{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,6,24]],"date-time":"2026-06-24T05:54:11Z","timestamp":1782280451243,"version":"3.54.5"},"posted":{"date-parts":[[2025,5,13]]},"group-title":"Cell Biology","reference-count":53,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,5,13]],"date-time":"2025-05-13T00:00:00Z","timestamp":1747094400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000968","name":"American Heart Association","doi-asserted-by":"crossref","award":["16SDG30200001"],"award-info":[{"award-number":["16SDG30200001"]}],"id":[{"id":"10.13039\/100000968","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000968","name":"American Heart Association","doi-asserted-by":"crossref","award":["23IPA1053083"],"award-info":[{"award-number":["23IPA1053083"]}],"id":[{"id":"10.13039\/100000968","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000060","name":"National Institute of Allergy and Infectious Diseases","doi-asserted-by":"crossref","award":["1R21AI156225"],"award-info":[{"award-number":["1R21AI156225"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100010269","name":"Wellcome Trust","doi-asserted-by":"crossref","award":["214229_Z_18_Z"],"award-info":[{"award-number":["214229_Z_18_Z"]}],"id":[{"id":"10.13039\/100010269","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100007131","name":"Texas Tech University","doi-asserted-by":"crossref","award":["N\/A"],"award-info":[{"award-number":["N\/A"]}],"id":[{"id":"10.13039\/100007131","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,11,11]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  The common fungal pathogen,\n                  <jats:italic>Candida albicans<\/jats:italic>\n                  , relies on the pore-forming toxin candidalysin to damage host cells. Cells counteract pore-forming toxins by Ca\n                  <jats:sup>2+<\/jats:sup>\n                  -dependent mechanisms, such as microvesicle shedding and annexin recruitment to resist cholesterol-dependent cytolysins like streptolysin O (SLO), or annexin involvement and patch repair in the case of aerolysin. However, the specific Ca\n                  <jats:sup>2+<\/jats:sup>\n                  -dependent repair pathways engaged in response to candidalysin remain poorly understood. Here, we determined the involvement of different Ca\n                  <jats:sup>2+<\/jats:sup>\n                  -dependent repair mechanisms to candidalysin and compared responses to SLO and aerolysin using flow cytometry and high-resolution microscopy. We report that candidalysin triggered Ca\n                  <jats:sup>2+<\/jats:sup>\n                  -dependent repair, but patch repair and ceramide failed to provide significant protection. MEK-dependent repair and annexins A1, A2 and A6 contributed partially to repairing damage caused by candidalysin. However, annexin translocation after candidalysin challenge was delayed compared to SLO or aerolysin challenge. Surprisingly, extracellular Cl\n                  <jats:sup>\u2212<\/jats:sup>\n                  improved cell survival after candidalysin challenge, but not after challenge with SLO or aerolysin. Finally, we found that candidalysin is removed via extracellular vesicle shedding. These findings reveal that Ca\n                  <jats:sup>2+<\/jats:sup>\n                  -dependent microvesicle shedding protects cells from candidalysin and can be engaged by multiple molecular mechanisms during membrane repair.\n                  <jats:fig id=\"figa1\" position=\"float\" orientation=\"portrait\" fig-type=\"figure\">\n                    <jats:label>Graphical Abstract.<\/jats:label>\n                    <jats:caption>\n                      <jats:title>Candidalysin is resisted by distinct repair mechanisms compared to bacterial PFTs.<\/jats:title>\n                      <jats:p>\n                        After pore formation and membrane damage by each toxin, multiple repair pathways are triggered downstream of Ca\u00b2\u207a flux. Candidalysin induces a protective Cl\n                        <jats:sup>\u2212<\/jats:sup>\n                        influx and activates MEK-dependent repair, which contributes to cell protection. Annexin translocation occurs slowly and provides minor protection, while patch repair is ineffective. In contrast, aerolysin does not benefit from Cl\n                        <jats:sup>\u2212<\/jats:sup>\n                        influx or MEK protection. Aerolysin triggers moderate annexin translocation and relies primarily on patch repair as the main protective mechanism. Streptolysin O elicits rapid annexin translocation and activates MEK signaling, both of which contribute to robust protection. Patch repair plays only a minor protective role against SLO. The figure was created using BioRender.\n                      <\/jats:p>\n                    <\/jats:caption>\n                    <jats:graphic xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" xlink:href=\"653080v2_figa1\" position=\"float\" orientation=\"portrait\"\/>\n                  <\/jats:fig>\n                <\/jats:p>","DOI":"10.1101\/2025.05.09.653080","type":"posted-content","created":{"date-parts":[[2025,5,13]],"date-time":"2025-05-13T08:05:22Z","timestamp":1747123522000},"source":"Crossref","is-referenced-by-count":0,"title":["The fungal peptide toxin candidalysin induces distinct membrane repair mechanisms compared to bacterial pore-forming toxins"],"prefix":"10.64898","author":[{"given":"Roshan","family":"Thapa","sequence":"first","affiliation":[{"name":"Department of Biological Sciences, Texas Tech University, Lubbock, TX 79409"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Victor","family":"Kayejo","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences, Texas Tech University, Lubbock, TX 79409"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Claire M.","family":"Lyon","sequence":"additional","affiliation":[{"name":"Centre for Host-Microbiome Interactions, Faculty of Dental, Oral & Craniofacial Sciences, King\u2019s College London, London, SE1 1UL, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bernhard","family":"Hube","sequence":"additional","affiliation":[{"name":"Department of Microbial Pathogenicity Mechanisms, Leibniz Institute for Natural Product Research and Infection Biology-Hans Knoell Institute (HKI) Jena, Germany"},{"name":"Institute of Microbiology, Friedrich Schiller University, Jena, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Julian R.","family":"Naglik","sequence":"additional","affiliation":[{"name":"Centre for Host-Microbiome Interactions, Faculty of Dental, Oral & Craniofacial Sciences, King\u2019s College London, London, SE1 1UL, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8637-8538","authenticated-orcid":false,"given":"Peter A.","family":"Keyel","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences, Texas Tech University, Lubbock, TX 79409"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026041121462345000_2025.05.09.653080v2.1","doi-asserted-by":"crossref","first-page":"e0002123","DOI":"10.1128\/mmbr.00021-23","article-title":"Candida albicans and Candida glabrata: global priority pathogens","volume":"88","year":"2024","journal-title":"Microbiology and molecular biology reviews : MMBR"},{"key":"2026041121462345000_2025.05.09.653080v2.2","doi-asserted-by":"crossref","unstructured":"Schille, T.B. , Sprague, J.L. , Naglik, J.R. , Brunke, S. & Hube, B . Commensalism and pathogenesis of Candida albicans at the mucosal interface. Nat Rev Microbiol (2025).","DOI":"10.1038\/s41579-025-01174-x"},{"key":"2026041121462345000_2025.05.09.653080v2.3","doi-asserted-by":"publisher","DOI":"10.1038\/nature17625"},{"key":"2026041121462345000_2025.05.09.653080v2.4","doi-asserted-by":"publisher","DOI":"10.1128\/mbio.03510-21"},{"key":"2026041121462345000_2025.05.09.653080v2.5","doi-asserted-by":"crossref","first-page":"669","DOI":"10.1038\/s41564-024-01606-z","article-title":"Secretion of the fungal toxin candidalysin is dependent on conserved precursor peptide sequences","volume":"9","year":"2024","journal-title":"Nature microbiology"},{"key":"2026041121462345000_2025.05.09.653080v2.6","doi-asserted-by":"crossref","unstructured":"Russell, C.M. et al. The Candida albicans virulence factor candidalysin polymerizes in solution to form membrane pores and damage epithelial cells. eLife 11 (2022).","DOI":"10.7554\/eLife.75490"},{"key":"2026041121462345000_2025.05.09.653080v2.7","doi-asserted-by":"publisher","DOI":"10.1016\/j.immuni.2021.08.009"},{"key":"2026041121462345000_2025.05.09.653080v2.8","doi-asserted-by":"crossref","first-page":"113240","DOI":"10.1016\/j.celrep.2023.113240","article-title":"Toll-like receptor 4 and CD11b expressed on microglia coordinate eradication of Candida albicans cerebral mycosis","volume":"42","year":"2023","journal-title":"Cell reports"},{"key":"2026041121462345000_2025.05.09.653080v2.9","doi-asserted-by":"crossref","first-page":"2553","DOI":"10.1038\/s41564-024-01794-8","article-title":"Sulfated glycosaminoglycans are host epithelial cell targets of the Candida albicans toxin candidalysin","volume":"9","year":"2024","journal-title":"Nature microbiology"},{"key":"2026041121462345000_2025.05.09.653080v2.10","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-024-46141-x"},{"key":"2026041121462345000_2025.05.09.653080v2.11","doi-asserted-by":"crossref","unstructured":"Thapa, R. , Ray, S. & Keyel, P.A . Interaction of Macrophages and Cholesterol-Dependent Cytolysins: The Impact on Immune Response and Cellular Survival. 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Chloride ions in health and disease. 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Visualization of bacterial toxin induced responses using live cell fluorescence microscopy. 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