. Several non-piliated, loss-of-function mutants were more sensitive to streptonigrin killing than the piliated parental strain. Consistent with the idea that higher available iron levels in the under- and non-piliated strains were responsible for the higher streptonigrin sensitivity, iron limitation by desferal chelation restored resistance to streptonigrin in these strains and the addition of iron restored the sensitivity to streptonigrin killing. The antioxidants tiron and dimethylthiourea rescued the pilE mutant from streptonigrin-mediated killing, suggesting that the elevated labile iron pool in non-piliated bacteria leads to streptonigrin-dependent reactive oxygen species production. These antioxidants did not affect LL-37-mediated killing. We confirmed that the pilE mutant is not more sensitive to other antibiotics showing that the streptonigrin phenotypes are not due to general bacterial envelope disruption. The total iron content of the cell was unaltered by piliation when measured using ICP-MS suggesting that only the labile iron pool is affected by piliation. These results support the hypothesis that piliation state affects N. gonorrhoeae iron homeostasis and influences sensitivity to various host-derived antimicrobial agents.

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.

The Gonococcal NlpD Protein Facilitates Cell Separation by Activating Peptidoglycan Cleavage by AmiC.

UNLABELLED: Key steps in bacterial cell division are the synthesis and subsequent hydrolysis of septal peptidoglycan (PG), which allow efficient separation of daughter cells. Extensive studies in the Gram-negative, rod-shaped bacterium Escherichia coli have revealed that this hydrolysis is highly regulated spatially and temporally. Neisseria gonorrhoeae is an obligate Gram-negative, diplococcal pathogen and is the only causative agent of the sexually transmitted infection gonorrhea. We investigated how cell separation proceeds in this diplococcal organism. We demonstrated that deletion of the nlpD gene in strain FA1090 leads to poor growth and to an altered colony and cell morphology. An isopropyl-beta-d-galactopyranoside (IPTG)-regulated nlpD complemented construct can restore these defects only when IPTG is supplied in the growth medium. Thin-section transmission electron microscopy (TEM) revealed that the nlpD mutant strain grew in large clumps containing live and dead bacteria, which was consistent with deficient cell separation. Biochemical analyses of purified NlpD protein showed that it was able to bind purified PG. Finally, we showed that, although NlpD has no hydrolase activity itself, NlpD potentiates the hydrolytic activity of AmiC. These results indicate that N. gonorrhoeae NlpD is required for proper cell growth and division through its interactions with the amidase AmiC. IMPORTANCE: N. gonorrhoeae is the sole causative agent of the sexually transmitted infection gonorrhea. The incidence of antibiotic-resistant gonococcal infections has risen sharply in recent years, and N. gonorrhoeae has been classified as a "superbug" by the CDC. Since there is a dearth of new antibiotics to combat gonococcal infections, elucidating the essential cellular process of N. gonorrhoeae may point to new targets for antimicrobial therapies. Cell division and separation is one such essential process. We identified and characterized the gonococcal nlpD gene and showed that it is essential for cell separation. In contrast to other pathogenic bacteria, the gonococcal system is streamlined and does not appear to have any redundancies.

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.

The Neisseria gonorrhoeae photolyase orthologue phrB is required for proper DNA supercoiling but does not function in photo-reactivation.

Neisseria gonorrhoeae (Gc) is an obligate human pathogen and the causative agent of the sexually transmitted infection, gonorrhoea. Despite the fact that the gonococcus is not normally exposed to UV irradiation or visible light, the bacterium expresses a phrB orthologue, which in other organisms encodes a DNA photolyase that repairs UV-induced pyrimidine dimers with energy provided by visible light. We show that a Gc phrB mutant is not more sensitive to UV irradiation, independent of visible light exposure, and that the Gc phrB cannot complement an Escherichia coli phrB mutant strain. The Gc phrB mutant had a reduced colony size that was not a result of a growth defect and the mutant cells exhibited an altered morphology. Although the phrB mutant exhibited increased sensitivity to oxidative killing; it showed increased survival on media containing nalidixic acid or rifampicin, but did not have an increased mutation rate to these antibiotics or spectinomycin and kasugamycin. The Gc phrB mutant showed increased negative DNA supercoiling, but while the protein bound double-stranded DNA, it did not express topoisomerase activity. We conclude that the Gc PhrB has a previously unrecognized role in maintaining DNA supercoiling that is important for normal cell physiology.

The DNA-binding activity of the Neisseria gonorrhoeae LexA orthologue NG1427 is modulated by oxidation.

Neisseria gonorrhoeae is a human-specific organism that is not usually exposed to UV light or chemicals but is likely to encounter reactive oxygen species during infection. Exposure of N. gonorrhoeae to sublethal hydrogen peroxide revealed that the ng1427 gene was upregulated sixfold. N. gonorrhoeae was thought to lack an SOS system, although NG1427 shows amino acid sequence similarity to the SOS response regulator LexA from Escherichia coli. Similar to LexA and other S24 peptidases, NG1427 undergoes autoproteolysis in vitro, which is facilitated by either the gonococcal or E. coli RecA proteins or high pH, and autoproteolysis requires the active and cleavage site residues conserved between LexA and NG1427. NG1427 controls a three gene regulon: itself; ng1428, a Neisseria-specific, putative integral membrane protein; and recN, a DNA repair gene known to be required for oxidative damage survival. Full NG1427 regulon de-repression requires RecA following methyl methanesulphonate or mitomycin C treatment, but is largely RecA-independent following hydrogen peroxide treatment. NG1427 binds specifically to the operator regions of the genes it controls, and DNA binding is abolished by oxidation of the single cysteine residue encoded in NG1427. We propose that NG1427 is inactivated independently of RecA by oxidation.

Less is more: Neisseria gonorrhoeae RecX protein stimulates recombination by inhibiting RecA.

Escherichia coli RecX (RecX(Ec)) is a negative regulator of RecA activities both in the bacterial cell and in vitro. In contrast, the Neisseria gonorrhoeae RecX protein (RecX(Ng)) enhances all RecA-related processes in N. gonorrhoeae. Surprisingly, the RecX(Ng) protein is not a RecA protein activator in vitro. Instead, RecX(Ng) is a much more potent inhibitor of all RecA(Ng) and RecA(Ec) activities than is the E. coli RecX ortholog. A series of RecX(Ng) mutant proteins representing a gradient of functional deficiencies provide a direct correlation between RecA(Ng) inhibition in vitro and the enhancement of RecA(Ng) function in N. gonorrhoeae. Unlike RecX(Ec), RecX(Ng) does not simply cap the growing ends of RecA filaments, but it directly facilitates a more rapid RecA filament disassembly. Thus, in N. gonorrhoeae, recombinational processes are facilitated by RecX(Ng) protein-mediated limitations on RecA(Ng) filament presence and/or length to achieve maximal function.

Complexes of RecA with LexA and RecX differentiate between active and inactive RecA nucleoprotein filaments.

The bacterial RecA protein has been the dominant model system for understanding homologous genetic recombination. Although a crystal structure of RecA was solved ten years ago, we still do not have a detailed understanding of how the helical filament formed by RecA on DNA catalyzes the recognition of homology and the exchange of strands between two DNA molecules. Recent structural and spectroscopic studies have suggested that subunits in the helical filament formed in the RecA crystal are rotated when compared to the active RecA-ATP-DNA filament. We examine RecA-DNA-ATP filaments complexed with LexA and RecX to shed more light on the active RecA filament. The LexA repressor and RecX, an inhibitor of RecA, both bind within the deep helical groove of the RecA filament. Residues on RecA that interact with LexA cannot be explained by the crystal filament, but can be properly positioned in an existing model for the active filament. We show that the strand exchange activity of RecA, which can be inhibited when RecX is present at very low stoichiometry, is due to RecX forming a block across the deep helical groove of the RecA filament, where strand exchange occurs. It has previously been shown that changes in the nucleotide bound to RecA are associated with large motions of RecA's C-terminal domain. Since RecX binds from the C-terminal domain of one subunit to the nucleotide-binding core of another subunit, a stabilization of RecA's C-terminal domain by RecX can likely explain the inhibition of RecA's ATPase activity by RecX.

Neisseria gonorrhoeae metalloprotease NGO1686 is required for full piliation, and piliation is required for resistance to H2O2- and neutrophil-mediated killing.

UNLABELLED: The sexually transmitted infection gonorrhea is caused exclusively by the human-specific pathogen Neisseria gonorrhoeae. Type IV pili are an essential virulence factor uniformly expressed on clinical gonococcal isolates and are required for several aspects of gonococcal pathogenesis, including adherence to host tissues, autoagglutination, twitching motility, and the uptake of DNA during transformation. Symptomatic gonococcal infection is characterized by the influx of neutrophils or polymorphonuclear leukocytes (PMNs) to the site of infection. PMNs are a key component of gonococcal pathogenesis, mediating the innate immune response through the use of oxidative and nonoxidative killing mechanisms. The M23B family zinc metallopeptidase NGO1686 is required for gonococci to survive oxidative killing by H2O2- and PMN-mediated killing through unknown mechanisms, but the only known target of NGO1686 is peptidoglycan. We report that the effect of NGO1686 on survival after exposure to H2O2 and PMNs is mediated through its role in elaborating pili and that nonpiliated mutants of N. gonorrhoeae are less resistant to killing by H2O2, LL-37, and PMNs than the corresponding piliated strains. These findings add to the various virulence-associated functions attributable to gonococcal pili and may explain the selection basis for piliation in clinical isolates of N. gonorrhoeae. IMPORTANCE: Successful infectious agents need to overcome host defense systems to establish infection. We show that the Neisseria pilus, a major virulence factor of this organism, which causes gonorrhea, helps protect the bacterium from two major killing mechanisms used by the host to combat infections. We also show that to express the pilus, an enzyme needs to partially degrade the cell wall of the bacterium.

Purification and characterization of the RecA protein from Neisseria gonorrhoeae.

The strict human pathogen Neisseria gonorrhoeae is the only causative agent of the sexually transmitted infection gonorrhea. The recA gene from N. gonorrhoeae is essential for DNA repair, natural DNA transformation, and pilin antigenic variation, all processes that are important for the pathogenesis and persistence of N. gonorrhoeae in the human population. To understand the biochemical features of N. gonorrhoeae RecA (RecA(Ng)), we overexpressed and purified the RecA(Ng) and SSB(Ng) proteins and compared their activities to those of the well-characterized E. coli RecA and SSB proteins in vitro. We observed that RecA(Ng) promoted more strand exchange at early time points than RecA(Ec) through DNA homologous substrates, and exhibited the highest ATPase activity of any RecA protein characterized to date. Further analysis of this robust ATPase activity revealed that RecA(Ng) is more efficient at displacing SSB from ssDNA and that RecA(Ng) shows higher ATPase activity during strand exchange than RecA(Ec). Using substrates created to mimic the cellular processes of DNA transformation and pilin antigenic variation we observed that RecA(Ec) catalyzed more strand exchange through a 100 bp heterologous insert, but that RecA(Ng) catalyzed more strand exchange through regions of microheterology. Together, these data suggest that the processes of ATP hydrolysis and DNA strand exchange may be coupled differently in RecA(Ng) than in RecA(Ec). This difference may explain the unusually high ATPase activity observed for RecA(Ng) with the strand exchange activity between RecA(Ng) and RecA(Ec) being more similar.

Neisseria gonorrhoeae DNA recombination and repair enzymes protect against oxidative damage caused by hydrogen peroxide.

The strict human pathogen Neisseria gonorrhoeae is exposed to oxidative damage during infection. N. gonorrhoeae has many defenses that have been demonstrated to counteract oxidative damage. However, recN is the only DNA repair and recombination gene upregulated in response to hydrogen peroxide (H(2)O(2)) by microarray analysis and subsequently shown to be important for oxidative damage protection. We therefore tested the importance of RecA and DNA recombination and repair enzymes in conferring resistance to H(2)O(2) damage. recA mutants, as well as RecBCD (recB, recC, and recD) and RecF-like pathway mutants (recJ, recO, and recQ), all showed decreased resistance to H(2)O(2). Holliday junction processing mutants (ruvA, ruvC, and recG) showed decreased resistance to H(2)O(2) resistance as well. Finally, we show that RecA protein levels did not increase as a result of H(2)O(2) treatment. We propose that RecA, recombinational DNA repair, and branch migration are all important for H(2)O(2) resistance in N. gonorrhoeae but that constitutive levels of these enzymes are sufficient for providing protection against oxidative damage by H(2)O(2).

The RecF protein antagonizes RecX function via direct interaction.

The RecX protein inhibits RecA filament extension, leading to net filament disassembly. The RecF protein physically interacts with the RecX protein and protects RecA from the inhibitory effects of RecX. In vitro, efficient RecA filament formation onto single-stranded DNA binding protein (SSB)-coated circular single-stranded DNA (ssDNA) in the presence of RecX occurs only when all of the RecFOR proteins are present. The RecOR proteins contribute only to RecA filament nucleation onto SSB-coated single-stranded DNA and are unable to counter the inhibitory effects of RecX on RecA filaments. RecF protein uniquely supports substantial RecA filament extension in the presence of RecX. In vivo, RecF protein counters a RecX-mediated inhibition of plasmid recombination. Thus, a significant positive contribution of RecF to RecA filament assembly is to antagonize the effects of the negative modulator RecX, specifically during the extension phase.

The transcriptome response of Neisseria gonorrhoeae to hydrogen peroxide reveals genes with previously uncharacterized roles in oxidative damage protection.

Symptomatic gonococcal infection, caused by the pathogen Neisseria gonorrhoeae (Gc), is characterized by the influx of polymorphonuclear leukocytes (PMNs) to the site of infection. Although PMNs possess several mechanisms of oxidative killing, intact Gc can be found associated with PMNs, suggesting that gonococcal defences against oxidative stress are crucial for its ability to evade killing by PMNs. We used microarrays to identify genes that were differentially expressed after transient exposure of Gc to hydrogen peroxide (H2O2). Of the 75 genes found to be upregulated after H2O2 treatment, over one-quarter, including two of the most highly upregulated genes (NGO1686 and NGO554), were predicted to encode proteins with unknown functions. Further characterization of a subset of these upregulated genes demonstrated that NGO1686, a putative zinc metalloprotease, protects against oxidative damage caused by both H2O2 and cumene hydroperoxide, and that NGO554, a Gc-specific protein, acts to protect against damage caused by high levels of H2O2. Our current study also ascribes a role in H2O2 damage protection to recN, a gene previously characterized for its role in DNA repair. A PMN survival assay demonstrated that the recN and NGO1686 mutants were more susceptible to killing than the parent strain FA1090. These results define for the first time the robust transcriptional response to H2O2 by this strict human pathogen and underscore the importance of this system for survival to host defences.

Differential cross-complementation patterns of Escherichia coli and Neisseria gonorrhoeae RecA proteins.

The Escherichia coli RecA protein is one of the best-studied enzymes, but less is understood about how RecA homologues of other species are similar to or different from the E. coli RecA. In the Gram-negative pathogen Neisseria gonorrhoeae (the gonococcus; Gc), the causative agent of gonorrhoea, RecA is involved in DNA transformation, pilin antigenic variation, and DNA repair. By expressing the recA genes from Gc and E. coli under control of lac regulatory sequences in E. coli, the authors have shown that the Gc RecA fully complements an E. coli recA mutant for homologous recombination, but only partially complements for survival to DNA damage. By expressing similar constructs in Gc, it was shown that the E. coli RecA complements for pilin antigenic variation, partially complements for DNA transformation, but does not complement for survival to DNA damage, suggesting that species-specific interactions are important for DNA repair, but not for homologous recombination. Co-expression of the E. coli recA and recX genes in Gc suggests that in this heterologous system RecX modulates RecA-mediated processes.

Escherichia coli RecX inhibits RecA recombinase and coprotease activities in vitro and in vivo.

In Escherichia coli the RecA protein plays a pivotal role in homologous recombination, DNA repair, and SOS repair and mutagenesis. A gene designated recX (or oraA) is present directly downstream of recA in E. coli; however, the function of RecX is unknown. In this work we demonstrated interaction of RecX and RecA in a yeast two-hybrid assay. In vitro, substoichiometric amounts of RecX strongly inhibited both RecA-mediated DNA strand exchange and RecA ATPase activity. In vivo, we showed that recX is under control of the LexA repressor and is up-regulated in response to DNA damage. A loss-of-function mutation in recX resulted in decreased resistance to UV irradiation; however, overexpression of RecX in trans resulted in a greater decrease in UV resistance. Overexpression of RecX inhibited induction of two din (damage-inducible) genes and cleavage of the UmuD and LexA repressor proteins; however, recX inactivation had no effect on any of these processes. Cells overexpressing RecX showed decreased levels of P1 transduction, whereas recX mutation had no effect on P1 transduction frequency. Our combined in vitro and in vivo data indicate that RecX can inhibit both RecA recombinase and coprotease activities.

The DinI and RecX proteins are competing modulators of RecA function.

The DinI and RecX proteins of Escherichia coli both modulate the function of RecA protein, but have very different effects. DinI protein stabilizes RecA filaments, preventing disassembly but permitting assembly. RecX protein blocks RecA filament extension, which can lead to net filament disassembly. We demonstrate that both proteins can interact with the RecA filament, and propose that each can replace the other. The DinI/RecX displacement reactions are slow, requiring multiple minutes even when a large excess of the challenging protein is present. The effects of RecX protein on RecA filaments are manifest at lower modulator concentrations than the effects of DinI protein. Together, the DinI and RecX proteins constitute a new regulatory network. The two proteins compete directly as mainly positive (DinI) and negative (RecX) modulators of RecA function.