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

Abstract

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 Cleavage Under Targets and Release Using Nuclease (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 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 Candida.

Importance: Understanding antifungal resistance in emerging Candida pathogens is essential to managing treatment failures and guiding the development of new therapeutic strategies. Like other Candida species, the environmental opportunistic fungal pathogen Clavispora (Candida) lusitaniae 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 C. lusitaniae Mrr1 also directly regulates CDR1, another major multi-drug transporter gene, along with MDR1. In strains with activated Mrr1, upregulation of MDR1 and CDR1 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 Candida species.