The gonococcal vaccine candidate antigen NGO1701 is a N. gonorrhoeae periplasmic copper storage protein

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 Δ csp 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 Δ csp by dot blot and ELISA, and the sera's ability to kill N. gonorrhoeae was abrogated against Δ csp . 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.

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 Δ csp 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 cbb 3 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 Δ csp 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 .