{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,6,24]],"date-time":"2026-06-24T05:48:25Z","timestamp":1782280105380,"version":"3.54.5"},"posted":{"date-parts":[[2025,5,6]]},"group-title":"Microbiology","reference-count":73,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,5,6]],"date-time":"2025-05-06T00:00:00Z","timestamp":1746489600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,5,5]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>\n                  Infections caused by the emerging pathogenic yeast\n                  <jats:italic>Clavispora (Candida) lusitaniae<\/jats:italic>\n                  can be difficult to manage due to multi-drug resistance. Resistance to the frontline antifungal fluconazole (FLZ) in\n                  <jats:italic>Candida<\/jats:italic>\n                  spp. is commonly acquired through gain-of-function (GOF) mutations in the gene encoding the transcription factor Mrr1. These activated Mrr1 variants enhance FLZ efflux via upregulation of the multi-drug transporter gene\n                  <jats:italic>MDR1<\/jats:italic>\n                  . Recently, it was reported that, unlike in the well-studied\n                  <jats:italic>Candida albicans<\/jats:italic>\n                  species,\n                  <jats:italic>C. lusitaniae<\/jats:italic>\n                  and\n                  <jats:italic>Candida parapsilosis<\/jats:italic>\n                  with activated Mrr1 also have high expression of\n                  <jats:italic>CDR1<\/jats:italic>\n                  , which encodes another multi-drug transporter with overlapping but distinct transported substrate profiles and Cdr1-dependent FLZ resistance. To better understand the mechanisms of Mrr1 regulation of\n                  <jats:italic>MDR1<\/jats:italic>\n                  and\n                  <jats:italic>CDR1<\/jats:italic>\n                  , and other co-regulated genes, we performed CUT&amp;RUN analysis of Mrr1 binding sites. Mrr1 bound the promoter regions of\n                  <jats:italic>MDR1<\/jats:italic>\n                  and\n                  <jats:italic>CDR1<\/jats:italic>\n                  as well as\n                  <jats:italic>FLU1<\/jats:italic>\n                  , which encodes another transporter capable of FLZ efflux. Mdr1 and Cdr1 independently contributed to the decreased susceptibility of the\n                  <jats:italic>\n                    MRR1\n                    <jats:sup>GOF<\/jats:sup>\n                  <\/jats:italic>\n                  strains against diverse clinical azoles and other antifungals, including 5-flucytosine. A consensus motif, CGGAGWTAR, enriched in Mrr1-bound\n                  <jats:italic>C. lusitaniae<\/jats:italic>\n                  DNA was also conserved upstream of\n                  <jats:italic>MDR1<\/jats:italic>\n                  and\n                  <jats:italic>CDR1<\/jats:italic>\n                  across species including\n                  <jats:italic>C. albicans<\/jats:italic>\n                  . CUT&amp;RUN and RNA-seq data were used to define the Mrr1 regulon which includes genes involved in transport, stress responses, and metabolism. Activated and inducible Mrr1 bound similar regions in the promoters of Mrr1 regulon genes. Our studies provide new evolutionary insights into the coordinated regulation of multi-drug transporters and potential mechanism(s) that aid secondary resistance acquisition in emerging\n                  <jats:italic>Candida<\/jats:italic>\n                  .\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>SIGNIFICANCE<\/jats:title>\n                  <jats:p>\n                    Understanding antifungal resistance in emerging\n                    <jats:italic>Candida<\/jats:italic>\n                    pathogens is essential to manage treatment failures and guide the development of new therapeutic strategies. Like other\n                    <jats:italic>Candida<\/jats:italic>\n                    species, the environmental opportunistic fungal pathogen\n                    <jats:italic>Clavispora<\/jats:italic>\n                    (\n                    <jats:italic>Candida<\/jats:italic>\n                    )\n                    <jats:italic>lusitaniae<\/jats:italic>\n                    can acquire resistance to the antifungal fluconazole by overexpression of the multi-drug efflux pump Mdr1 through gain-of-function mutations in the gene encoding the transcription factor Mrr1. Here, we show that\n                    <jats:italic>C. lusitaniae<\/jats:italic>\n                    Mrr1 also directly regulates\n                    <jats:italic>CDR1,<\/jats:italic>\n                    another major multi-drug transporter gene, along with\n                    <jats:italic>MDR1.<\/jats:italic>\n                    In strains with activated Mrr1, upregulation of\n                    <jats:italic>MDR1<\/jats:italic>\n                    and\n                    <jats:italic>CDR1<\/jats:italic>\n                    protects against diverse antifungals potentially aiding the rise of other resistance mutations. Mrr1 also regulates several stress response and metabolism genes thereby providing new perspectives into the physiology of drug-resistant strains. The identification of an Mrr1 binding motif that is conserved across strains and species will advance future efforts to understand multi-drug resistance across\n                    <jats:italic>Candida<\/jats:italic>\n                    species.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.1101\/2025.05.04.652153","type":"posted-content","created":{"date-parts":[[2025,5,6]],"date-time":"2025-05-06T17:35:12Z","timestamp":1746552912000},"source":"Crossref","is-referenced-by-count":0,"title":["Coordinated regulation of Mdr1- and Cdr1-mediated protection from antifungals by the Mrr1 transcription factor in emerging\n                  <i>Candida<\/i>\n                  spp"],"prefix":"10.64898","author":[{"given":"Dhanabala-Subhiksha","family":"Rajesh-Khanna","sequence":"first","affiliation":[{"name":"Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carolina G.","family":"Pi\u00f1a P\u00e1ez","sequence":"additional","affiliation":[{"name":"Department of Microbiology & Plant Pathology and Institute for Integrative Genome Biology, University of California\u2014Riverside, Riverside, California, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elora G.","family":"Dolan","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kiran S.","family":"Mirpuri","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7591-0020","authenticated-orcid":false,"given":"Jason E.","family":"Staijch","sequence":"additional","affiliation":[{"name":"Department of Microbiology & Plant Pathology and Institute for Integrative Genome Biology, University of California\u2014Riverside, Riverside, California, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6366-2971","authenticated-orcid":false,"given":"Deborah A.","family":"Hogan","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026041120430784000_2025.05.04.652153v1.1","doi-asserted-by":"publisher","DOI":"10.1016\/S1473-3099(23)00692-8"},{"key":"2026041120430784000_2025.05.04.652153v1.2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1003550"},{"key":"2026041120430784000_2025.05.04.652153v1.3","doi-asserted-by":"crossref","unstructured":"Casagrande Pierantoni D , Giuliano S , Conti A , Corte L , Angelini J , Cardinali G , Tascini C. 2024. 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