Coordinated regulation of Mdr1- and Cdr1-mediated protection from antifungals by the Mrr1 transcription factor in emerging Candida spp.

Infections caused by the emerging pathogenic yeast Clavispora (Candida) lusitaniae can be difficult to manage due to multi-drug resistance. Resistance to the frontline antifungal fluconazole (FLZ) in Candida 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 MDR1. Recently, it was reported that, unlike in the well-studied Candida albicans species, C. lusitaniae and Candida parapsilosis with activated Mrr1 also have high expression of CDR1, 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 MDR1 and CDR1, and other co-regulated genes, we performed CUT&RUN analysis of Mrr1 binding sites. Mrr1 bound the promoter regions of MDR1 and CDR1 as well as FLU1, which encodes another transporter capable of FLZ efflux. Mdr1 and Cdr1 independently contributed to the decreased susceptibility of the MRR1(GOF) strains against diverse clinical azoles and other antifungals, including 5-flucytosine. A consensus motif, CGGAGWTAR, enriched in Mrr1-bound C. lusitaniae DNA was also conserved upstream of MDR1 and CDR1 across species including C. albicans. CUT&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 Candida.