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Resistance to the frontline antifungal fluconazole (FLZ) in\n                      <jats:italic toggle=\"yes\">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 toggle=\"yes\">MDR1<\/jats:italic>\n                      . Recently, it was reported that, unlike in the well-studied\n                      <jats:italic toggle=\"yes\">Candida albicans<\/jats:italic>\n                      species,\n                      <jats:italic toggle=\"yes\">C. lusitaniae<\/jats:italic>\n                      and\n                      <jats:italic toggle=\"yes\">Candida parapsilosis<\/jats:italic>\n                      with activated Mrr1 also have high expression of\n                      <jats:italic toggle=\"yes\">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 toggle=\"yes\">MDR1<\/jats:italic>\n                      and\n                      <jats:italic toggle=\"yes\">CDR1<\/jats:italic>\n                      , and other co-regulated genes, we performed Cleavage Under Targets and Release Using Nuclease (CUT&amp;RUN) analysis of Mrr1 binding sites. Mrr1 bound the promoter regions of\n                      <jats:italic toggle=\"yes\">MDR1<\/jats:italic>\n                      and\n                      <jats:italic toggle=\"yes\">CDR1<\/jats:italic>\n                      , as well as\n                      <jats:italic toggle=\"yes\">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 toggle=\"yes\">\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 toggle=\"yes\">C. lusitaniae<\/jats:italic>\n                      DNA was also conserved upstream of\n                      <jats:italic toggle=\"yes\">MDR1<\/jats:italic>\n                      and\n                      <jats:italic toggle=\"yes\">CDR1<\/jats:italic>\n                      across species, including\n                      <jats:italic toggle=\"yes\">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 response, 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 toggle=\"yes\">Candida<\/jats:italic>\n                      .\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>\n                      Understanding antifungal resistance in emerging\n                      <jats:italic toggle=\"yes\">Candida<\/jats:italic>\n                      pathogens is essential to managing treatment failures and guiding the development of new therapeutic strategies. Like other\n                      <jats:italic toggle=\"yes\">Candida<\/jats:italic>\n                      species, the environmental opportunistic fungal pathogen\n                      <jats:italic toggle=\"yes\">Clavispora<\/jats:italic>\n                      (\n                      <jats:italic toggle=\"yes\">Candida<\/jats:italic>\n                      )\n                      <jats:italic toggle=\"yes\">lusitaniae<\/jats:italic>\n                      can acquire resistance to the antifungal fluconazole by overexpression of the multi-drug efflux pump Mdr1 through gain-of-function (GOF) mutations in the gene encoding the transcription factor Mrr1. Here, we show that\n                      <jats:italic toggle=\"yes\">C. lusitaniae<\/jats:italic>\n                      Mrr1 also directly regulates\n                      <jats:italic toggle=\"yes\">CDR1<\/jats:italic>\n                      , another major multi-drug transporter gene, along with\n                      <jats:italic toggle=\"yes\">MDR1<\/jats:italic>\n                      . In strains with activated Mrr1, upregulation of\n                      <jats:italic toggle=\"yes\">MDR1<\/jats:italic>\n                      and\n                      <jats:italic toggle=\"yes\">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 toggle=\"yes\">Candida<\/jats:italic>\n                      species.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/mbio.01323-25","type":"journal-article","created":{"date-parts":[[2025,10,7]],"date-time":"2025-10-07T13:02:54Z","timestamp":1759842174000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":4,"title":["Coordinated regulation of Mdr1- and Cdr1-mediated protection from antifungals by the Mrr1 transcription factor in emerging\n                    <i>Candida<\/i>\n                    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