{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,9,10]],"date-time":"2026-09-10T22:40:06Z","timestamp":1789080006048,"version":"build-2803163510"},"posted":{"date-parts":[[2025,5,7]]},"group-title":"Immunology","reference-count":47,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,5,7]],"date-time":"2025-05-07T00:00:00Z","timestamp":1746576000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"funder":[{"name":"NIH\/NIAID","award":["1R01AI166537-03"],"award-info":[{"award-number":["1R01AI166537-03"]}]},{"award":["MAESTRO 2021\/42\/A\/NZ1\/00214"],"award-info":[{"award-number":["MAESTRO 2021\/42\/A\/NZ1\/00214"]}],"id":[{"id":"https:\/\/ror.org\/03ha2q922","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,5,7]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  The increasing worldwide trend of antibiotic-resistant\n                  <jats:italic>Neisseria gonorrhoeae<\/jats:italic>\n                  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\n                  <jats:italic>Methylosinus trichosporium<\/jats:italic>\n                  OB3b. In this study, we confirmed that purified NGO1701 binds 15 Cu(I) ions per monomer\n                  <jats:italic>in vitro<\/jats:italic>\n                  , supporting its function as Csp in\n                  <jats:italic>N. gonorrhoeae<\/jats:italic>\n                  . Using a\n                  <jats:italic>ngo1701<\/jats:italic>\n                  deletion mutant generated in\n                  <jats:italic>N. gonorrhoeae<\/jats:italic>\n                  F62, we investigated its role in bacteria physiology. We showed that ablation of Csp was not limiting for bacterial growth and fitness\n                  <jats:italic>in vitro<\/jats:italic>\n                  , but the \u0394\n                  <jats:italic>csp<\/jats:italic>\n                  strain became significantly more susceptible to copper mediated toxicity. This phenotype was rescued by\n                  <jats:italic>csp<\/jats:italic>\n                  gene complementation, indicating a role in protection against copper toxicity. Our results indicate that Csp participates in periplasmic copper homeostasis in\n                  <jats:italic>N. gonorrhoeae,<\/jats:italic>\n                  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\n                  <jats:italic>in vitro<\/jats:italic>\n                  , since no difference in the ability of\n                  <jats:italic>N. gonorrhoeae<\/jats:italic>\n                  to adhere\/invade epithelial cells or induce IL-8 secretion was reported among wild type,\n                  <jats:italic>csp<\/jats:italic>\n                  deletion mutant and complemented strains. Furthermore, sera from mice immunized with NGO1701 failed to recognize \u0394\n                  <jats:italic>csp<\/jats:italic>\n                  by dot blot and ELISA, and the sera\u2019s ability to kill\n                  <jats:italic>N. gonorrhoeae<\/jats:italic>\n                  was abrogated against \u0394\n                  <jats:italic>csp<\/jats:italic>\n                  . 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                  <jats:italic>N. meningitidis<\/jats:italic>\n                  , thus making it a gonococcal-specific target.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>Author Summary<\/jats:title>\n                  <jats:p>\n                    Copper is essential for bacterial metabolism but can be toxic in excess. Here, we identify NGO1701 as a copper storage protein (Csp) in\n                    <jats:italic>Neisseria gonorrhoeae<\/jats:italic>\n                    , capable of sequestering Cu(I) ions. Deletion of\n                    <jats:italic>csp<\/jats:italic>\n                    led to increased copper sensitivity, while overexpression restored resistance, suggesting a role in copper homeostasis. The \u0394\n                    <jats:italic>csp<\/jats:italic>\n                    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\n                    <jats:italic>cbb<\/jats:italic>\n                    3 oxidase and nitric oxide reductase, which support bacterial survival under host-imposed stress. Although Csp is not required for\n                    <jats:italic>N. gonorrhoeae<\/jats:italic>\n                    host cell interactions, it is a strong immune target. Immune recognition of\n                    <jats:italic>N. gonorrhoeae<\/jats:italic>\n                    \u0394\n                    <jats:italic>csp<\/jats:italic>\n                    by anti- NGO1701 mouse sera was nearly abolished and the serum bactericidal activity was abrogated compared to\n                    <jats:italic>N. gonorrhoeae<\/jats:italic>\n                    F62 wild type bacteria, highlighting Csp\u2019s potential as a target for therapeutic or vaccine strategies against\n                    <jats:italic>N. gonorrhoeae<\/jats:italic>\n                    .\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.1101\/2025.05.01.651437","type":"posted-content","created":{"date-parts":[[2025,5,7]],"date-time":"2025-05-07T22:15:16Z","timestamp":1746656116000},"source":"Crossref","is-referenced-by-count":2,"title":["The gonococcal vaccine candidate antigen NGO1701 is a\n                  <i>N. gonorrhoeae<\/i>\n                  periplasmic copper storage protein"],"prefix":"10.64898","author":[{"given":"Shea K.","family":"Roe","sequence":"first","affiliation":[{"name":"Department of Immunology, Tufts University School of Medicine, Boston MA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rafa\u0142","family":"Mazgaj","sequence":"additional","affiliation":[{"name":"Institute of Biochemistry & Biophysics, Polish Academy of Science, Warsaw 02-106, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tianmou","family":"Zhu","sequence":"additional","affiliation":[{"name":"Department of Immunology, Tufts University School of Medicine, Boston MA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mariam","family":"Esmaeeli","sequence":"additional","affiliation":[{"name":"Institute of Biochemistry & Biophysics, Polish Academy of Science, Warsaw 02-106, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lisa A.","family":"Lewis","sequence":"additional","affiliation":[{"name":"Division of Infectious Diseases, University of Massachusetts Chan Medical School, Worcester, MA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Caroline","family":"Genco","sequence":"additional","affiliation":[{"name":"Department of Immunology, Tufts University School of Medicine, Boston MA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5577-7357","authenticated-orcid":false,"given":"Kevin J.","family":"Waldron","sequence":"additional","affiliation":[{"name":"Institute of Biochemistry & Biophysics, Polish Academy of Science, Warsaw 02-106, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3106-0711","authenticated-orcid":false,"given":"Paola","family":"Massari","sequence":"additional","affiliation":[{"name":"Department of Immunology, Tufts University School of Medicine, Boston MA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026041120511077000_2025.05.01.651437v1.1","unstructured":"CDC. 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