https://orcid.org/0000-0003-3106-0711
Figures
Abstract
The increasing worldwide trend of antibiotic-resistant Neisseria gonorrhoeae strains highlights the urgent need for new therapeutic strategies against this sexually transmitted pathogen, including a gonococcal vaccine. We previously designed a bioinformatics-based candidate selection pipeline (CASS) and identified potential novel gonococcal vaccine targets among hypothetical proteins expressed during natural human infection. One of these candidates, NGO1701, is a predicted periplasmic four-helix bundle protein with amino acid sequence homology to the copper storage protein 1 (Csp1) from Methylosinus trichosporium OB3b. In this study, we confirmed that purified NGO1701 binds 15 Cu(I) ions per monomer in vitro, supporting its function as Csp in N. gonorrhoeae. Using a ngo1701 deletion mutant generated in N. gonorrhoeae F62, we investigated its role in bacteria physiology. We showed that ablation of Csp was not limiting for bacterial growth and fitness in vitro, but the Δcsp strain became significantly more susceptible to copper mediated toxicity. This phenotype was rescued by csp gene complementation, indicating a role in protection against copper toxicity. Our results indicate that Csp participates in periplasmic copper homeostasis in N. gonorrhoeae, buffering excess copper to reduce toxicity and playing a putative role in copper delivery to important copper-enzymes. Csp does not appear to be involved in bacterial host cell interaction and activation in vitro, since no difference in the ability of N. gonorrhoeae to adhere/invade epithelial cells or induce IL-8 secretion was reported among wild type, csp deletion mutant and complemented strains. Furthermore, sera from mice immunized with NGO1701 failed to recognize Δcsp by dot blot and ELISA, and the sera’s ability to kill N. gonorrhoeae was abrogated against Δcsp. However, both functions were restored after gene complementation, supporting the relevance of Csp as a potential vaccine target. Allelic analysis of Neisseria species revealed that this gene is absent in N. meningitidis, thus making it a gonococcal-specific target.
Author summary
Copper is essential for bacterial metabolism but can be toxic in excess. Here, we identify NGO1701 as a copper storage protein (Csp) in Neisseria gonorrhoeae, capable of sequestering Cu(I) ions. Deletion of csp led to increased copper sensitivity, while overexpression restored resistance, suggesting a role in copper homeostasis. The Δcsp mutant also showed reduced growth in cobalt and manganese, likely due to metal interference by copper toxicity. Beyond detoxification, Csp may supply copper to essential cuproenzymes like cytochrome cbb3 oxidase and nitric oxide reductase, which support bacterial survival under host-imposed stress. Although Csp is not required for N. gonorrhoeae host cell interactions, it is a strong immune target. Immune recognition of N. gonorrhoeae Δcsp by anti-NGO1701 mouse sera was nearly abolished and the serum bactericidal activity was abrogated compared to N. gonorrhoeae F62 wild type bacteria, highlighting Csp’s potential as a target for therapeutic or vaccine strategies against N. gonorrhoeae.
Citation: Roe SK, Mazgaj R, Zhu T, Esmaeeli M, Lewis LA, Genco C, et al. (2025) The gonococcal vaccine candidate antigen NGO1701 is a N. gonorrhoeae periplasmic copper storage protein. PLoS Pathog 21(10): e1013559. https://doi.org/10.1371/journal.ppat.1013559
Editor: Nicholas J. Mantis, Wadsworth Center, New York State, UNITED STATES OF AMERICA
Received: March 20, 2025; Accepted: September 22, 2025; Published: October 9, 2025
This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Data Availability: All relevant data are within the manuscript and its Supporting information files.
Funding: This work was supported by NIH/NIAID grant 1R01AI166537-03 to PM, and by a MAESTRO grant from the National Science Center (NCN), Poland (2021/42/A/NZ1/00214) to KJW. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Neisseria gonorrhoeae is a Gram-negative bacterium and the etiological agent of gonorrhea, a human sexually transmitted disease (STD). The incidence of gonorrhea has increased substantially over the past decade, with approximately 82.6 million cases reported annually worldwide and over 600,000 cases occurring in the United States [1]. While in men gonococcal infections are predominantly symptomatic, facilitating diagnosis and treatment, women are often asymptomatic or present with nonspecific symptoms. This can delay diagnosis and treatment, and lead to severe reproductive tract sequelae, such as endometritis, pelvic inflammatory disease (PID), ectopic pregnancy, and infertility. Disseminated gonococcal infections (DGI) and co-infections with Chlamydia trachomatis, Treponema pallidum and HIV are also well-documented [1]. Rising development of antimicrobial resistance has significantly complicated treatment of gonorrhea; fluoroquinolone resistance and rising levels of cefixime mean inhibitory concentrations (MICs) have excluded these antibiotics from treatment guidelines in the United States. Ceftriaxone is currently the sole first-line treatment suggested by the CDC, but resistance to this antibiotic has already been reported outside the U.S. [2–4]. The emergence and global spread of multidrug-resistant N. gonorrhoeae strains is a stark warning of the possibility of untreatable gonorrhea making new therapeutic options essential. A safe and effective vaccine would be the ideal solution.
Although both gonococcal and meningococcal outer membrane vesicles are being currently evaluated for efficacy against gonorrhea, gonococcal antigens continue to be explored [5], with the most advanced being the lipooligosaccharide (LOS) epitope recognized by the 2C7 Mab (and thus referred to as 2C7 epitope) [6,7]. Preclinical research based on reverse vaccinology, transcriptomics and bioinformatics has led to discovery of conserved and surface-exposed antigens. Our group has developed a Candidate Antigen Selection Strategy (CASS) pipeline designed by integrating gonococcal protein expression in mucosal samples from men and women naturally infected with N. gonorrhoeae with predictions of protein surface exposure, immunogenicity, conservation and structure features [8–10]. With this approach, we identified several gonococcal hypothetical proteins as potential new vaccine targets. Three of these candidates (NGO0690, NGO0948 and NGO1701) were tested in mice and showed induction of antibodies with bactericidal activity, were recognized by IgG antibodies in sera from men and women naturally infected with N. gonorrhoeae, and where evaluated as a multi-antigen vaccine in mice [8,11].
NGO1701 is a homologue of Csp1, a member of the family of copper storage proteins (Csps) characterized in the methanotrophic bacterium Methylosinus trichosporium OB3b [12]. Genes encoding cytosolic Csp homologues are found in diverse bacterial genomes, including pathogenic bacteria [13], while Csps that possess the twin arginine translocase (TAT) targeting sequence for secretion out of the cytoplasm are rare outside of the methanotrophic bacteria. Csps adopt a four-helix bundle fold that forms a tube structure lined with Cys residues that coordinate Cu(I) ions for copper storage and supply to copper-requiring enzymes. However, the influence of copper-buffering by cytosolic Csps in bacterial resistance to copper toxicity remains unclear, and a role for secreted Csps in copper resistance is yet untested. Crucially, no function of a Csp protein has been demonstrated in any pathogenic bacterium, despite copper homeostasis being essential to pathogens, and copper toxicity being a known weapon in the nutritional immunity arsenal of immune cells used to fight invading pathogens [14].
In this study, we investigated the function of NGO1701 in N. gonorrhoeae, which we here designate as the gonococcal Csp. Consistent with its homology to characterized Csp homologues, we demonstrated its ability to bind large quantities of copper in vitro. By generating a csp deletion mutant strain, we showed its role in N. gonorrhoeae resistance to copper toxicity and in periplasmic copper homeostasis. Together, our studies define an important function for Csp in N. gonorrhoeae and strengthen the evidence base for this protein as a target antigen for future anti-gonorrhea vaccine development.
Results
Sequence analysis and putative function of NGO1701
We previously reported that NGO1701 (WP_003689877.1) has sequence homology to a TAT_Cys_rich four helix bundle copper-binding protein of the DUF326 superfamily [8]. It was previously shown that a similar protein, Csp1 (copper storage protein 1) is expressed by the methanotrophic bacterium M. trichosporium OB3b bacteria [12], which functions in storage of Cu(I) ions for supply to its copper-dependent methane monooxygenase enzyme [12]. A sequence alignment of NGO1701 with Csp1 (MtCsp1) and its paralogue Csp2 (MtCsp2) from M. trichosporium revealed that they share 38% and 34% sequence identity with the N. gonorrhoeae homologue, respectively (Fig 1). Crucially, all the Cys residues shown to coordinate Cu(I) ions within MtCsp1 are conserved in the N. gonorrhoeae protein. A model of the N. gonorrhoeae protein, based on the determined structure of MtCsp1 [12], predicted it would adopt an identical fold (Fig 2A) in which all the 15 Cys thiols would be localized within the core of the four-helix bundle (Fig 2B). The model also predicted the protein would form a biological tetramer, like other Csp homologues [13] and consistent with the recently predicted topology of the putative Csp from N. subflava [15] (WP_137041264.1). Notably, the predicted tetramer resulted in localization of all predicted antibody epitopes on the surface of the complex exposed to solvent (S1A and S1B Fig), validating this structural model. Based on this sequence and structural homology, we hypothesized that NGO1701 encoded a Csp family protein.
Alignment of the NGO1701 protein sequence with two related sequences from M. trichosporium OB3b (MtCsp1 and MtCsp2) [12] with Clustal Omega. Putative twin arginine translocase (TAT) targeting pre-peptides are shown in violet, Cys residues highlighted in yellow, and Cu(I)-coordinating His and Met residues highlighted in orange. In NGO1701, additional N-terminal His residues potentially involved in copper coordination are shown in blue. Positions exhibiting complete sequence identity are marked with an asterisk (*), and those showing conservation between groups of strongly similar (scores > 0.5) or weakly similar (< 0.5) amino acids based on the PAM250 matrix, are indicated by the: and. symbols, respectively.
(A-D) Structural models of a N. gonorrhoeae Csp monomer produced using IntFOLD, based on the published structure of MtCsp1 (PDB: 5FJD). A) Side-on, and B) end-on views of the apo-monomer, illustrating the predicted four-helix bundle structure (blue ribbon), with the metal-coordinating Cys residues in ball-and-stick representation, colored by element (yellow, S; red, O; blue, N). C) Side-on, and D) end-on view of the same model loaded with 13 copper ions (bronze), using the same color scheme, modelled with the help of RING 4.0. (E-H) SEC analysis of purified recombinant apo-Csp E) before and after titration with F) 10 or G) 20 mole equivalents of Cu(I). Copper (blue) and protein concentration (black), calculated from sulfur content, were determined by ICP-OES. H) Absorbance of the samples at 280 nm in E-G) eluted from the SEC column in the absence (black) or presence of 10 (light blue) or 20 (dark blue) mole equivalents of Cu(I) [12].
The N. gonorrhoeae Csp protein binds copper ions with high stoichiometry
To determine whether the N. gonorrhoeae Csp homologue has the potential to function in an analogous manner to MtCsp1, we first tested whether the protein shares its key biochemical property, i.e., the ability to bind large numbers of Cu(I) ions. Recombinant Csp, carrying a poly-His tag, was purified from E. coli BL21 cultured in LB medium (S1C Fig) and was found to be devoid of bound copper (<0.1 mole equivalent detected) or any other metal ion by ICP-OES (Fig 2E). Incubation of recombinant Csp with 10 or 20 mole equivalents of Cu(I) in vitro, followed by separation of unbound Cu by SEC (Fig 2F and 2G, respectively), demonstrated that Csp co-migrated with approximately 15 equivalents of copper, a suggested estimate of high stoichiometry. A dramatic increase in the protein’s absorbance at 280 nm after incubation with Cu(I) (Fig 2H) was observed, suggestive of the Cu(I) ions being coordinated by Cys residues within the Csp tube, as predicted (Fig 2C–2D). This coordination gives rise to strong ligand-to-metal charge transfer absorption in this region of the spectrum, where intensity is proportional to the number of Cys-Cu(I) bonds, as previously demonstrated for MtCsp1 [12]. Thus, the N. gonorrhoeae Csp binds a large number of Cu(I) ions, consistent with its homology to MtCsp1, its predicted structure, and with a proposed role in N. gonorrhoeae copper homeostasis.
Allelic analysis and conservation of Csp in Neisseria sp.
We previously showed that the gene encoding Csp is conserved in N. gonorrhoeae [8]. Since our original analysis, the number of gonococcal genomes available in the PubMLST database has increased to 28404 [16]. A new analysis of the csp (NEIS2720) gene sequence confirmed conservation in 55 alleles identified, with alleles 1 and 2 representing the most frequent alleles (22938 strains or 80% of total strains, and 4719 strains or 16% of total strains, respectively) (Table 1). Other alleles were present with low frequency (less than 2%), and alleles that could not be assigned were present in 70 N. gonorrhoeae strains (~0.25% of all strains), due to incomplete sequence data because the genes were located at the end of contigs. No polymorphic sites were present in allele 1, and a serine to alanine (S127A) change in allele 2, which was also detected in some of the less frequently represented alleles. This polar to non-polar amino acid change was located within the sequence of the predicted linear epitope 4 (K123 through K133) [11]; its potential impact on the protein structure or its immunological properties remains to be determined. Nonetheless, csp allelic analysis indicated protein sequence conservation across the vast majority of gonococcal strains. Analysis of presence and conservation of the csp gene was also examined in other Neisseriae genomes (Table 1). No record of csp presence was found in N. meningitidis (45281 strain genomes deposited in PubMLST). The gene was present in N. lactamica (65 alleles in 1200 strains), in N. polysaccharea (22 alleles in 86 strains and no alleles assigned to ~ 25% of the deposited strains), N. bergeri (6 alleles in 63 strains, 54% unassigned) and N. cinerea (14 assigned alleles in 58 strains, 67% unassigned) (Table 1). Csp was not present in the 184 N. subflava strain genomes in PubMLST, but it has been recently reported in this commensal [15]. An alanine to serine change in position 128 (A128S) was frequent in N. lactamica alleles, and, in all commensals, an alanine to methionine change in position 130 or 131, and a valine to alanine change in position 150 or 151 (falling within the predicted linear epitope 5 region of gonococcal Csp [11]) were also common. Notably, none of these mutations altered the residues predicted to bind metal ions in Csp. Thus, despite being present in multiple species, Csp has potential as a N. gonorrhoeae-specific vaccine target.
Construction and immune characterization of N. gonorrhoeae csp deletion mutant and complemented strains
To investigate the function of Csp in N. gonorrhoeae, a ngo1701 deletion mutant strain was generated (Δcsp) in N. gonorrhoeae F62, and a corresponding ngo1701 complemented strain (csp_c). Presence and expression of Csp was examined by dot blot using sera from mice previously immunized with purified NGO1701 [8]. N. gonorrhoeae F62, Δcsp and csp_c (uninduced) were grown on GC plates and in GCB medium; to induce Csp expression, csp_c was grown on plates containing 100 µM IPTG and inoculated in broth containing increasing concentrations of IPTG (0.25 mM, 0.5 mM or 1 mM) or no IPTG. Cultures were grown for 2 h, diluted to OD600 of 0.33 and equivalent volumes (5 µl, approx.7.5x105 bacteria/dot) were spotted on nitrocellulose filters. Csp expression was detected in uninduced csp_c and csp_c grown on GC plates containing IPTG (Fig 3A, dots 2 and 3) (pGCC4 is a known leaky plasmid [17]), and it was increased by addition of IPTG also in liquid culture (Fig 3A, dots 4–6) (Csp expression in uninduced csp_c suggests that the cloned DNA may contain a promoter element that drives transcription of the csp independently of the lac promoter). A final IPTG concentration of 0.25 mM was chosen for all subsequent experiments and bacteria grown in these conditions are referred to as csp_c + , while bacteria grown in liquid medium without IPTG are referred to as csp_c. A low-level immunoreactivity of Δcsp with anti-NGO1701 mouse sera was observed (Fig 3A, dot 7), and of all strains with sera from mice immunized with alum alone (Fig 3B, dots 1–7), which were consistent with non-specific sera reactivity with bacteria. Purified recombinant Csp (50 ng) was only recognized by the anti-NGO1701 mouse sera (Fig 3A and 3B, dots 8), indicating antigen specificity.
Bacterial suspensions (OD600 0.33) were spotted on nitrocellulose (5 μl/dot) and examined by dot blot with A) pooled sera from mice immunized with NGO1701 and alum or B) alum alone [8] (1:1000 dilution). 1) N. gonorrhoeae F62 wildtype; 2) uninduced csp_c; 3) csp_c plated on GC agar plates containing 100 μM IPTG and grown in GCB without IPTG; 4) csp_c plated as above and grown in GCB with 0.25 mM IPTG, 5) 0.5 mM IPTG or 6) 1 mM IPTG; 7) Δcsp; 8) purified recombinant Csp (50 ng). C) Total IgG antibodies (µg/ml ± SEM) measured by ELISA of mouse sera as above against N. gonorrhoeae F62 wildtype, Δcsp, csp_c and csp_c+ (0.25 mM IPTG). Alum alone sera (gray bars), anti-NGO1701 and alum (black bars). Sera were tested in triplicate or quadruplicate. **, p = 0.002 and 0.007, *** p = 0.0001 and **** p < 0.0001 by one way ANOVA with Tukey’s comparison test. D) Total IgM antibodies as in C). * p = 0.03, ** p = 0.001 and *** p = 0.0002 by one way ANOVA with Tukey’s comparison test.
A whole-cell ELISA was used to quantify antibody recognition of Csp. Similar levels of IgG antibodies recognizing N. gonorrhoeae F62 wildtype and csp_c were detected, confirming baseline Csp expression in both strains, and higher IgG antibody levels against csp_c+ confirmed protein induction (Fig 3C). Consistent with the dot blot results, immune recognition of Δcsp was significantly lower (Fig 3C), comparable to non-specific immunoreactivity of sera from mice immunized with alum alone (Fig 3C, gray bars). Total IgM antibody levels were also measured, showing significantly higher reactivity against N. gonorrhoeae F62, csp_c and csp_c+ than against Δcsp (Fig 3D, black bars), which was, again, comparable to IgM levels in sera from mice immunized with alum alone (Fig 3D, gray bars).
Phenotypic characterization of Csp in N. gonorrhoeae in vitro
We investigated the role of Csp in gonococcal physiology by examining the growth kinetics of N. gonorrhoeae F62 wildtype, Δcsp and csp complemented cells. No difference among the strains was observed in colony size or morphology by phase-contrast light microscopy of plated bacteria (Fig 4A–D). Growth in liquid medium was monitored for 6 h; no major difference was observed in growth of Δcsp by optical density (Fig 4E, squares), except for a slightly lower OD600 at 5 h compared to the wildtype strain (Fig 4E circles) and at 6 h compared to csp_c (Fig 4E, triangles). In addition, no significant difference in the number of Δcsp colony forming units (CFU)/ml was observed compared to N. gonorrhoeae F62 wildtype (Fig 4F, squares and circles, respectively) or csp_c (Fig 4F, triangles) throughout the 6 h growth curve. Growth kinetics of csp_c+ showed a significantly higher number of CFU/ml at 4, 5 and 6 h (Fig 4F, diamonds) but no significant difference in OD600 (Fig 4E, diamonds). These results suggested that Csp does not play a role in N. gonorrhoeae viability and fitness in vitro, but that IPTG induction of Csp improved bacterial growth.
Light microscopy images showing colony morphology of A) N. gonorrhoeae F62 wildtype, B) Δcsp, C) csp_c and D) csp_c+ on GC agar plates. E) OD600 values (average ± SEM) and F) Colony forming units (CFUs)/ml (average ± SEM) from >10 individual experiments per each strain. N. gonorrhoeae F62 wildtype (circles), Δcsp (squares), csp_c (triangles) and csp_c+ (diamonds). * p < 0.05 (csp_c and csp_c + vs wildtype, 4 h) and ** p < 0.005 (Δcsp vs wildtype, 5 h), and **** p < 0.0001 by 2way ANOVA with Dunnett’s multiple comparisons test.
Copper-mediated Csp function in N. gonorrhoeae
A putative role of Csp in copper homeostasis in N. gonorrhoeae was examined by analyzing growth of the mutant bacteria in the presence of increasing concentrations of copper sulfate (CuSO4). In N. gonorrhoeae F62 wildtype, a decrease in OD was observed starting at 100 µM copper, compared to control cultures with no copper added (Fig 5A, triangles and circles, respectively). Significantly lower OD600 values were measured with 200 µM copper at the 4, 5 and 6 h time points (Fig 5A, diamonds), and growth stopped in the early exponential phase when bacteria were exposed to 500 µM copper (Fig 5A, squares). This growth defect was also reflected in the number of CFU/ml of the bacterial suspensions in the presence of copper (Fig 5E). Copper-mediated toxicity was amplified by deletion of Csp. Growth of Δcsp in the presence of 200 µM and 500 µM copper (Fig 5B and 5F, diamonds and squares) was significantly lower compared to Δcsp grown in the absence of copper (Fig 5B and 5F, circles) and to N. gonorrhoeae F62 wildtype at the same copper concentrations. Bacterial survival was rescued in csp_c to levels comparable to the wildtype strain (Fig 5C and 5G) and further improved for csp_c+ (Fig 5D and 5H).
N. gonorrhoeae F62 wildtype, Δcsp, csp_c and csp_c+ were grown for 6 h in the presence of 100 µM copper sulfate (CuSO4) (triangles), 200 µM (diamonds), 500 µM (squares) or without copper (circles). A-D) OD600 values (average ± SEM) from a minimum of three independent experiments for each strain. Statistical significance was determined by 2-way ANOVA with Dunnett’s multiple comparisons test vs no copper and is indicated as * for any p value < 0.05. E-H) CFUs/ml (average ± SEM) as above. Statistical significance was determined by multiple Mann-Whitney test with Holm-Sidak correction set for p = 0.05 vs no copper for each strain, indicated by *.
Additional evidence for the role of Csp in N. gonorrhoeae resistance to copper was obtained using a disc diffusion assay. Bacterial suspensions were plated as a lawn on GC agar plates and exposed to paper discs impregnated with different concentrations of sterile CuSO4 solution. A dose-dependent zone of inhibition around the discs was visible for N. gonorrhoeae F62 wildtype. The highest copper concentration (200 mM) (Fig 6A, top) led to a wider area devoid of bacteria, which became smaller as the copper concentration decreased (Fig 6A). No zone of inhibition was visible around the control disc soaked with water (Fig 6A, center). Δcsp showed a wider zone of inhibition at all copper concentrations (Fig 6B), confirming increased sensitivity to copper toxicity. Resistance to copper of both csp_c and csp_c + was restored to levels similar to the wildtype strain (Fig 6C and 6D, respectively). The size of the zone of inhibition for each strain was quantified (Fig 6E), confirming higher sensitivity of Δcsp to copper toxicity. Collectively, these results supported a role for Csp in copper resistance in N. gonorrhoeae.
Representative images of the zone of inhibition around discs presoaked with increasing concentrations of CuSO4 (counterclockwise from top: 200 mM, 100 mM, 50 mM and 20 mM) or sterile water (center). A) N. gonorrhoeae F62 wildtype, B) Δcsp, C) csp_c and D) csp_c + . E) The diameter of the zone of inhibition was measured in mm and expressed as average ± SD from triplicate experiments. N. gonorrhoeae F62 wildtype (circles), Δcsp (squares), csp_c (triangles) and csp_c+ (diamonds). * p < 0.05, ** p < 0.005, *** p < 0.0005 and **** p < 0.0001 by 2way ANOVA with Dunnett’s multiple comparisons test vs Δcsp.
Other transition metals important for bacterial growth and functions were examined. No difference in sensitivity to nickel (NiSO4, 200 μM) between N. gonorrhoeae F62 and Δcsp was observed, with similar OD600 values and CFU numbers throughout the 6 h growth curves (S2A and S2B Fig). This suggested no role for Csp in resistance to nickel toxicity. Similarly, no differences in bacterial growth were observed when Δcsp was exposed to either 100 µM ferric nitrate (iron-replete) or 100 μM desferal (iron-deplete) compared to N. gonorrhoeae F62 wildtype nor to excess zinc (50 μM ZnCl2) (S1 File). Sensitivity to manganese (25 µM MnSO4) (S2C and S2D Fig) and cobalt (50 µM CoCl2) (S2E and S2F Fig) appeared to be slightly increased by Csp deletion, with a statistically significant lower OD600 and number of CFUs than wildtype N. gonorrhoeae F62 starting at the 5 h or 3 h time point, respectively. Resistance to cobalt was restored in csp_c and csp_c+ to (S2E and S2F Fig), suggesting that Csp might also be involved in buffering excess of these metals. However, incubation of recombinant Csp with even 1 mole equivalent of Co(II) led to protein precipitation, suggesting that these ions are unlikely to be able to enter the Csp tube. We conclude, therefore, that these other metal phenotypes are likely to be indirect, independent of direct buffering of these metal ions by Csp inside N. gonorrhoeae cells. Together, these results suggested that Csp was not likely to be involved in buffering of metal ions other than Cu(I), supporting a copper-specific function for Csp in N. gonorrhoeae.
Intracellular copper measurement
Copper accumulation by N. gonorrhoeae F62 wildtype and Δcsp was also assessed. Bacteria were digested, and the elemental composition of the resulting lysate was determined by ICP-OES. After adjusting the metal content for sulfur content to normalize for biomass, the results showed that Δcsp contained approximately 60% of the cellular copper content of N. gonorrhoeae F62 wildtype after 4 h culture under basal copper conditions (Fig 7). No significant decreases were detected in cellular content of zinc (Fig 7), manganese, iron or magnesium (S3 Fig). No difference in cellular copper content was detected between N. gonorrhoeae F62 wildtype and Δcsp cells cultured for 4 h in the presence of 200 μM copper (Fig 7), perhaps reflecting a minor contribution of Csp in the periplasm (which represents a small component of the total bacterial cell’s volume) to total copper sequestration under these conditions. These data support a model in which Csp stores copper in N. gonorrhoeae, presumably for supply of this cofactor to copper-dependent enzymes in the cell envelope.
N. gonorrhoeae F62 wildtype and Δcsp were grown for 4 h in GCB without added copper (basal [Cu] medium) or in the presence of 200 μM copper sulphate. After removing surface-adsorbed metal, bacterial pellets were digested and examined for copper (Cu, blue), zinc (Zn, grey) and sulfur (S) content by ICP-OES. Metal content was normalized to S content to account for differences in biomass. P represents the results of an unpaired t test.
Csp does not play a direct role in N. gonorrhoeae interaction with host epithelial cells
To explore whether Csp may play a role in the ability of N. gonorrhoeae to adhere to and invade host cells, we used HeLa cells, a human epithelial reproductive tract cell line previously shown to support gonococcal invasion [18–20]. HeLa cells were incubated with live N. gonorrhoeae F62, Δcsp, csp_c or csp_c+ at a multiplicity of infection (MOI) of 100 for 2 h; monolayers were washed, lysed and plated for colony counting to quantify the number of cell-associated bacteria. No difference was detected among the strains (S1 File). To evaluate internalized bacteria, a gentamicin protection assay was used. HeLa cells were incubated with N. gonorrhoeae as above, unattached bacteria were removed, and medium containing gentamicin was added to kill adherent bacteria. After overnight incubation without antibiotic, the total number of intracellular bacteria was also quantified by colony counting. No difference was observed among the strains, suggesting that Csp did not play a direct role in gonococcal adhesion/invasion processes. Next, to evaluate whether presence and expression of Csp affected the ability of N. gonorrhoeae to induce host cell activation, production of the pro-inflammatory cytokine IL-8 was used as read-out. HeLa cells (104/ml) were incubated overnight with purified Csp (10 µg/ml), or with N. gonorrhoeae F62, Δcsp, csp_c or csp_c+ strains at MOIs of 10 and 100 (formalin-inactivated to prevent bacterial proliferation during the incubation), and IL-8 secretion was measured by ELISA of the co-culture supernatants. All strains induced similar levels of IL-8 secretion in a dose-dependent manner according to the bacterial MOI (S4A and S4B Fig). Although a small, statistically significant lower IL-8 production was observed in response to Δcsp compared to N. gonorrhoeae F62 wildtype, csp_c and csp_c + , this was unlikely to be biologically significant. Purified Csp (10 µg/ml) also induced low levels of IL-8 secretion (S4A and S4B Fig, gray bars). Overall, these results did not suggest a role for Csp in N. gonorrhoeae interaction with host cells in vitro.
Serum bactericidal activity (SBA)
Serum bactericidal activity (SBA) is universally accepted as a correlate of protection against meningococcal infection [21]; although it has not been established as an unequivocal correlate of protection against N. gonorrhea, SBA is considered a close surrogate of protection in vitro against this pathogen [22,23]. We previously showed that anti-NGO1701 mouse sera have bactericidal activity against several N. gonorrhoeae strains, with killing titers ranging from 1/5–1/40, depending on the strain [8]. When anti-NGO1701 mouse sera were tested against N. gonorrhoeae Δcsp, killing was almost completely abrogated compared to N. gonorrhoeae F62 wildtype (Fig 8A and 8B), even at the highest sera concentration used (1/10). SBA was restored against csp_c (Fig 8C) and csp_c+ (Fig 8D). These results supported our previous studies and the potential of Csp as a vaccine candidate antigen against N. gonorrhoeae.
Survival of A) N. gonorrhoeae F62 wildtype, B) Δcsp, C) csp_c and D) csp_c+ incubated with sera from mice immunized with alum alone (gray bars) or NGO1701 and alum (white bars), expressed as % CFU (T30/T0) ± SEM from triplicate experiments. Bacteria alone, (black bars). * p < 0.05, ** p < 0.009 and **** p < 0.0001 by ordinary one-way ANOVA with Dunnett’s multiple comparisons test vs adjuvant alone sera. Sera dilutions are shown on the x-axis.
Discussion
Rising antimicrobial resistance in N. gonorrhoeae has sparked interest in developing a vaccine against this sexually transmitted infection to mitigate the global disease burden. Initial clinical trial studies with a killed whole-cell vaccine or purified antigens (pilin, porins) failed to confer protection against heterologous re-infection in a human volunteer male urethral infection model. This was attributed to antigenic variability and induction of blocking antibodies (reviewed in [5]). Recently, outer membrane vesicle (OMV)-based vaccines developed for N. meningitidis have suggested cross-protective potential against N. gonorrhoeae [24,25], supported by induction of bactericidal antibodies and protection in preclinical studies [26–28]. Gonococcal OMVs have also been tested, showing serogroup-specific protection in a mouse model of gonococcal vaginal colonization [29]. However, the protein content of the OMVs is just beginning to be characterized and it is still unclear how many and which targets are responsible for the observed protection [30,31].
NGO1701 is a gonococcal hypothetical protein that we identified as a potential vaccine antigen through a novel transcriptomics-based approach (CASS) [8]. Its periplasmic localization was predicted based on its possession of a pre-sequence that will target it to the TAT system for secretion, and this prediction is supported by its detection in extracts of the cell envelope [32] as well as its recognition by antibodies herein. Immunization of mice with this protein (alone, or combined with two other CASS candidates, NGO0690 and NGO0948 (BamC)) induced antibodies with bactericidal activity [8,11]; serum bactericidal activity (SBA) is regarded as the closest in vitro surrogate of protection assay for N. gonorrhoeae [22]. In ongoing experiments aimed at evaluating a trivalent vaccine composed of these three antigens with Alum+MPLA as adjuvants, we have observed a significant reduction in bacterial burden and a faster gonococcal clearance in a mouse model of gonococcal vaginal colonization [33]. However, due to lack of information about NGO1701 function in N. gonorrhoeae, it is impossible to predict whether expression of this protein might be modified by N. gonorrhoeae as a mechanism of immune escape, while maintaining bacterial fitness in vitro and in vivo.
We have initiated the characterization of NGO1701, which we here designated as the N. gonorrhoeae copper storage protein (Csp). Consistent with sequence homology to characterized Csp proteins (Fig 1), we found that purified recombinant N. gonorrhoeae Csp was able to sequester approximately 15 Cu(I) ions per protein monomer (Fig 2). Copper was likely coordinated to Cys residues localized to the interior cavity of the putative four-helix bundle structure as copper binding resulted in a strong increase in absorbance at 280 nm. This is consistent with the presence of ligand-to-metal charge transfer absorptions on the formation of Cys-Cu(I) bonds, as was observed in MtCsp1. Like the previously characterized Csp1 in M. trichosporium OB3b, the gonococcal Csp possesses a putative TAT signal sequence, consistent with its presence in the cell envelope but how it localizes to the bacterial surface, enabling recognition by antibodies (Fig 3), is unclear. This also raises a question from where the Cu(I) that Csp stores comes from within N. gonorrhoeae, which is also unclear in M. trichosporium. TAT translocation could indicate that Csp acquires copper inside the reducing cytosol prior to transport, or alternatively, folding prior to TAT transport may function to protect its Cys residues once inside the periplasm, and instead it may receive Cu(I) effluxed from the cytosol by the copper transporter CopA [34,35] or from an as yet undiscovered reductase. A cellular function for Csp proteins in sequestration of excess copper to detoxify this highly redox active metal to prevent deleterious side-reactions has been tested in some systems but has yielded contradictory results. Heterologous expression of a cytosolic Csp in E. coli slightly increased growth [13], whereas a strain of Streptomyces lividans lacking its cytosolic Csp showed slightly reduced growth under toxic copper conditions [36]. However, a mutant strain of B. subtilis lacking its cytosolic Csp did not show inhibited growth in elevated copper [37]. Here, we have established that N. gonorrhoeae growth in high copper conditions correlates with Csp expression, with a Δcsp strain exhibiting growth inhibition and a csp_c+ strain with induced expression of Csp growing better relative to wildtype cells (Figs 4–6). Interestingly, we also observed reduced growth of Δcsp in the presence of other metals such as cobalt and manganese (S4 Fig) but, unlike Cu(I), we observed no sequestration of Co(II) or Mn(II) ions by the recombinant Csp protein. It seems likely that these phenotypes are caused by displacement of copper ions by excess cobalt or manganese within the cell envelope. Although the observed differences in growth under high copper conditions were small, they are notable given that Csp is secreted out of the cytosol. This indicates that sequestration of excess Cu(I) ions by Csp can protect some vulnerable target(s) in the cell envelope under copper stress conditions. This may be important during infection as copper detoxification is essential for pathogens to overcome copper toxicity during infection [14,38,39].
On the other hand, prior studies have indicated a homeostatic role for Csp proteins in the storage of copper cofactors for supply to copper-dependent enzymes. In M. trichosporium, Csp storage influences copper availability to the copper-sensing regulator that controls the switch between its copper-dependent and iron-dependent isozymes of methane monooxygenase [12], which performs the critical reaction in its utilization of methane. The cytosolic Csp in Bacillus subtilis has been suggested to supply copper to the multicopper oxidase enzyme CotA during sporulation [37]. N. gonorrhoeae lacking Csp accumulated significantly fewer copper atoms when cultured under basal copper conditions, suggesting a function for Csp in normal growth (Fig 7). N. gonorrhoeae possesses several important copper-dependent enzymes in the cell envelope, including cytochrome cbb3 oxidase, an azurin homologue, and nitric oxide reductase [40,41]. The latter is both periplasmic and surface-localized, plays a role in virulence, and its metalation involves the cuprochaperone AccA [42]. Therefore, a working hypothesis is that Csp in the N. gonorrhoeae envelope can supply copper to one or more of these target copper-enzymes. Metals are important for gonococcal responses to host nutritional immunity and virulence but, since no considerable growth defect (i.e., colony morphology, size, optical density, CFU numbers and replication time) was observed for Δcsp compared to wildtype N. gonorrhoeae in standard culture conditions (Fig 4), it is possible that Csp is not critical for bacterial fitness. Whether Csp plays a role in N. gonorrhoeae virulence awaits explicit experimental testing. So far, we have not observed an effect of Csp on the ability of N. gonorrhoeae to adhere to and invade a human cervical epithelial cell line model, HeLa cells, widely used to study bacterial host cell interactions. While this result was not unexpected (there is no indication that Csp may act as an adhesin), future studies may examine other reproductive tract epithelial cell lines and immune cells, particularly macrophages. Similarly, no effect of Csp presence or expression by N. gonorrhoeae was observed on epithelial cell inflammatory responses in vitro (S4 Fig). Unfortunately, analysis of the effect of copper on N. gonorrhoeae virulence in a host cell co-culture model is limited by the toxicity of copper in these systems; we observed a dose-dependent and time-dependent cytotoxic effect of copper on HeLa cells starting at 100 µM, a concentration for which only a small difference in survival was reported between Δcsp and N. gonorrhoeae F62 wild type. However, csp is indeed expressed in vivo by gonococci recovered from men and women naturally infected with N. gonorrhoeae [10,43,44], which was one of the attributes for its identification as a potential vaccine target. The ability of N. gonorrhoeae Δcsp to survive in vivo is currently being tested in a mouse model of vaginal colonization to correlate bacterial fitness with infection.
The bactericidal activity of anti-NGO1701 mouse sera is abrogated against Δcsp (Fig 8) and thus, if Csp expression by N. gonorrhoeae in vivo is turned down or suppressed as a vaccine escape strategy, the immune responses induced by vaccination with this antigen alone may become less relevant. However, including Csp in a multivalent subunit vaccine may contribute to broader protection and decrease the chances of vaccine failure. Understanding the precise role of Csp in gonococcal physiology and homeostasis, and especially its putative function in supply of copper to envelope copper-enzymes essential for virulence will enable better assessment of its future value as a therapeutic and vaccine target.
Materials and methods
Csp sequence analysis and structure modeling
The sequence of characterized M. trichosporium OB3b (MtCsp1 and MtCsp2) [12] Csp proteins were downloaded from NCBI and aligned with N. gonorrhoeae NGO1701 (new locus tag NGO_RS08465) using Clustal Omega [45]. Annotations were added manually based on prior characterization studies [12,13]. Structural modelling used IntFOLD and GalaxyHomomer [46,47], with modelling of copper ions using RING 4.0 [48] and were visualized using Pymol [49] (available at: http://www.pymol.org/pymol). Allelic analysis of Csp (NEIS2720) in all Neisseria sp. genomes available in the PubMLST database [16] was carried out to evaluate csp gene sequence presence and conservation.
Expression of recombinant Csp in E. coli
N. gonorrhoeae Csp (Accession number: WP_003689877.1) was cloned into a pET17b plasmid with an ampicillin-resistance cassette and a C-terminal 6x His-tag as previously described [8]. Recombinant Csp was expressed in E. coli BL21 (DE3) cells in selective (50 μg/mL ampicillin) LB medium through 4 h induction with 1 mM IPTG, and initially purified by nickel affinity chromatography as previously described [8]. The purified protein was resolved by size exclusion chromatography (SEC) on a Superdex 75 10/300 GL column (Cytiva) in 20 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA. Purified protein was resolved by reducing SDS-PAGE on 16% (w/v) acrylamide gels and Coomassie staining confirmed the presence of a single band (Thermo).
Construction of csp deletion mutant and complemented strains in N. gonorrhoeae F62
N. gonorrhoeae F62 wildtype (Pil + /Opa+) organisms were propagated from frozen glycerol stocks on GC medium base agar plates (Difco) supplemented with 1% (v/v) IsoVitaleX (GC plates) at 37°C with 5% CO2 overnight. Colonies were swabbed with a sterile loop and inoculated in liquid cultures in GC broth (Difco) supplemented with 1% IsoVitaleX (GCB) and grown at 37ºC with shaking at 120 rpm for 2 h. Growth was measured spectrophotometrically at OD600 (OD600 = 1 corresponding to ~ 1–2 x 109 bacteria/ml). For generating a csp deletion mutant strain (Δcsp), the ngo1701 gene was amplified by PCR using primers designed based on the available sequence of ngo1701 from FA1090 (NCBI accession # NC_002946) (S1 Table). The chromosomal DNA region 428-bp upstream of ngo1701 was amplified using upstream_F and upstream_R primers, and the region 262-bp downstream of ngo1701 using downstream_F and downstream_R primers. Primers encoded EcoRI and HindIII sites, respectively, allowing the insertion of a kanamycin (Kan) resistance cassette (kan) containing the same restriction endonucleases. The Kan cassette (975-bp) was amplified from the pGCC4 plasmid (Addgene, Watertown, MA) using kan_F and kan_R primers, digested with EcoRI and HindIII (New England Biolabs, Ipswich, MA) and ligated with ngo1701 upstream and downstream products single-digested with the same restriction enzymes. The resulting linear construct, ngo1701::kan, was transformed into naturally competent N. gonorrhoeae F62 and integrated into the chromosome by homologous recombination [50] to generate the Δcsp strain. Transformants were selected on GC plates containing Kan (50 μg/ml) and grown as described above. Correct insertion of ngo1701::kan into the chromosome was verified by PCR using a ngo1701 upstream confirmation primer (ngo1701_F-check) and a primer (Kan_F/R) complementary to the Kan cassette (pGCC4 mid Kan), and by Sanger sequencing. All primers were synthesized by Integrated DNA Technologies (IDT) (Coralville, IA). For construction of the ngo1701 complemented strain (csp_c), the Neisseria insertional complementation system (NICS) was used [51] with pGCC4 to insert a functional copy of csp in the Δcsp strain, ectopically at an unlinked chromosomal locus between the lctP and aspC genes, under control of an isopropyl--D-thiogalactoside (IPTG)-regulated lac promoter, and with an Erythromycin-resistance cassette. Cloning of the pGCC4::ngo1701 complementation plasmid was outsourced to Genscript (Piscataway, NJ). Briefly, the csp gene, including 131-bp upstream of the ATG start codon and 113 bp downstream of the csp stop codon, was synthesized located between the PacI and FseI restriction sites to create the pGCC4::ngo1701 complementation plasmid. All plasmids were confirmed by whole plasmid sequencing (Plasmidsaurus Inc.). The linearized pGCC4::ngo1701 complementation plasmid was transformed into N. gonorrhoeae F62 wildtype, and transformants were selected on GC plates containing erythromycin (1.25 µg/ml). Genomic DNA obtained from N. gonorrhoeae F62 harboring ngo1701 at the complementation site (AspC-LctP) was used to transform Δcsp. Transformant colonies were selected on GC plates containing erythromycin (1.25 µg/ml) and kanamycin (50 µg/ml). The presence and proper insertion of the complementing copy of ngo1701 at the ectopic site between lctP and aspC was confirmed by PCR using LctP_F and AspC_R primers or LctP_F and Laclq_R primers. PCR was also used to confirm mutation of the native chromosomal copy of ngo1701. The nucleotide sequence of the complementing copy of ngo1701 in N. gonorrhoeae csp_c was confirmed by sequence analysis of the amplified PCR product (Genewiz, Azenta Life Sciences).
Expression of Csp in N. gonorrhoeae csp_c and dot blot
The csp_c strain was grown on GC plates containing 100 µM IPTG. Bacteria were swabbed from the plates and inoculated in GCB containing 0.1, 0.25, 0.5 or 1 mM IPTG, or no IPTG. The N. gonorrhoeae F62 and the Δcsp strains were grown in GCB as previously described. Liquid cultures were grown for 2 h, diluted to OD600 = 0.33 and 5 μl aliquots (~ 1.6 - 3.3 x 106 bacteria total) were directly spotted onto nitrocellulose membranes. Purified recombinant Csp (5 µl, 50 ng total) was used as a positive control. Membranes were blocked with PBS/5% BSA and incubated overnight at 4°C with sera from mice immunized with alum alone or with NGO1701 and alum [8] (1:1000 dilution). Immunoreactivity was detected with an anti-mouse IgG secondary AP-conjugated antibody (Southern Biotech, Birmingham, AL, USA) and NBT/BCIP (5-bromo-4-chloro-3-indolyl phosphate/Nitroblue Tetrazolium) chromogenic substrate (Bio-Rad, Hercules, CA, USA). A concentration of 0.25mM IPTG in liquid culture was chosen for all subsequent experiments and bacteria grown in this condition were designated as csp_c + .
Elemental analysis by inductively coupled plasma optical emission spectrometry (ICP-OES)
Recombinant Csp samples were digested in 500 μL concentrated (65% v/v) nitric acid (Merck) overnight at room temperature. Heat-killed bacterial samples were digested in 500 μL concentrated nitric acid at 70°C for at least 48 h. The digested samples were diluted 10-fold with 1% nitric acid solution containing 50 μg/L Ir as internal standard. Matrix-matched standard solutions containing manganese (Mn; monitored at wavelength 257.610 nm), iron (Fe; 259.94 nm), cobalt (Co; 228.616 nm), copper (Cu; 324.754 nm), zinc (Zn; 206.2 nm), nickel (Ni; 231.604 nm), calcium (Ca; 317.933 nm) and sulfur (S; 180.731 nm) were prepared in an identical manner for generation of a calibration curve. All standard and sample solutions were analyzed by inductively coupled plasma optical emission spectrometry (ICP-OES) on a Thermo iCAP PRO instrument (RF power 1250 W, with nebulizer gas flow 0.5 L/min, auxiliary gas flow 0.5 L/min, and cool gas flow 13.5 L/min argon). Elemental concentrations in each sample were calculated by comparison with the standard curve with Qtegra ISDS Software (Thermo).
Csp in vitro copper binding assay
Purified Csp (500 μL, estimated at 10 μM by nanodrop) was digested in nitric acid and analyzed by ICP-OES. Protein concentrations were then accurately calculated from the quantitation of S (Csp contains 14 Cys and 4 Met residues). Purified Csp was buffer-exchanged into 20 mM Tris, pH 7.5, 150 mM NaCl to remove EDTA from the purification process using Amicon filter-concentrators (10 kDa MWCO). Aliquots (25 μM) were then titrated with increasing equivalents Cu(I) by addition of a 1:20 mixture of CuSO4 and ascorbate. After each addition of 1 mole equivalent Cu(I), samples were incubated for 5 minutes at room temperature. After titration with 10 and 20 mole equivalents, aliquots (500 μL) were resolved by SEC on Superdex 75 in the same buffer. Fractions were analyzed by ICP-OES as above.
Growth curves
Bacteria were grown as described above. Suspensions were grown for 2 h, diluted to OD600 ~ 0.02 (~ 2–4 x 107 bacteria/ml) and grown for additional 6 h. Growth was monitored hourly spectrophotometrically at OD600 and by plating serial culture dilutions on GC plates in triplicate for counting of colony forming units (CFU).
Mouse antibody ELISA
Bacterial suspensions (N. gonorrhoeae F62 wildtype, Δcsp, csp_c and csp_c+) were grown as described above, centrifuged and resuspended at OD600 of ~ 2 (corresponding to ~ 2–4 x 109 bacteria/ml) followed by formalin fixing as previously described [8]. ELISA plates were coated with 100 µl of bacterial suspensions diluted to obtain ~ 1.5 x 108 bacteria/ml overnight at 4°C. Plates were then washed, blocked with 1% BSA in PBS/0.05% Tween-20 (PBS/T) for 2 h at room temperature (r.t.), and incubated with serial dilutions of pooled mouse sera (alum alone or anti-NGO1701) overnight at 4°C. The next day, plates were washed, incubated for 2h at r.t. with AP-conjugated secondary anti-mouse total IgG or IgM antibodies (Southern Biotech), followed by 1-step PNPP (p-nitrophenyl phosphate) reagent (Thermo Fisher Scientific) and spectrophotometric detection at OD405. Sera were tested in triplicate or quadruplicate wells. Total IgG and IgM were quantified in µg/ml using antibody reference standard curves (Southern Biotech) and a linear regression function [8].
Metal sensitivity
Bacterial suspensions as described above were diluted to OD600 ~ 0.02 (Time 0) and grown for additional 6 h in the presence of 100, 200 or 500 µM copper sulphate (CuSO4). Growth was monitored hourly spectrophotometrically at OD600, and by plating serial dilutions of the cultures in triplicate on GC plates for colony counting. Each growth curve was repeated at least twice. Other metals tested included cobalt chloride (CoCl2, 50 µM); manganese sulphate (MnSO4, 25 µM); nickel sulphate (NiSO4, 200 µM), zinc chloride (ZnCl2, 50 µM), ferric nitrate (Fe(NO3)3, 100 µM) and Desferal (100 μM) for iron-depleted conditions.
Disc diffusion assay
Bacterial suspensions as above were diluted to OD600 ~ 0.2 (2–4 x 108 bacteria/ml) and plated as a lawn on GC plates using a sterile cotton swab. Paper discs (Oxoid) pre-soaked with 10 µl of CuSO4 solution at concentrations of 20, 50, 100 or 200 mM were placed on the lawn and plates were incubated overnight [35]. Discs soaked with sterile water were used as negative control. The next day, the zone of clearing around each disc was measured in mm. Disc diffusion experiments were repeated three times per each strain.
Measurement of intracellular copper content
Bacterial suspensions as above were diluted to OD600 of ~ 0.1 and incubated with 200 µM copper sulphate for 4 h. Bacteria were pelleted, washed with 5 ml of cold PBS, followed by another wash with PBS containing 1 mM EDTA to remove any additional residual metal. Bacteria were heat-killed at 65°C for 30 minutes and pellets were digested in 500 μL concentrated (65% v/v) nitric acid (Merck) at 70°C for at least 48 h. Once digestion was complete, the digests were centrifuged at 10,000 g for 10 minutes and the supernatants diluted 10-fold with 1% nitric acid solution and analyzed by ICP-OES as described above. Metal contents were normalized for small differences in biomass according to the S measurements to yield metal:sulfur ratios to enable comparisons.
HeLa cells stimulation and invasion assay
HeLa cells (ATCC CCL-2) were grown in DMEM medium (Gibco) containing 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin in a 5% CO2 incubator at 37°C. For stimulation, 104 cells/well were seeded in 96-well plates and let adhere overnight. The next day, formalin-killed N. gonorrhoeae F62, Δcsp, csp_c and csp_c+ (~ 1–2 x 107 total bacteria in 100 µl) and purified Csp (10 μg/ml) were added to the wells and incubated overnight. Medium alone was used as negative control. Supernatants were collected, and secretion of IL-8 was quantified by ELISA using OptEIA ELISA kits (BD Biosciences) as specified by the manufacturer. For bacterial adherence and internalization assays, HeLa cells (5x104 cells/ml) were seeded in 24-well plates as above. The next day, wells were washed with PBS, followed by addition of live N. gonorrhoeae F62, Δcsp, csp_c or csp_c+ suspensions at MOI 100 (multiplicity of infection) in fresh medium without antibiotics. The co-cultures were incubated for 2 h at 37°C, after which wells were washed with PBS to remove free and non-adherent bacteria. To evaluate bacterial adherence, a set of wells was incubated with 200 μl of PBS containing 1% saponin for 10 minutes at room temperature to lyse the cells followed by vigorous pipetting. Cell lysates were serially diluted and plated on GC plates for subsequent colony counting. Invasion was evaluated by gentamicin protection assay [52]. Briefly, fresh medium containing 100 µg/ml of gentamicin (Sigma) was added to another set of wells for 1 h at 37° C to kill adherent bacteria. Wells were washed with PBS, followed by addition of fresh medium without gentamicin overnight. The next day, cells were lysed as described above to release intracellular bacteria, and the lysates were plated as above.
Serum bactericidal activity assay (SBA)
SBA was carried out in 96-well U-bottom plates as previously described [8,11]. Bacterial suspensions (OD600 of ~ 0.2) were serially diluted to 2–4 x 104 bacteria/ml in HBSS supplemented with 0.15 mM CaCl2, 1 mM MgCl2 (HBSS++) and 2% BSA [11]. Bacteria were incubated with serial dilutions of heat-inactivated (56 °C for 30 min) pooled mouse sera (alum alone or anti-NGO1701) for 20 min at 37 °C. IgG/IgM-depleted normal human serum (NHS) (10%) (Pel-Freez Biologicals, Rogers, AR) was added as a source of complement, and 5 µl of bacterial suspensions were immediately plated in triplicate on GC plates (Time 0). The remaining suspensions were incubated for 30 minutes at 37 °C and aliquots were plated as above (Time 30). The next day, bacterial killing was evaluated by CFU counting. Survival was expressed as percent of CFUs at T30/T0, and bactericidal titers were defined as the reciprocal of the lowest serum dilution with ≥ 50% killing after 30 minutes. Controls included bacteria alone and bacteria incubated with NHS alone.
Statistical analysis
Statistical significance was examined with GraphPad Prism 10.4.1 by Ordinary one-way ANOVA with Tukey’s comparison test, by 2way ANOVA with Tukey’s multiple comparisons test or with Dunnett’s multiple comparisons test, by multiple Mann-Whitney test with Holm-Sidak correction method (p = 0.05) or by unpaired t test as indicated in the Figure legends. Differences were considered significant at a minimum p value of 0.05.
Supporting information
S1 Fig. Structure model of the Csp tetramer.
A) Side view of a cartoon model of Csp based on the Csp1 structural model created with GalaxyHomomer (one pair of symmetry-related monomers shown in dark blue and the other pair in light blue). B) Front view (90° rotation relative to A). The colored regions illustrate the predicted linear epitopes (LE) 1–5 and conformational epitopes (CE) 1–3 [11], located on the outside of the tetrameric structure, validating the model and confirming this tetrameric form as the likely structure in solution. C) SDS-PAGE and Coomassie staining of purified recombinant Csp fractions from size exclusion chromatography following Ni-NTA column chromatography. Fractions were resolved on a 16% polyacrylamide gel.
https://doi.org/10.1371/journal.ppat.1013559.s001
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S2 Fig. N. gonorrhoeae growth and metal toxicity.
OD600 values (average ± SEM) from three independent growth curves in the presence of A) 200 µM nickel sulfate (NiSO4), C) 25 µM manganese sulfate (MnSO4) or E) 50 µM cobalt chloride (CoCl2). * p ≤ 0.05 vs no treatment by 2-way ANOVA with Dunnett’s multiple comparisons test. B-D-F) CFUs/ml (average ± SEM) as above. Statistical significance was determined by multiple Mann-Whitney test with Holm-Sidak correction set for p = 0.05 vs no treatment for each strain, indicated by *. N. gonorrhoeae F62 wildtype (circles), Δcsp (squares), csp_c (triangles) and csp_c+ (diamonds).
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S3 Fig. Accumulation of other metals in N. gonorrhoeae Δcsp is not affected by Cu. N. gonorrhoeae F62 wildtype and Δcsp were grown for 4 h in GBC without added copper (basal [Cu] medium) or in the presence of 200 μM copper sulphate.
After removing residual surface-adsorbed metal, bacterial pellets were digested and analyzed for Ca (black), Mn (teal), Fe (pink), Mg (blue) and S (sulfur) by ICP-OES. Metal content was normalized to S content to account for differences in biomass. P represents the results of an unpaired t test.
https://doi.org/10.1371/journal.ppat.1013559.s003
(TIF)
S4 Fig. IL-8 secretion by HeLa cells.
Cells were incubated with purified Csp (10 μg/ml) (gray bars), N. gonorrhoeae F62 wildtype (black bars), Δcsp (dotted bars), csp_c (thin striped bars) or csp_c+ (thick striped bars) at A) MOI 10 or B) MOI 100 for 18 h. IL-8 secretion was measured in cell culture supernatants by ELISA and expressed as pg/ml ± SEM from triplicate wells. * p < 0.05, **, p < 0.005, ***, p < 0.0005 and ****, p < 0.0001 by Ordinary one way ANOVA with Tukey’s multiple comparison test.
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S1 File. Quantitative data used in calculations corresponding to primary Figs 1–8 and S1–S4 and Table 1, zinc, iron and Desferal bacterial growth curves, and HeLa cells adhesion/invasion experiments described in the text.
Numeric values used to generate graphs, means, and standard deviations are indicated in each tab corresponding to the relevant figure panel.
https://doi.org/10.1371/journal.ppat.1013559.s006
(XLSX)
Acknowledgments
The authors thank Dr. Karrera Djoko (Durham University, UK) for critical reading of the manuscript.
This work was prepared while Lisa Lewis was employed at the University of Massachusetts Chan Medical School. The opinions expressed in this article are the author’s own and do not reflect the view of the National Institutes of Health, the Department of Health and Human Services, or the United States government.
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