TOR–Calcineurin-mediated regulation of sphingolipid metabolism governs amphotericin susceptibility in Candida glabrata

TOR–Calcineurin-mediated regulation of sphingolipid metabolism governs amphotericin susceptibility in Candida glabrata | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article TOR–Calcineurin-mediated regulation of sphingolipid metabolism governs amphotericin susceptibility in Candida glabrata Mohit Kumar, Sonam Kumari, Mohd. Wasi, Basharat Ali, Praveen Kumar, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7939343/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Understanding the molecular determinants of antifungal drug susceptibility is essential for addressing rising resistance in fungal pathogens. Here, we uncover a central role for sphingolipid (SL) metabolism in modulating susceptibility to amphotericin B (AmB) in Candida glabrata . Through targeted lipidomics, we show that C. glabrata lacks the glucosylceramide biosynthetic branch and is enriched in long-chain phytoceramides. Deleting the inositol phosphosphingolipid phospholipase C gene ( Cgisc1 ) resulted in elevated AmB sensitivity, independent of ergosterol biosynthesis, and was associated with altered SL profiles and disrupted ceramide chain-length ratios. We further demonstrate that this phenotype is regulated by the TOR and calcineurin signalling pathways. Pharmacological inhibition and genetic interaction studies reveal that TOR-dependent regulators (ORM2) and calcineurin effectors (CK2 subunits CKB1/2) modulate SL biosynthesis and restore AmB tolerance when perturbed in a Cgisc1Δ background. These findings define a signaling lipid regulator that governs membrane composition and AmB sensitivity, providing a framework for targeting sphingolipid pathways to potentiate antifungal therapies. Mycology Candida glabrata CgIsc1 Sphingolipids TOR pathway Calcineurin Amphotericin B Full Text Additional Declarations The authors declare no competing interests. Supplementary Files SupplementaryData.xlsx Supplementary file details: Supplementary Data 1: Sphingolipidomics analysis of wild-type (WT) and Cgisc1Δ strains. Data are presented as molar percentage (Mol%) of total sphingolipid species. Supplementary Data 2: Sphingolipids of WT and Cgisc1󠄉Δ in different spotting conditions used in reversal experiments Supplementary Data 3: Comparative phospholipid composition of WT and Cgisc1Δ strains. Supplementary Data 4: Sphingolipids comparison between Cgisc1󠄉Δ and Cgisc1󠄉Δ/Cgorm2Δ deletions. Supplementary Data 5: Sphingolipids comparison between Cgisc1󠄉Δ and Cgisc1󠄉Δ/Cgckb1Δ deletions. Supplementary Data 6: Sphingolipids comparison between Cgisc1󠄉Δ and Cgisc1󠄉Δ/Cgckb2Δ deletions. SupplementaryFigandTable.docx Supplementary Fig 1: SL distribution and molecular species composition in C. glabrata . Supplementary Fig 2: Comparison of sterol changes in the Cgisc1 Δ mutant compared to WT Supplementary Fig 3: Raw Western blot Images Supplementary Table 1: Doubling time and relative fitness of SLs deletion strains using growth curve analysis Supplementary Table 2: Strains used in the study Supplementary Table 3: Plasmids Used in the study Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7939343","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":534429472,"identity":"ab4f63f7-2317-43b6-a3a8-d2416b6e3d7f","order_by":0,"name":"Mohit Kumar","email":"","orcid":"","institution":"Amity Institute of Biotechnology, Amity University Gurgaon, Haryana, India","correspondingAuthor":false,"prefix":"","firstName":"Mohit","middleName":"","lastName":"Kumar","suffix":""},{"id":534429473,"identity":"06702739-2cc7-4784-b9b6-e4f9c5562429","order_by":1,"name":"Sonam Kumari","email":"","orcid":"","institution":"Indian Council of Medical Research, New Delhi, India","correspondingAuthor":false,"prefix":"","firstName":"Sonam","middleName":"","lastName":"Kumari","suffix":""},{"id":534429474,"identity":"2e580290-6f2d-443a-8072-956a9c975913","order_by":2,"name":"Mohd. 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Data are presented as molar percentage (Mol%) of total sphingolipid species.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Data 2\u003c/strong\u003e: Sphingolipids of WT and Cgisc1󠄉Δ in different spotting conditions used in \u003cstrong\u003ereversal experiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Data 3\u003c/strong\u003e: Comparative phospholipid composition of WT and Cgisc1Δ strains.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Data 4:\u003c/strong\u003e Sphingolipids comparison between Cgisc1󠄉Δ and Cgisc1󠄉Δ/Cgorm2Δ deletions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Data 5\u003c/strong\u003e: Sphingolipids comparison between Cgisc1󠄉Δ and Cgisc1󠄉Δ/Cgckb1Δ deletions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Data 6:\u003c/strong\u003e Sphingolipids comparison between Cgisc1󠄉Δ and Cgisc1󠄉Δ/Cgckb2Δ deletions.\u003c/p\u003e","description":"","filename":"SupplementaryData.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7939343/v1/3c0074664e4d9c4780576e13.xlsx"},{"id":94451209,"identity":"1af0e7ec-09a3-4794-aae0-c29ec37eeaff","added_by":"auto","created_at":"2025-10-27 14:39:57","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":69514734,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Fig 1: \u003c/strong\u003eSL distribution and molecular species composition in \u003cem\u003eC. glabrata\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Fig 2: \u003c/strong\u003eComparison of sterol changes in the \u003cem\u003eCgisc1\u003c/em\u003eΔ\u003cem\u003e \u003c/em\u003emutant \u003cem\u003ecompared\u003c/em\u003e to WT\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Fig 3:\u003c/strong\u003e Raw Western blot Images\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Table 1:\u003c/strong\u003e Doubling time and relative fitness of SLs deletion strains using growth curve analysis\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Table 2: Strains used in the study\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Table 3: Plasmids Used in the study\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"SupplementaryFigandTable.docx","url":"https://assets-eu.researchsquare.com/files/rs-7939343/v1/c33958f4daadc4d5e90a968b.docx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eTOR–Calcineurin-mediated regulation of sphingolipid metabolism\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e governs\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e amphotericin \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003esusceptibility in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCandida glabrata\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Amity University","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Candida glabrata, CgIsc1, Sphingolipids, TOR pathway, Calcineurin, Amphotericin B","lastPublishedDoi":"10.21203/rs.3.rs-7939343/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7939343/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eUnderstanding the molecular determinants of antifungal drug susceptibility is essential for addressing rising resistance in fungal pathogens. Here, we uncover a central role for sphingolipid (SL) metabolism in modulating susceptibility to amphotericin B (AmB) in \u003cem\u003eCandida glabrata\u003c/em\u003e. Through targeted lipidomics, we show that \u003cem\u003eC. glabrata\u003c/em\u003e lacks the glucosylceramide biosynthetic branch and is enriched in long-chain phytoceramides. Deleting the inositol phosphosphingolipid phospholipase C gene (\u003cem\u003eCgisc1\u003c/em\u003e) resulted in elevated AmB sensitivity, independent of ergosterol biosynthesis, and was associated with altered SL profiles and disrupted ceramide chain-length ratios. We further demonstrate that this phenotype is regulated by the TOR and calcineurin signalling pathways. Pharmacological inhibition and genetic interaction studies reveal that TOR-dependent regulators (ORM2) and calcineurin effectors (CK2 subunits CKB1/2) modulate SL biosynthesis and restore AmB tolerance when perturbed in a \u003cem\u003eCgisc1Δ\u003c/em\u003e background. These findings define a signaling lipid regulator that governs membrane composition and AmB sensitivity, providing a framework for targeting sphingolipid pathways to potentiate antifungal therapies.\u003c/p\u003e","manuscriptTitle":"TOR–Calcineurin-mediated regulation of sphingolipid metabolism governs amphotericin susceptibility in Candida glabrata","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-27 11:32:01","doi":"10.21203/rs.3.rs-7939343/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c73522b8-7d10-438f-be2e-df81dc1c4df5","owner":[],"postedDate":"October 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":56824921,"name":"Mycology"}],"tags":[],"updatedAt":"2025-10-27T11:32:01+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-27 11:32:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7939343","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7939343","identity":"rs-7939343","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}