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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Mol Microbiol. Author manuscript; available in PMC Dec 31, 2008.
Published in final edited form as:
PMCID: PMC2612779

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 defenses 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 up-regulated after H2O2 treatment, over one-quarter, including two of the most highly up-regulated genes (NGO1686 and NGO554), were predicted to encode proteins with unknown functions. Further characterization of a subset of these up-regulated 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 hydrogen peroxide by this strict human pathogen and underscore the importance of this system for survival to host defences.

Keywords: Neisseria gonorrhoeae, hydrogen peroxide, oxidative damage, microarray, recN, nlpD


All aerobically-grown cells are exposed to toxic reactive oxygen species (ROS) that are evolved via the process of oxidative phosphorylation during normal cellular respiration. These ROS include hydrogen peroxide (H2O2), hydroxyl radical (OH), and superoxide anion (O2•-), and are capable of damaging many different biomolecules, including proteins, membrane lipids, carbohydrates, and nucleotide bases, through a variety of different reactions (Fang, 2004). Neisseria gonorrhoeae (Gc), the only causative agent of the disease gonorrhea, is an obligate human pathogen that colonizes mucosal surfaces, most commonly those of the urogenital tract. In addition to self-generated ROS, Gc is also exposed to ROS during infection. In the human body, H2O2-producing commensal lactobacilli inhabit the same niche as Gc, and these lactobacilli may inhibit the growth of Gc (St Amant et al., 2002; Zheng et al., 1994). The hallmark of symptomatic gonococcal infection is a massive influx of activated polymorphonuclear leukocytes (PMN) into the urethra resulting in a purulent discharge (Shafer and Rest, 1989). PMNs kill microorganisms through the combined activity of antimicrobial proteins and reactive oxygen species (ROS) (Segal, 2005). PMNs are capable of delivering a potent bactericidal burst, generating substantial amounts of ROS; however, viable Gc can be found associated with PMNs both bound to the cell surface and intracellularly, suggesting that PMNs may be unable to kill Gc (Shafer and Rest, 1989; Simons et al., 2005). Thus, the ability of Gc to avoid the PMN-derived oxidative burst and subsequent killing may be an important virulence factor, and several gonococcal genes have been identified that protect against oxidative damage. The catalase (kat) (Soler-Garcia and Jerse, 2004), peptide methionide sulfoxide reductase (msrAB) (Skaar et al., 2002b), cytochrome c peroxidase (ccp) (Seib et al., 2004; Turner et al., 2003), bacterioferritin (bfrA) (Chen and Morse, 1999), and cytochrome c oxidase (sco) (Seib et al., 2003) genes, and a Mn(II) uptake system (mntC) (Tseng et al., 2001) all encode products that offer various degrees of protection against different types of oxidative damaging agents in vitro. Thus, Gc is exposed to both internally and externally-generated sources of oxidative stress and is likely to employ a number of mechanisms to protect itself from oxidative damage.

Study of the proteome and transcriptome response of bacteria to oxidative damage was begun nearly 20 years ago when the induction of approximately 30 Salmonella proteins after H2O2 treatment was observed by 2-D gel electrophoresis (Christman et al., 1985; Morgan et al., 1986). Further characterization of this response has revealed that the positive transcriptional regulators SoxR and OxyR sense O2•- and H2O2 stress, respectively, and coordinate the expression of a number of antioxidant genes (Storz and Imlay, 1999). Recently, microarray analyses have been employed to study gene expression in response to oxidative damage in several different bacteria (Mostertz et al., 2004; Palma et al., 2004; Porwollik et al., 2003; Zheng et al., 2001). These studies have revealed that, in addition to those genes with defined antioxidant functions, many encode proteins with no established function. Studies of the pathogenic Neisseria suggest that their response to oxidative damage is fundamentally different than that of E. coli. First, the OxyR protein of N. gonorrhoeae acts as a repressor of catalase (kat), which is the opposite of the E. coli paradigm (Tseng et al., 2003). Second, studies of the response to oxidative damage in N. meningitidis revealed a response that differs from that which has been observed in other bacterial species (Grifantini et al., 2004).

In this study we used Neisseria microarrays to detect alterations in global gene expression by N. gonorrhoeae in response to the oxidative damaging agent H2O2. We demonstrate a significant regulation of >150 genes in response to oxidative challenge. Several of the up-regulated genes have been previously shown to be important for protection against oxidative damage in Gc; however, many of the up-regulated genes encode proteins with either no demonstrated function or with an untested role in oxidative damage protection. To investigate the roles of some of these gene products, we created deletion mutants in two genes with no established function in Gc and studied their roles, as well as that of recN, in protection against oxidative killing. All three genes encode proteins that protected Gc against oxidative killing by H2O2. Furthermore, we demonstrated that mutants in recN and NGO1686 show greater susceptibility to killing by PMNs. Thus, this study illustrates the utility of microarrays for both elucidating the antioxidant response of Gc and uncovering potential novel virulence factors.


Transcriptional changes induced by H2O2

To begin to understand how N. gonorrhoeae responds to oxidative stress, we mapped the global transcriptional response of Gc to H2O2. We used microarrays to measure changes in steady-state levels of Gc strain FA1090 mRNA resulting from a 15 min treatment with 5 mM H2O2. This length of exposure was predicted to be sufficient to detect changes in mRNA levels induced by treatment based on a doubling time of 45-60 min for Gc. Since the main source of variability in microarray experiments is biological variability (Davies et al., unpublished data; Lee et al., 2000), we performed four independent biological replicates by growing and treating cultures with H2O2 on four separate days. Pairs of differentially labeled untreated and H2O2-treated cDNAs from each biological replicate were hybridized to two microarray slides, containing each gene spotted in triplicate: for one slide the untreated cDNA was labeled with Alexa555 and the H2O2-treated was labeled with Alexa647, and for the second slide the fluorescent labels were reversed (dye-swap). Thus, a maximum of 24 data points was averaged to determine changes in gene expression as a result of H2O2 treatment. 75 genes were up-regulated ≥2.5-fold, with P-values ≤0.01 by the Student’s t-test and representation in at least 6 of 8 slides (microarray annotation supplied by Davies et. al., unpublished data) (Table 1). 80 genes were down-regulated by the same criteria and are included in Supplementary material (Table S1). Many of the down-regulated genes encode functions related to transcription and translation, which has been previously observed (Mostertz et al., 2004), but the only down-regulated genes that will be discussed in detail are three putative transcriptional regulators. Genes with predicted functions particularly germane to oxidative damage and the regulation of the response to oxidative damage are detailed below.

Table 1
Genes up-regulated in response to hydrogen peroxide

Oxidative response/repair genes

Peroxide treatment led to the up-regulation of 7 genes whose products are predicted or known to be involved in protecting against oxidative damage (Table 1). In Gc, mutations in the ccp (Seib et al., 2004; Turner et al., 2003) and msrAB (Skaar et al., 2002b) genes result in decreased survival to oxidative damaging agents. ccp encodes a cytochrome c peroxidase and catalyzes the reduction of hydrogen peroxide to water; msrAB encodes a peptide methionine sulfoxide reductase enzyme which catalyzes the reduction of damaged (oxidized) methionine residues on proteins. Thioredoxins and glutaredoxins function in maintaining a reduced cytosol, which is essential for the control of protein function (Carmel-Harel and Storz, 2000), and the ald gene, predicted to encode alcohol dehydrogenase, also shows sequence similarity to Zn-dependent oxidoreductases (as annotated in STDGEN; http://www.stdgen.lanl.gov/). Finally, we observed up-regulation of the superoxide dismutase gene (sodB), whose product catalyzes the dismutation of superoxide into H2O2. In E. coli, the sodB gene has a minor role in protection against oxidative stress (Kargalioglu and Imlay, 1994); however, a Gc sodB mutant has no diminished resistance to paraquat or xanthine/xanthine oxidase (Tseng et al., 2001). Thus, we observed up-regulation of many genes whose products have been demonstrated to function as antioxidants in bacteria.

Heat shock genes

We observed induction of a number of heat shock genes and molecular chaperones after H2O2 treatment (groEL, groES, grpE, dnaJ, dnaK, clpB, lonC, ftsH, and secB). Overlap of the heat shock regulon with other stresses, including H2O2, was originally observed through 2-D gel analysis of proteins in S. typhimurium (Morgan et al., 1986) and has been repeatedly shown through transcriptional genomic analysis (Mostertz et al., 2004; Palma et al., 2004; Zheng et al., 2001). This suggests that the genes encoding these proteins respond to multiple stimuli.

Iron-related and Fur-regulated genes

Fifteen genes that are related to iron acquisition (Perkins-Balding et al., 2004), or are known to be regulated by Fur in Gc (Sebastian et al., 2002), were up-regulated by treatment with H2O2. The majority of the products of these genes (tbpA, tbpB, frpB, fbpA, fetB, exbB, exbD, hpuA, tonB, pigA, and fur) are involved in iron acquisition. The fumC, nifS, and nifU genes encode the TCA cycle enzyme fumarate hydratase and two proteins involved in the synthesis of ([Fe-S]) clusters, respectively. The Fur-regulated (Sebastian et al., 2002) recN gene is involved in the repair of damaged DNA in both E. coli (Picksley et al., 1984) and Gc (Skaar et al., 2002a), but its exact biochemical function is unknown. Although it is unclear exactly why recN transcription increases as a result of exposure to H2O2, this has been observed in other bacteria (Porwollik et al., 2003; Zheng et al., 2001), including N. meningitidis (Grifantini et al., 2003).

Transcriptional regulators

Four probable transcriptional regulators were up-regulated in response to H2O2 treatment. Fur has been previously discussed above, NGO2115 belongs to the AraC family of positive transcriptional regulators, NGO1427 shows similarity to the cI repressor protein of phage λ, and NGO637 belongs to a family of predicted transcriptional regulators. Additionally, three of the down-regulated genes encode proteins involved in the regulation of transcription in other bacteria: lrp (NGO1294), merR (NGO602), and hydH (NGO1867). The hydH gene of E. coli encodes the sensor protein of a two-component regulatory system that responds to lead and zinc (Leonhartsberger et al., 2001). The merR gene shows similarity to the MerR family of proteins, members of which coordinate responses to a variety of stimuli, including oxidative stress and heavy metals (Brown et al., 2003), and the lrp gene encodes a global regulator that modulates the transition to stationary phase and regulates the transcription of over 400 genes including many involved in the response to nutrient limitation and osmotic stress in E. coli (Tani et al., 2002). Apart from Fur, none of these transcriptional regulators have been studied in Gc, so the contribution of each of these regulators to the H2O2 response is presently unknown.

Genes with unknown function

Approximately one quarter of the genes up-regulated in the microarray analysis (20 of 75), including two of the three most highly up-regulated genes, were annotated as encoding proteins with no known functions. Over one third (8/20) of these hypothetical proteins contained N-terminal signal sequences, suggesting that they could be secreted from the cytoplasm (NGO554, NGO1686, NGO1947, NGO1428, NGO555, NGO1948, NGO382, and NGO1900). Many of the genes with proven antioxidant functions in Gc are localized to the membrane or periplasm (msrAB (Skaar et al., 2002b), ccp (Seib et al., 2004; Turner et al., 2003), mntC (Tseng et al., 2001), and sco (Seib et al., 2003)), further supporting the hypothesis that these hypothetical proteins could be functioning as antioxidants.

Q-PCR validates microarray data and reveals kat regulation

To validate our results we performed quantitative real-time PCR (Q-PCR) on a subset of the genes identified in our microarray analysis, measuring the expression levels of msrAB, recN, NGO554, and NGO1686, as well as the omp3 gene, whose expression was unaffected by hydrogen peroxide treatment in the microarray (data not shown), in H2O2-treated cells and untreated controls. Increases in expression determined by micrarray and Q-PCR are as follows: msrAB increased 3.7-fold by microarray and 2.5 by Q-PCR [standard error (SE)=0.75]; recN increased 3.0-fold by microarray and 2.3-fold by Q-PCR (SE=0.30); NGO554 increased 70.6-fold by microarray and 36.9-fold by Q-PCR (SE=19.5); NGO1686 increased 19.3-fold by microarray and 17.15-fold by Q-PCR (SE=8.8). Overall, a consistent differential expression was found using the two methodologies. We saw no difference in omp3 message levels in response to H2O2 treatment by Q-PCR (data not shown). Earlier studies on N. gonorrhoeae have shown that the expression of catalase enzyme is up-regulated by treatment with H2O2 (Zheng et al., 1992); however, we did not detect the kat transcript in our microarray analysis, which was surprising given the high level of catalase activity in Gc (Hassett et al., 1990). We therefore measured the levels of kat mRNA in H2O2-treated cells and untreated controls by Q-PCR. A 51.6-fold-(SE=42.28) up-regulation of the kat gene was observed in H2O2-treated cells.

Highly up-regulated genes of unknown function and recN have roles in protection against oxidative damage

Based on the unpublished annotation initially used in conjunction with our microarray analysis (Davies et al., unpublished data), two of the genes most highly up-regulated in response to H2O2, NGO554 (70-fold up-regulation) and NGO1686 (19-fold up-regulation) were predicted to encode proteins with no known function. Further analysis of these proteins using information contained in the STDGEN database (http://www.stdgen.lanl.gov/), including COGS and BLAST (Altschul et al., 1997) analyses, as well as annotation provided by David Dyer at the University of Oklahoma (http://www.genome.ou.edu/gono.html), revealed that both of these proteins are predicted to contain cleavable signal sequences, suggesting that they may be secreted from the cytoplasm, and that both of these proteins show limited sequence similarity to proteins of known function.

The carboxy terminal half of the predicted 46-kDa NGO1686 protein showed sequence similarity to the M23B family of zinc metalloendopeptidases as classified by the MEROPS protease database (http://merops.sanger.ac.uk). Certain members of this family, such as lysostaphin, have been demonstrated to cleave bacterial cell wall peptidoglycans, but very few members of this family have been functionally characterized (Rawlings et al., 2004). The M23B family of proteases contains a HxH motif (residues 373-375 of NGO1686), where the first histidine is the active site and the second histidine binds the zinc ligand. An additional HxxxD motif found in this family (residues 295-299 of NGO1686) is also believed to bind zinc. BLAST searches using default settings revealed that the NGO1686 protein showed the highest degree of sequence similarity to uncharacterized probable peptidases from Chromobacterium violaceum (gb AE002098.2; 1e-75) and Nitrosomonas europaea (emb BX321861.1; 2e-47). NGO1686 also showed sequence similarity to the partially characterized E. coli NlpD (SwissProt P33648)(Ichikawa et al., 1994; Lange and Hengge-Aronis, 1994) and Corynebacterium glutamicum MepA (Möker et al., 2004) proteins, both of which showed 35% sequence identity and approximately 50% sequence similarity to the carboxy terminal third of the NGO1686 protein. However, the fact that the NGO1686 protein showed sequence similarity to small regions of MepA and NlpD suggests that the NGO1686 protein is not a functional homolog of these proteins, but is simply a zinc metalloprotease

The most highly up-regulated gene (70-fold up-regulation) encoded the NGO554 protein, a Gc-specific predicted 33-kDa protein that showed no similarity to any proteins in the database, including no meningococcal proteins from the three sequenced genomes. Therefore, we wondered whether the NGO554 protein could be a virulence factor that was unique to Gc. Despite extensive sequence analysis, the only structural feature identified in this protein was a predicted cleavable signal sequence, suggesting that the NGO554 protein could be secreted from the cytoplasm. Since the roles of these proteins in the response to oxidative damage, or their potential functions as antioxidants, were not patently obvious, we decided to investigate their functions in protection against oxidative damage.

We insertionally inactivated these two genes by deleting an internal portion of the gene and inserting an antibiotic resistance cassette in its place (see Experimental Procedures), creating strains FA1090ΔNGO1686 (Δ1686) and FA1090ΔNGO554 (Δ554). Neither mutant exhibited a growth defect in liquid; however, the Δ1686 mutant exhibited altered colony morphology when cultivated on solid agar medium, which was not a result of altered pilus or Opa expression (data not shown). We then tested the sensitivity of the mutants to the oxidative damaging agents H2O2, cumene hydroperoxide (an organic peroxide), paraquat (which generates intracellular superoxide), and diamide (which damages thiol groups), relative to the parent strain FA1090. The Δ1686 mutant showed a statistically significant 30 to 300-fold increase in sensitivity to H2O2 at the 10, 20, and 50 mM doses (Figure 1). The Δ1686 mutant also showed a significant 4-fold increase in sensitivity to cumene hydroperoxide at the 0.01% dose (data not shown), but showed no increase in sensitivity to either paraquat or diamide (data not shown), suggesting that the NGO1686 gene product specifically protects against oxidative damage caused by peroxides. In contrast, the Δ554 mutant showed over a 100-fold statistically significant increase in sensitivity to the highest dose (50 mM) of H2O2 (Figure 1), and showed no increase in sensitivity to any of the other oxidative damaging agents tested (data not shown). Oxidative damage assays using a previously-characterized recN mutant (Skaar et al., 2002a) revealed that the recN mutant was significantly increased 5-fold to the 20 mM dose and 60-fold to the 50 mM dose of H2O2 (Figure 1), but showed no increase in sensitivity to the other tested oxidative damaging agents (data not shown). To ensure that the observed differences in sensitivity to oxidative damaging agents were not due to polar effects on downstream genes, we introduced functional copies of the NGO1686 and NGO554 genes at unlinked chromosomal loci either under control of the gene’s exogenous promoter (NGO1686), or under control of lac regulatory sequences (NGO554) (see Experimental Procedures), creating strains Δ1686/1686+ and Δ554/554+. In both cases we were able to complement the observed increases in sensitivity by supplying a functional copy of the gene ectopically, as strains Δ1686/1686+ and Δ554/554+ were statistically indistinguishable from the parental strain FA1090 and statistically different from the mutant strain in both H2O2 sensitivity (Figure 1) and cumene hydroperoxide sensitivity (data not shown). These results confirmed that the phenotypes of the mutants were directly due to inactivation of the NGO1686 and NGO554 genes. We were also able to restore normal colony morphology to the Δ1686 mutant strain by supplying a functional copy of the gene ectopically (data not shown). The altered colony morphology of the Δ1686 mutant could be indicative of the NGO1686 protein playing a role in cell wall peptidoglycan metabolism, as has been suggested to be the case with its closest characterized homologs (Ichikawa et al., 1994; Lange and Hengge-Aronis, 1994; Möker et al., 2004). However, since the Δ1686 mutant showed no alteration in MIC (minimal inhibitory concentration) to a panel of antibiotics with a variety of cellular targets (data not shown), as well as no decreased resistance to the oxidative damaging agents paraquat and diamide (data not shown). It is unlikely that the phenotype of increased sensitivity is due to increased membrane permeability but instead suggests a more specific role for the NGO1686 protein in oxidative damage protection.

Figure 1
H2O2 resistance of strains with mutations in genes up-regulated by H2O2 treatment. Cells were treated with varying doses of H2O2 for 15 min and serially diluted into media containing catalase. The relative survival at each dose was calculated as the viable ...

recN and Δ1686 mutants show reduced survival in a PMN killing assay

Gonococcal infection results in the recruitment of large numbers of PMNs to the site of infection. PMNs perpetrate a bactericidal respiratory burst, generating substantial amounts of reactive oxygen species (ROS), and also possess a variety of non-oxidative killing mechanisms. The fact that viable Gc can be found associated with PMNs suggests that Gc can survive the interaction with PMNs. To assess the contributions of recN, NGO1686, and NGO554 to survival in a biologically relevant model system, we measured the abilities of strain FA1090 and the corresponding mutants to survive following exposure to PMNs. Freshly isolated adherent human PMNs were co-incubated with Gc strain FA1090 or one of the corresponding mutants at a temperature permissive for bacterial adherence but not internalization. The PMN-bacteria mixture was then warmed to 37°C, and at various times thereafter the number of viable, cell-associated CFU was assessed for each strain. Both the recN and Δ1686 mutants, but not the Δ554 mutant, showed a statistically significant, approximately 2-fold decrease in survival after exposure to PMNs relative to the parent strain FA1090 (Figure 2 and data not shown). The Δ1686/1686+ strain complemented the mutation and was statistically indistinguishable from the FA1090 parent strain (data not shown). Taken together, these data suggest that both recN and NGO1686 are important for the ability of Gc to survive PMN-mediated killing and can thus be considered potential virulence factors.

Figure 2
Susceptibility of strains to PMN-mediated killing. Adherent, IL-8-primed PMNs were synchronously infected with Gc strains at a multiplicity of infection of 1. At various times post-infection, PMNs were washed and lysed in 1% saponin prior to serial dilution. ...


The data presented in this study comprise the first examination of the global effects of H2O2 on gene expression in the strict human pathogen N. gonorrhoeae. In addition to revealing the up-regulation of several genes whose products have been previously shown to be involved in resistance to oxidative stress, we have identified many novel genes with robust responses to H2O2 treatment. Further characterization of the genes responding strongly to H2O2 demonstrated that NGO1686, a predicted zinc metalloprotease, protects against damage by two types of peroxides, and that NGO554, a Gc-specific protein, acts to protect against damage caused by high levels of H2O2. Our current study has also ascribed a role in oxidative damage protection to recN, a Fur-regulated gene (Sebastian et al., 2002) involved in DNA repair in Gc (Skaar et al., 2002a). A PMN survival assay allowed us to test these defined mutants in a biologically relevant system, which has revealed the importance of both recN and NGO1686 in resistance to PMN-mediated killing. Finally, our analysis has further underscored the link between iron, Fur, and oxidative stress resistance in bacteria, specifically in the pathogenic Neisseria.

Although we observed that the expression of seven transcriptional regulators was altered by H2O2 treatment, the only one of these regulators that has been studied in detail in Gc is Fur. In some bacteria, Fur-like proteins have been shown to directly regulate the response to oxidative stress (Boylan et al., 2003; Mongkolsuk and Helmann, 2002). In E. coli and B. subtilis, fur expression increases after H2O2 treatment (Mostertz et al., 2004; Zheng et al., 1999), and in E. coli (Zheng et al., 2001), B. subtilis (Mostertz et al., 2004), P. aeruginosa (Palma et al., 2004), N. meningitidis (Grifantini et al., 2004), and S. typhimurium (Porwollik et al., 2003) various members of the Fur regulon were derepressed after H2O2 treatement. One explanation for this observation is that H2O2 directly inactivates the Fur protein, derepressing the Fur regulon. The resulting increases in intracellular Fur levels could lead to Fe2+ sequestration by Fur, thus limiting the participation of Fe2+ in Fenton biochemistry (Fe2+ + H2O2 → Fe3+ + OH- + OH), which results in generation of the highly damaging hydroxyl radical (OH). This Fur-dependent Fe2+ sequestration would be advantageous for the bacterium after exposure to H2O2, and has been suggested to occur in E. coli (Zheng et al., 1999).

In our current study, 15 iron-related or Fur-regulated genes were up-regulated in response to H2O2 in N. gonorrhoeae. Ducey et al. recently published a microarray analysis of the response of Gc strain FA1090 to iron (Ducey et al., 2005) which has substantially expanded the list of iron-regulated genes in Gc. Although a chemically defined medium was used for their analysis, while our present study was conducted in rich medium, there is an overlap in the genes regulated by iron and those up-regulated by H2O2 treatment. Of the 75 peroxide up-regulated genes, 24 were shown to be regulated by iron: 18 were repressed by iron (ald, grxB, fetA, tbp1, tbp2, pigA, fbpA, fetB, recN, exbB, aldA, NGO2115, NGO2065, NGO554, NGO108, NGO322, NGO1690, and NGO1174) and 6 were activated by iron (NGO376, NGO775, NGO1901, NGO1686, NGO1948, and NGO865) (Ducey et al., 2005). However, only 13 of these 24 genes contain a putative Fur box, as denoted by Ducey et al (Ducey et al., 2005), suggesting that the mechanism of this portion of the peroxide response is not simply due to activation or repression by Fur. This finding adds to the number of bacterial species where an overlap exists between the iron and hydrogen peroxide response regulons. In none of these bacteria has the mechanism of this overlap been elucidated, but our data suggests that it is not simply due to Fur.

Studies of the closely-related N. gonorrhoeae (Gc) and N. meningitidis (Nm) have revealed that, although their genomes are very similar, they differ greatly in their pathogenicities. In contrast to what we observed in Gc, treatment of Nm with H2O2 only results in up-regulation of 10 genes. In Nm, Fur has recently been shown to regulate expression of an operon involved in resistance to oxidative damage (NMB1436-8) (Grifantini et al., 2004), and this operon is also present in Gc (data not shown). In a NMB1436-8 mutant a larger number of genes were up-regulated in response to H2O2, but only 20 of those genes were found in the Gc peroxide up-regulated dataset (Grifantini et al., 2004). Although it is difficult to directly compare these results with ours, since we treated Gc with 5 mM H2O2 for 15 min, whereas Grifantini et al. treated Nm with 200 μM H2O2 for 30 min, these data suggest that Gc and Nm have different transcriptional responses to oxidative damage. Further characterization of the response of both Gc and Nm to different types of oxidative damage could begin to elucidate the similarities and differences in the oxidative damage responses of these closely related but pathogenically distinct bacteria.

Of the 75 genes up-regulated in our microarray analysis, we chose to study three in more detail for their resistance to oxidative damaging agents and PMN-mediated killing. Both the recN and Δ1686 mutants showed increased sensitivity to H2O2 and killing by PMNs, whereas the Δ554 mutant only showed decreased resistance to H2O2. RecN plays a role in DNA repair in both E. coli (Picksley et al., 1984) and Gc (Skaar et al., 2002a) and additionally has a role in competence in B. subtilis (Kidane and Graumann, 2005). Although RecN has SMC domains (Skaar et al., 2002a) and is an ATP-dependent ssDNA binding protein (Kidane and Graumann, 2005), the exact biochemical role of RecN is unknown in any bacterium. RecN is known to be important for the repair of DNA double-strand breaks in both E. coli and B. subtilis (Imlay and Linn, 1987; Kidane et al., 2004; Picksley et al., 1984), and a Gc recN mutant is extremely sensitive to DNA double stand breaks caused by nalidixic acid (Skaar et al., 2002a). recN is up-regulated in response to H2O2 treatment in E. coli (Zheng et al., 2001) and S. typhimurium (Porwollik et al., 2003), and although it has been suggested that this is the result of induction of the SOS response by H2O2-damaged DNA, this cannot be the case in Gc, which lacks an SOS response (Black et al., 1998). Since RecN is likely to act in the repair of oxidatively-damaged DNA, it is interesting that recN is the only DNA repair gene up-regulated in the microarray and may suggest a specific role for RecN in oxidative damage repair.

The other H2O2-regulated gene shown to have a role in protection against oxidative damage and PMN killing is NGO1686. The NGO1686 protein belongs to the M23B family of zinc metalloendopeptidases, is likely to be exported from the cytoplasm, and provides protection against PMN killing, as well as oxidative damage by two types of peroxides. Some members of the M23B family function to cleave peptidoglycan, and limited phenotypes have been described for other members of this family which are consistent with a role in cell wall maintenance, but none of the members of this family of proteins have been tested for a role in protection against oxidative damage. Furthermore, since no functions have been ascribed to the proteins in the database most closely related to NGO1686, we cannot use these sequence homologs as a means to predict the mechanism by which NGO1686 protects against oxidative damage.

Although the specific mechanisms by which the RecN and NGO1686 proteins protect against oxidative damage are unknown, we have clearly shown that both genes are up-regulated in response to H2O2 and that both are important for survival to H2O2 and PMNs. Importantly, this is the first demonstration of gonococcal genes that affect PMN survival in the literature. The most straightforward hypothesis supported by our present data is that Gc strain FA1090 is protected from the oxidative burst of PMNs by a variety of antioxidant proteins, among them being NGO1686 and RecN, and that their removal from the antioxidant arsenal results in increased sensitivity to PMN-mediated killing. However, an alternate hypothesis is that these proteins have differing functions in their interactions with pure H2O2 and PMNs, perhaps conferring survival to the non-oxidative killing mechanisms of PMNs, such as antimicrobial peptides, with H2O2 serving as a signal for their up-regulation in their interaction with PMNs. Intriguingly, this could suggest potential roles for the Gc- and Neisseria-specific hypothetical proteins found in our analysis in the complex interaction between these strict human pathogens and the human innate immune response.


Bacterial strains, growth conditions, and chemicals

Escherichia coli One Shot TOP10 cells (Invitrogen) were grown on Luria-Bertani (LB) broth or agar (Difco) at 37°C to propagate plasmids. Gc strains were grown at 37°C on Gc medium base (GCB; Difco) plus Kellogg supplement I [22.2 mM glucose, 0.68 mM glutamine, 0.45 mM cocarboxylase] and II [1.23 mM Fe(NO3)3]; (Kellogg et al., 1963) at 37°C in 5% CO2 or in Gc liquid (GCBL) medium [1.5% proteose peptone no. 3 (Difco), 0.4% K2HPO4, 0.1% KH2PO4, 0.1% NaCl] with Kellogg supplements I, II, and 0.042% sodium bicarbonate. Unless explicitly stated otherwise, GCB and GCBL always contained Kellogg supplements I and II. Unless otherwise indicated, liquid-grown Gc strains were prepared as follows: Gc was grown from freezer stocks for approximately 20 h and ~10 colonies were passaged onto GCB. After 12 h, colonies were collected with a Dacron swab (Puritan), resuspended in GCBL at OD550[congruent with]0.1, grown 12h, diluted to OD550[congruent with]0.3, grown 2.5-3 h, and diluted to OD550[congruent with]0.06. This culture was grown in a flask on a shaking incubator to mid-log phase (OD600[congruent with]0.5). All strains used in this study showed similar growth rates. All FA1090 strains used in this study contained the 1-81-S2 variant pilE sequence (Seifert et al., 1994). Antibiotics (Sigma) were used at the following concentrations for E. coli: kanamycin (Kan) 40 μg/ml; erythromycin (Erm), 275 μg/ml; chloramphenicol (Cam), 25 μg/ml. For Gc, the concentrations were: Cam, 0.75 μg/ml, Erm, 0.75 μg/ml, Kan, 40 μg/ml. Antibiotic MICs were determined using E-test strips (AB BIODISK). All chemicals were obtained from Sigma unless otherwise indicated.

DNA manipulations and analysis

Standard procedures were performed as described (Sambrook et al., 1989). Plasmid DNA from E. coli, genomic DNA from Gc, and PCR products were isolated using Qiagen kits. All modifying and restriction enzymes were obtained from New England Biolabs, unless otherwise indicated, and used as specified. For Southern blot analysis, DNA was transferred to Magnagraph nylon membranes (Micron Separations, Inc.) and hybridized with Dig-labeled probes generated and used as specified by the manufacturer (Roche). Gonococcal pilE sequences were determined as described (Stohl and Seifert, 2001), except for sequencing reactions as below. Sequencing reactions were performed using CEQ Dye Terminator Cycle Sequencing Quick Start Kit and CEQ 2000XL automated sequencer (Beckman Coulter) according to the manufacturer’s instructions. DNA analysis was performed using VectorNTI software (Informax, Inc).

H2O2 treatment of Gc, RNA isolation, cDNA synthesis and fluorescence labeling

Gc strain FA1090 was grown as above to mid-log phase and diluted 1:10 into GCBL. The culture was split into two flasks, and half of the culture was treated with 5 mM H2O2 for 15 min (with shaking for both flasks) after which both cultures were treated with a final concentration of 10μg/ml catalase for 1 min to degrade the H2O2. Cultures were quickly cooled in a dry ice-ethanol bath, harvested by centrifugation for 15 min (4,000 × g), and pellets were stored at -80°C. RNA was isolated using the RNAeasy Miniprep kit (Qiagen) with the additional on-column DNAse digestion step, and quality and quantity of the RNA was assessed by agarose gel electrophoresis, spectrophotometry, and Bioanalyzer (Agilent). cDNA was synthesized as follows: 20 μg total RNA of each sample was mixed with 30 μg random hexamers (Roche), heated to 70°C for 10 min and cooled on ice for 10 min. 0.5 μl RNAsin (Promega), 6 μl Superscript II buffer (Life Technologies, Inc.), 3 μl DTT, a final concentration of: 0.50 mM for dATP, dCTP, and dGTP, 0.15 mM for dTTP (Invitrogen), and 0.30 mM for aminoallyl-dUTP (Sigma), and 2 μl Superscript II reverse transcriptase (Invitrogen) was added and incubated at 42°C for 2.5 h. Reactions were terminated and the cDNA labeled as recommended by Molecular Probes as follows: reactions were heated to 95°C for 5 min and immediately placed on ice. 0.42 volumes of 1 M NaOH were added and incubated at 65°C for 15 min. Solution was neutralized by addition of an equal volume (equal to amount of NaOH added) 1 M HCl and 0.11 volume of 1 M Tris-HCl (pH 7.0). Amine-modified cDNA was purified using the Qiaquick PCR purification kit according to the manufacturer’s instructions, except performing two additional was steps were performed, followed by ethanol precipitation of cDNA with added glycogen (Roche). The cDNAs were indirectly labeled by coupling to either AlexaFluor dye 555 or 647 (Molecular Probes) as follows: cDNA was dissolved in 5 uL DNA, 3 μl sodium bicarbonate (25 mg/ml) and the Alexa dye dissolved in 2 μl DMSO (Sigma) was added to the mixture and incubated at room temperature for 1 h in the dark. The reaction was purified using the Qiaquick PCR purification kit and precipitated as above. Labeled pellets were resuspended in 4 μl water and used within 1 h.

Microarray hybridization and data analysis

We used a pan-Neisseria microarray for our analysis, which will be described in detail elsewhere (Davies et al., unpublished). Briefly, the microarray contains 2704 PCR products corresponding to the most conserved regions of the 6294 potential coding sequences from N. gonorrhoeae strains FA1090 and MS11, N. meningitidis strains MC58 and Z2491, and a variety of control DNA spots, all of which are spotted in triplicate. The printed microarray contains 99.6% of all the FA1090 annotated features. Microarrays were incubated with 32 μl prehybridization solution [(25% formamide, 5X SSC, 0.1% SDS, 10 mg/ml BSA (Sigma), 1 mg/ml salmon sperm DNA (Eppendorf)] for 45 min at 42°C inside a Corning Hybridization Chamber containing a small amount of 5 x SSC to maintain constant moisture. Slides were washed in water and dried by centrifugation (5 min, 2,000 × g). For hybridization of the microarray slides, 4 μl of each differentially-labeled Alexafluor 555 and 647 probe was added to 24 μl hybridization solution (25% formamide, 5X SSC, 0.1% SDS, 1 mg/ml salmon sperm DNA), the mixture was heated to 95°C for 5 min, applied to the microarray surface, a coverslip (Corning) was added, and allowed to hybridize for 16 h at 42°C as above. Slides were sequentially washed as follows in Coplin jars: Slides were placed in 2 x SSC, 0.1% SDS until the coverslip detached, and subsequently incubated 42°C for 5 min in fresh solution. Slides were washed for 10 min at room temperature in 0.1 x SSC, 0.1% SDS, 4 × 1 min in 0.1 x SSC, 10 s in 0.01 x SSC, and dried by centrifugation. Slides were scanned and signal intensities were quantitated using a Packard ScanArray 4000XL laser scanner and ScanArray Express version 2.1.8 software. Spots were analyzed by adaptive circle quantitation, local background was subtracted for each spot, and Lowess normalization was applied to calculate the log2 Alexa647/Alexa555 median fluorescence ratios. Results were exported to Excel and statistical analyses were performed.


cDNA was synthesized and purified using RNA from two biological replicates as described above except that a final concentration of 0.5 mM was used for each dNTP. PCR products to be used as standards in Q-PCR reactions were generated using the following pairs MSRAB-Q1 (5′-TGACCGAAGAGCAATACC-3′), MSRAB -Q2(5′-GAGCCGGAATCATATTTGTC-3′); RECN-Q1 (5′-CTTTGGATGCGATTGGTCTG-3′), RECN-Q2 (5′-GCGGCGGATACTGAGTTC-3′); NGO1686-Q1 (5′-GGTGTCGGCGGCATACATTG-3′), NGO1686-Q2 (5′-GCCTCCTGCACCCAATATG-3′); NGO554-Q1 (5′-ATTTCCTACCCTTTGGTTCG-3′), NGO554-Q2 (5′-CCGTTTACATTGGTGTTCTG-3′); OMP3FOR (5′-AGCAGGCTCCTCAATATGTT3′), OMP3REV (5′-CTTGAGTCATTTGCGCTTGA-3′)(Sebastian et al., 2002; Sechman et al., 2005); KAT-Q1 (5′-TCGCCCGTTTCACCACCGTG-3′), KAT-Q2 (5′-TTTGTGGCGGAACGCATATTG-3′). PCR products were subsequently gel purified (Qiaquick), and serial dilutions were included in Q-PCR runs to generate standard curves for quantitation of transcripts. Q-PCR was performed on a LightCycler instrument (Roche). All assays contained 2 μl of LightCycler DNA master SYBR Green mix (Roche), 1.6 μl of MgCl2 (for a final concentration of 3 mM), 2 μl of each primer (0.5 mM final concentration), 2 μl of template, and PCR-grade sterile water for a final volume of 20 μl. The annealing temperature was 52°C for NGO1686, NGO554, recN, and msrAB; 55°C for omp3; and 58°C for kat. Fluorescence in channels F1 and F2 was acquired at the end of every extension step, and the F2/F1 ratio was analyzed. All Q-PCR assays were performed at least twice using cDNA from two biological replicates.

Construction of mutant strains

To individually mutate the NGO554 and NGO1686 genes, internal fragments of the genes were deleted and antibiotic resistance cassettes (KanR or ErmR, respectively) were inserted in their place. We used primer pair 554UPFWD (5′-GGCGCGCCACGTACCGCTTGAAACATATGG-3′) and 554UPREV (5′-GCTAGCGTAGGAAATCGGATTCGGCAGC-3′), which introduces a NheI site (underlined), and primer pair 554DOWNFWD (5′-TTAATTAAGGCCAATTCTACGGCAATGG-3′) and 554DOWNREV (5′-GAGCTCGACAGGTAGTTTTCCACGATGG-3′), which introduces a PacI site (underlined), to PCR-amplify fragments containing the 5′ and 3′ ends of the gene, respectively, using TaqPlusLong polymerase (Stratagene). PCR products were gel purified and ligated to pCR2.1-TOPO (Invitrogen) to create pNGO554UP and pNGO554DOWN, respectively. The kanamycin resistance cassette from EZ::TN <Kan-2> (Epicentre Technologies) was amplified with Pfu polymerase using primer pair KAN-2FWD (5′-GCTAGCCGTCTGAACTCAAAATCTCTGATGTTACATTGC-3′), which introduces a NheI site (underlined) and a gonococcal uptake sequence (in bold), and KAN-2REV (5′-TTAATTAAGTTGATGAGAGCTTTGTTGTAGG-3′), which introduces a PacI site (underlined). The PCR product was gel purified and ligated into pCR-Blunt (Invitrogen) to create pBluntGUSKan-2. To clone the three fragments together, the Kan-2 cassette was excised from pBluntGUSKan-2 by NheI/SacI digestion and ligated to pNGO554UP digested with the same enzymes to yield the plasmid pNGO554UPKan-2. This construct was subsequently digested with SacI/PacI and ligated to a fragment from pNGO554DOWN containing the 3′ end of the gene to yield pNGO554Kan-2.

We used primer pair NGO1686-1 (5′-GCGCTGTCCCAATTCAACACC-3′) and NGO1686-2Pme (5′-CATGTTTAAACCGGTTACCGTGTCGCAATCGG-3′), which introduces a PmeI site (underlined) and primer pair NGO1686-4 (5′-CCACAAATAACGTGCGTAAAATGCCG-3′) NGO1686-3Pme (5′-ATCGTTTAAACGTCAAGGTCGAAGCGG-3′), which introduces a PmeI site (underlined) to PCR-amplify fragments containing the 3′ and 5′ ends of the gene, respectively, using Pfu polymerase. PCR products were gel purified and ligated to PCR-Blunt (Invitrogen) to create pBlunt1686DOWN and pBlunt1686UP, respectively. To clone the two fragments together, pBlunt1686DOWN was digested with PmeI/BamHI, treated with Klenow and CIP, pBlunt1686UP was digested with PmeI/EcoRV to liberate the insert, and the two fragments were ligated together to yield pBlunt1686. This plasmid was digested with PmeI, treated with CIP, and a non-polar erm resistance cassette prepared as described (Stohl and Seifert, 2001) was cloned into the site to yield pBlunt1686Erm. All mutant constructs were sequenced to verify that only the desired mutations had been introduced. Each mutant allele was recombined into the Gc chromosome by spot transformation as described (Stein et al., 1988; Stohl and Seifert, 2001) and verified by Southern blotting and PCR analysis.

To create strains to complement the mutations, we used the NICS system (Mehr and Seifert, 1998; Mehr, 2000) to insert a functional copy of the gene ectopically at an unlinked chromosomal locus between the lctP and aspC genes, either under control of its endogenous promoter (constructs in plasmid pGCC5) or lac regulatory sequences (constructs in plasmid pGCC4), and linked to either a Cam resistance cassette (pGCC5) or an Erm resistance cassette (pGCC4). Plasmid pGCC5/1686 was created by PCR amplification using Pfu polymerase of the NGO1686 coding region and its predicted promoter with primers NGO1686-Pac (5′-GTAGCTTAATTAACCGCCGTCAGCACATTTGCCTG-3′), which introduces a PacI site (underlined) and NGO1686-6 (5′-TTTTCAGACGGCATTGTTTTCGTCGC-3′), with subsequent directional cloning into PacI/PmeI-digested pGCC5. Plasmid pGCC4/554 was created by PCR amplification with Pfu polymerase of the 554 coding region using primer pair PAC554FWD (5′-TTAATTAACCATCATATCTTTTCTTTAAAGG-3′), which introduces a PacI site (underlined), and 554REVPME (5′-GGTTAACCGAAAACACCGGCATTCC-3′), which introduces a PmeI site (underlined), with subsequent directional cloning into PacI/PmeI-digested pGCC4. Both complement constructs were sequenced to verify no mutations had been introduced, recombined into the appropriate Gc mutant strain with selection on appropriate antibiotics, and verified by PCR and Southern analysis.

Oxidative damage assays

To prepare Gc for oxidative damage assays, all strains were grown in GCBL to mid-log phase (OD600[congruent with]0.5) as described above and used immediately. For H2O2 sensitivity assays, cells were diluted 1:10 into GCBL and 5 ml aliquots were placed into 15 ml Falcon tubes. H2O2 was added to the tubes at final concentrations of 0, 5, 10, 20, or 50 mM and tubes were placed in a drum rotator for 15 min. Cultures were immediately serially diluted into GCBL containing 10μg/ml catalase, and plated onto GCB agar. Colonies were counted after ~20 h growth and survival of each strain at the 5, 10, 20, and 50 mM dose of H2O2 was calculated relative to survival at the 0 mM dose. For cumene hydroperoxide and diamide sensitivity assays, cultures were also diluted 1:10 into GCBL and aliquoted into Falcon tubes incubated at 37°C. Cumene hydroperoxide was added to final concentrations of 0.005% and 0.01%, and diamide was added to final concentrations of 10 and 20 mM , cultures were incubated for 15 and 30 min, respectively, immediately diluted into GCBL, and plated. Relative survival was calculated as above. For paraquat sensitivity assays, cells were diluted to a final concentration of ~5 × 106 CFU/ml in GCBL containing only Kellogg supplement I and 0.042% sodium bicarbonate in 15 ml Falcon tubes. An aliquot was removed to quantify starting CFUs, paraquat was added to 0.1mM final concentration, and tubes were placed in a drum rotor. After 20 and 40 min, aliquots were taken and immediately serially diluted into GCBL and plated onto GCB agar. Survival was calculated relative to the starting CFUs.

Polymorphonuclear leukocyte (PMN) killing assay

Heparinized blood was drawn from healthy volunteers, following a protocol approved by the Northwestern University Institutional Review Board. PMN were isolated by dextran sedimentation followed by purification over a Ficoll-Hypaque gradient (Amersham) as previously described (Simons et al., 2005). PMN were resuspended in Dulbecco’s PBS (without Ca2+ and Mg2+) (Mediatech) containing 0.1% dextrose (Fisher). Synchronized PMN infection was carried out using a protocol adapted from Brinkmann et al. (Brinkmann et al., 2004). PMN were resuspended in RPMI (Gibco) containing 10% heat-inactivated fetal bovine serum (Gibco) and 10 nM human interleukin-8 (R&D Systems) at 2.5 × 106 PMN/ml, and 0.4 ml of the suspension was allowed to adhere to plastic coverslips (Sarstedt) in sterile 24-well tissue culture-treated plates at 37°C for 30 min. Gc were added to each well at a multiplicity of infection of 1 and the plate was centrifuged at 400 × g for 4 min at 10°C. Cells were washed once and replaced in RPMI with serum and warmed to 37°C. At various times post-infection, cells were washed and lysed in 1% saponin in PBS prior to serial dilution and plate count. The number of viable CFU at each time point was expressed as the percent of CFU present at the 0 min time point (after 10°C centrifugation). Data are expressed as the mean ± SEM of at least three replicate wells, and experiments were repeated at least three times. Significance was determined at a 95% confidence interval by the two-tailed Student’s t test.

Supplementary Material

Supp Table


We thank Deborah Tobiason for reading and editing this manuscript. We thank Nadereh Jafari in the Center for Genetic Medicine Microarray Core Facility (NU) for help in experimental design and technical assistance, Trish Dyck in the Robert H. Lurie Cancer Center (NU) for assistance with statistical analysis, Mike Apicella and members of his laboratory for technical assistance in working with PMNs, and John Davies (Monash University, Australia), and members of the Neisseria Array Consortium for supplying microarray slides, and David Dyer (University of Oklahoma) for supplying the FA1090 genome annotation. This work was supported by the following grants: R01 AI44239 and R01/R37 AI33493 (HSS), and F32 AI056681 (AKC).


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