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

UNLABELLED

The increasing worldwide trend of antibiotic-resistant 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 OB3b. In this study, we confirmed that purified NGO1701 binds 15 Cu(I) ions per monomer , supporting its function as Csp in . Using a deletion mutant generated in F62, we investigated its role in bacteria physiology. We showed that ablation of Csp was not limiting for bacterial growth and fitness , but the Δ strain became significantly more susceptible to copper mediated toxicity. This phenotype was rescued by gene complementation, indicating a role in protection against copper toxicity. Our results indicate that Csp participates in periplasmic copper homeostasis in 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 , since no difference in the ability of to adhere/invade epithelial cells or induce IL-8 secretion was reported among wild type, deletion mutant and complemented strains. Furthermore, sera from mice immunized with NGO1701 failed to recognize Δ by dot blot and ELISA, and the sera's ability to kill was abrogated against Δ . 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 , 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 , capable of sequestering Cu(I) ions. Deletion of led to increased copper sensitivity, while overexpression restored resistance, suggesting a role in copper homeostasis. The Δ 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 3 oxidase and nitric oxide reductase, which support bacterial survival under host-imposed stress. Although Csp is not required for host cell interactions, it is a strong immune target. Immune recognition of Δ by anti- NGO1701 mouse sera was nearly abolished and the serum bactericidal activity was abrogated compared to F62 wild type bacteria, highlighting Csp's potential as a target for therapeutic or vaccine strategies against .