Identification of a conserved protein involved in anaerobic unsaturated fatty acid synthesis in Neiserria gonorrhoeae: implications for facultative and obligate anaerobes that lack FabA.

Transcriptome analysis of the facultative anaerobe, Neisseria gonorrhoeae, revealed that many genes of unknown function were induced under anaerobic conditions. Mutation of one such gene, NGO1024, encoding a protein belonging to the 2-nitropropane dioxygenase-like superfamily of proteins, was found to result in an inability of gonococci to grow anaerobically. Anaerobic growth of an NG1024 mutant was restored upon supplementation with unsaturated fatty acids (UFA), but not with the saturated fatty acid palmitate. Gonococcal fatty acid profiles confirmed that NGO1024 was involved in UFA synthesis anaerobically, but not aerobically, demonstrating that gonococci contain two distinct pathways for the production of UFAs, with a yet unidentified aerobic mechanism, and an anaerobic mechanism involving NGO1024. Expression of genes involved in classical anaerobic UFA synthesis, fabA, fabM and fabB, was toxic in gonococci and unable to complement a NGO1024 mutation, suggesting that the chemistry involved in gonococcal anaerobic UFA synthesis is distinct from that of the classical pathway. NGO1024 homologues, which we suggest naming UfaA, form a distinct lineage within the 2-nitropropane dioxygenase-like superfamily, and are found in many facultative and obligate anaerobes that produce UFAs but lack fabA, suggesting that UfaA is part of a widespread pathway involved in UFA synthesis.

The role of catalase in gonococcal resistance to peroxynitrite.

We have reported that Neisseria gonorrhoeae is extremely resistant to reactive nitrogen species (RNS) including peroxynitrite (PN). Recent literature suggests that catalase can provide protection against commercial preparations of PN. Though wild-type gonococci were shown to be highly resistant to 2 mM PN, Neisseria meningitidis and a gonococcal katA mutant were both shown to be extremely sensitive to 2 mM PN. Analysis of translational fusions to lacZ of the catalase promoters from N. gonorrhoeae and N. meningitidis demonstrated that basal katA expression from gonococci is 80-fold higher than in meningococci, though meningococcal katA retains a greater capacity to be activated by OxyR. This activation capacity was shown to be due to a single base pair difference in the -10 transcription element between the two kat promoters. PN resistance was initially shown to be associated with increasing catalase expression; however, commercial preparations of PN were later revealed to contain higher levels of contaminating hydrogen peroxide (H2O2) than expected. Removal of H2O2 from PN preparations with manganese dioxide markedly reduced PN toxicity in a gonococcal katA mutant. Simultaneous treatment with non-lethal concentrations of PN and H2O2 was highly lethal, indicating that these agents act synergistically. When treatment was separated by 5 min, high levels of bacterial killing occurred only when PN was added first. Our results suggest that killing of N. gonorrhoeae ΔkatA by commercial PN preparations is likely due to H2O2, that H2O2 is more toxic in the presence of PN, and that PN, on its own, may not be as toxic as previously believed.

Deep sequencing-based analysis of the anaerobic stimulon in Neisseria gonorrhoeae.

BACKGROUND: Maintenance of an anaerobic denitrification system in the obligate human pathogen, Neisseria gonorrhoeae, suggests that an anaerobic lifestyle may be important during the course of infection. Furthermore, mounting evidence suggests that reduction of host-produced nitric oxide has several immunomodulary effects on the host. However, at this point there have been no studies analyzing the complete gonococcal transcriptome response to anaerobiosis. Here we performed deep sequencing to compare the gonococcal transcriptomes of aerobically and anaerobically grown cells. Using the information derived from this sequencing, we discuss the implications of the robust transcriptional response to anaerobic growth. RESULTS: We determined that 198 chromosomal genes were differentially expressed (~10% of the genome) in response to anaerobic conditions. We also observed a large induction of genes encoded within the cryptic plasmid, pJD1. Validation of RNA-seq data using translational-lacZ fusions or RT-PCR demonstrated the RNA-seq results to be very reproducible. Surprisingly, many genes of prophage origin were induced anaerobically, as well as several transcriptional regulators previously unknown to be involved in anaerobic growth. We also confirmed expression and regulation of a small RNA, likely a functional equivalent of fnrS in the Enterobacteriaceae family. We also determined that many genes found to be responsive to anaerobiosis have also been shown to be responsive to iron and/or oxidative stress. CONCLUSIONS: Gonococci will be subject to many forms of environmental stress, including oxygen-limitation, during the course of infection. Here we determined that the anaerobic stimulon in gonococci was larger than previous studies would suggest. Many new targets for future research have been uncovered, and the results derived from this study may have helped to elucidate factors or mechanisms of virulence that may have otherwise been overlooked.

Functional analysis of NsrR, a nitric oxide-sensing Rrf2 repressor in Neisseria gonorrhoeae.

Nitric oxide (NO) has been shown to be an important component of the human immune response, and as such, it is important to understand how pathogenic organisms respond to its presence. In Neisseria gonorrhoeae, recent work has revealed that NsrR, an Rrf2-type transcriptional repressor, can sense NO and control the expression of genes responsible for NO metabolism. A highly pure extract of epitope-tagged NsrR was isolated and mass spectroscopic analysis suggested that the protein contained a [2Fe-2S] cluster. NsrR/DNA interactions were thoroughly analysed in vitro. Using EMSA analysis, NsrR::FLAG was shown to interact with predicted operators in the norB, aniA and nsrR upstream regions with a K(d) of 7, 19 and 35 nM respectively. DNase I footprint analysis was performed on the upstream regions of norB and nsrR, where NsrR was shown to protect the predicted 29 bp binding sites. The presence of exogenously added NO inhibited DNA binding by NsrR. Alanine substitution of C90, C97 or C103 in NsrR abrogated repression of norB::lacZ and inhibited DNA binding, consistent with their presumed role in co-ordination of a NO-sensitive Fe-S centre required for DNA binding.

Nitrite reductase NirS is required for type III secretion system expression and virulence in the human monocyte cell line THP-1 by Pseudomonas aeruginosa.

The nitrate dissimilation pathway is important for anaerobic growth in Pseudomonas aeruginosa. In addition, this pathway contributes to P. aeruginosa virulence by using the nematode Caenorhabditis elegans as a model host, as well as biofilm formation and motility. We used a set of nitrate dissimilation pathway mutants to evaluate the virulence of P. aeruginosa PA14 in a model of P. aeruginosa-phagocyte interaction by using the human monocytic cell line THP-1. Both membrane nitrate reductase and nitrite reductase enzyme complexes were important for cytotoxicity during the interaction of P. aeruginosa PA14 with THP-1 cells. Furthermore, deletion mutations in genes encoding membrane nitrate reductase (Delta narGH) and nitrite reductase (Delta nirS) produced defects in the expression of type III secretion system (T3SS) components, extracellular protease, and elastase. Interestingly, exotoxin A expression was unaffected in these mutants. Addition of exogenous nitric oxide (NO)-generating compounds to Delta nirS mutant cultures restored the production of T3SS phospholipase ExoU, whereas nitrite addition had no effect. These data suggest that NO generated via nitrite reductase NirS contributes to the regulation of expression of selected virulence factors in P. aeruginosa PA14.

Biochemical and genomic analysis of the denitrification pathway within the genus Neisseria.

Since Neisseria gonorrhoeae and Neisseria meningitidis are obligate human pathogens, a comparison with commensal species of the same genus could reveal differences important in pathogenesis. The recent completion of commensal Neisseria genome draft assemblies allowed us to perform a comparison of the genes involved in the catalysis, assembly and regulation of the denitrification pathway, which has been implicated in the virulence of several bacteria. All species contained a highly conserved nitric oxide reductase (NorB) and a nitrite reductase (AniA or NirK) that was highly conserved in the catalytic but divergent in the N-terminal lipid modification and C-terminal glycosylation domains. Only Neisseria mucosa contained a nitrate reductase (Nar), and only Neisseria lactamica, Neisseria cinerea, Neisseria subflava, Neisseria flavescens and Neisseria sicca contained a nitrous oxide reductase (Nos) complex. The regulators of the denitrification genes, FNR, NarQP and NsrR, were highly conserved, except for the GAF domain of NarQ. Biochemical examination of laboratory strains revealed that all of the neisserial species tested except N. mucosa had a two- to fourfold lower nitrite reductase activity than N. gonorrhoeae, while N. meningitidis and most of the commensal Neisseria species had a two- to fourfold higher nitric oxide (NO) reductase activity. For N. meningitidis and most of the commensal Neisseria, there was a greater than fourfold reduction in the NO steady-state level in the presence of nitrite as compared with N. gonorrhoeae. All of the species tested generated an NO steady-state level in the presence of an NO donor that was similar to that of N. gonorrhoeae. The greatest difference between the Neisseria species was the lack of a functional Nos system in the pathogenic species N. gonorrhoeae and N. meningitidis.

Resistance to peroxynitrite in Neisseria gonorrhoeae.

Neisseria gonorrhoeae encodes a number of important genes that aid in survival during times of oxidative stress. The same immune cells capable of oxygen-dependent killing mechanisms also have the capacity to generate reactive nitrogen species (RNS) that may function antimicrobially. F62 and eight additional gonococcal strains displayed a high level of resistance to peroxynitrite, while Neisseria meningitidis and Escherichia coli showed a four- to seven-log and a four-log decrease in viability, respectively. Mutation of gonococcal orthologues that are known or suspected to be involved in RNS defence in other bacteria (ahpC, dnrN and msrA) resulted in no loss of viability, suggesting that N. gonorrhoeae has a novel mechanism of resistance to peroxynitrite. Whole-cell extracts of F62 prevented the oxidation of dihydrorhodamine, and decomposition of peroxynitrite was not dependent on ahpC, dnrN or msrA. F62 grown in co-culture with E. coli strain DH10B was shown to protect E. coli viability 10-fold. Also, peroxynitrite treatment of F62 did not result in accumulation of nitrated proteins, suggesting that an active peroxynitrite reductase is responsible for peroxynitrite decomposition rather than a protein sink for amino acid modification.

Differences in nitric oxide steady states between arginine, hypoxanthine, uracil auxotrophs (AHU) and non-AHU strains of Neisseria gonorrhoeae during anaerobic respiration in the presence of nitrite.

Neisseria gonorrhoeae can grow by anaerobic respiration using nitrite as an alternative electron acceptor. Under these growth conditions, N. gonorrhoeae produces and degrades nitric oxide (NO), an important host defense molecule. Laboratory strain F62 has been shown to establish and maintain a NO steady-state level that is a function of the nitrite reductase/NO reductase ratio and is independent of cell number. The nitrite reductase activities (122-197 nmol NO2 reduced x min(-1) x OD600(-1)) and NO reductase activities (88-155 nmol NO reduced x min(-1) x OD600(-1)) in a variety of gonococcal clinical isolates were similar to the specific activities seen in F62 (241 nmol NO2 reduced x min(-1) x OD600(-1) and 88 nmol NO reduced x min(-1) x OD600(-1), respectively). In seven gonococcal strains, the NO steady-state levels established in the presence of nitrite were similar to that of F62 (801-2121 nmol x L-1 NO), while six of the strains, identified as arginine, hypoxanthine, and uracil auxotrophs (AHU), that cause asymptomatic infection in men had either two- to threefold (373-579 nmol x L-1 NO) or about 100-fold (13-24 nmol x L-1 NO) lower NO steady-state concentrations. All tested strains in the presence of a NO donor, 2,2'-(hydroxynitrosohydrazono)bis-ethanimine/NO, quickly lowered and maintained NO levels in the noninflammatory range of NO (<300 nmol x L-1). The generation of a NO steady-state concentration was directly affected by alterations in respiratory control in both F62 and an AHU strain, although differences in membrane function are suspected to be responsible for NO steady-state level differences in AHU strains.

Laboratory maintenance of Neisseria gonorrhoeae.

Neisseria gonorrhoeae is a human pathogen of mucosal surfaces, thus laboratory manipulations must include appropriate safety measures. The growth requirements and behavior of the gonococcus are significantly different from many bacteria, necessitating modifications of common laboratory techniques. A fastidious organism, N. gonorrhoeae requires enriched media in a CO2 atmosphere at 35 degrees to 37 degrees C for growth. In addition, N. gonorrhoeae expresses potent autolysins whose activity increases following glucose depletion during stationary phase, leading to cell death. Long believed to be an obligate aerobe, the gonococcus is capable of anaerobic growth when provided with a suitable electron acceptor. This unit provides information for both aerobic and anaerobic growth, basic long-term and daily maintenance of gonococcal cultures, as well as safety considerations for laboratory studies.

cis- and trans-acting elements involved in regulation of norB (norZ), the gene encoding nitric oxide reductase in Neisseria gonorrhoeae.

The ability of Neisseria gonorrhoeae to reduce nitric oxide (NO) may have important immunomodulatory effects on the host during infection. Therefore, a comprehensive understanding of the regulatory mechanism of the nitric oxide reductase gene (norB) needs to be elucidated. To accomplish this, we analysed the functional regions of the norB upstream region. The promoter contains an extended -10 motif (TGNTACAAT) that is required for high-level expression. Deletion and substitution analysis of the norB upstream region revealed that no sequence upstream of the -10 motif is involved in norB regulation under anaerobic conditions or in the presence of NO. However, replacement of a 29 bp inverted repeat sequence immediately downstream of the extended -10 motif gave high levels of aerobic expression of a norB : : lacZ fusion. Insertional inactivation of gonococcal nsrR, predicted to bind to this inverted repeat sequence, resulted in the loss of norB repression and eliminated NO induction capacity. Single-copy complementation of nsrR in trans restored regulation of both norB transcription and NorB activity by NO. In Escherichia coli, expression of a gonococcal nsrR gene repressed gonococcal norB; induction of norB occurred in the presence of exogenously added NO. NsrR also regulates aniA and dnrN, as well as its own expression. We also determined that Fur regulates norB by a novel indirect activation method, by preventing the binding of a gonococcal ArsR homologue, a second repressor whose putative binding site overlaps the Fur binding site.

Determinants of nitric oxide steady-state levels during anaerobic respiration by Neisseria gonorrhoeae.

Nitric oxide (NO) is an important host defence molecule that varies its immune stimulatory effects depending on the concentrations at which it is produced, with low concentrations (< 1 microM) promoting an anti-inflammatory host response while higher concentrations (>1 microM) lead to inflammatory responses. Neisseria gonorrhoeae grows anaerobically by anaerobic respiration using nitrite reductase (Nir) to convert nitrite to NO and nitric oxide reductase (Nor) to convert NO to nitrous oxide. As N. gonorrhoeae can both produce and degrade NO, we have begun a study of NO metabolism in this bacterium to understand how gonococcal manipulation of NO concentration may influence the inflammatory response during infection. N. gonorrhoeae has an apparent Nir Km of 33 microM nitrite and an apparent Nor Km of 1.2 microM NO. The maximum specific activities for Nir and Nor were 135 nmoles nitrite reduced per minute per OD600 (pH 6.7) and 270 nmoles NO reduced per minute per OD600 (pH 7.5) respectively. N. gonorrhoeae established a steady-state concentration of NO after nitrite addition that was dependent on the nitrite concentration until saturation at 1 mM nitrite. The NO steady-state level decreased as pH increased, and the ratio of activities of Nir and Nor correlated to the NO steady-state level. When the NO donor DETA/NO was used to simulate host NO production, N. gonorrhoeae also established a NO steady-state level. The concentration of NO at steady state was found to be a function of the concentration of NO generated by DETA/NO, with N. gonorrhoeae reducing the NO from proinflammatory (>1 microM) to anti-inflammatory (approximately 100 nM) concentrations. The implications of the ability of N. gonorrhoeae to maintain an anti-inflammatory NO concentration is discussed in relation to asymptomatic infection in women.

L12 enhances gonococcal transcytosis of polarized Hec1B cells via the lutropin receptor.

We previously reported that gonococci convert to a more invasive phenotype (Inv(+)GC) following contact with cells expressing the lutropin receptor (LHr) and that Inv(+)GC express a novel adhesin that interacts with LHr. We propose that this adhesion allows Inv(+)GC to activate LHr and induce gonococcal transcytosis, usurping normal LHr function in fallopian and endometrial epithelium, which is to transport fetal chorionic gonadotropin (hCG) into the mother. Infected polarized Hec1B monolayers, grown on collagen-coated transwells, showed that the passage of GC across the monolayer occurred rapidly, within 30 min, and proceeded at a constant rate with Inv(+)GC passage three-fold faster than GC grown in tissue culture media alone (Inv(-)GC). Electron microscopy found that Inv(+)GC triggered pseudopod formation around the bacterium, with GC found throughout the Hec1B targets within 30 min, while Inv(-)GC did neither. Pre-treatment of Inv(-)GC with recombinant ribosomal protein L12, a gonococcal "hCG-like" protein previously shown to increase invasion, also increased Inv(-)GC transcytosis to the rate of Inv(+)GC. This enhancement was completely abolished by addition of luteinizing hormone, a cognate ligand of LHr. This is convincing evidence that surface expressed L12 mediates gonococcal invasion and transcytosis via LHr, a mechanism that could be important in the development of invasive gonococcal disease in women.