Functional characterization of Candida auris DHA1 transporters conferring flucytosine resistance.

Candida (Candidozyma) auris has recently been designated a critical priority pathogen by the World Health Organization (WHO) in its Fungal Priority Pathogens List (FPPL). With resistance now widespread against frontline antifungals and limited availability of alternative therapeutics, combination regimens incorporating the nucleoside analog flucytosine (5-fluorocytosine; 5-FC) have gained renewed attention. While transporter-mediated resistance is well established for azole antifungals, knowledge on the molecular basis underlying reduced susceptibility to 5-FC in C. auris remains limited. Here, we aimed to functionally characterize two C. auris DHA1 transporters and to evaluate whether their inhibition may modulate 5-FC efficacy. Building on earlier reports showing elevated expression of two major facilitator superfamily (MFS) transporters belonging to the Drug/H+ antiporter-1 (DHA1) family upon 5-FC exposure, we now provide functional evidence supporting their contribution to 5-FC resistance. Heterologous expression of these transporters, B9J08_002663 (CauQdr2) and B9J08_004113 (CauMdr1.2) in a Saccharomyces cerevisiae system resulted in a marked increase in 5-FC resistance, with a > 2-fold shift in IC₅₀ values compared to the host strain. Moreover, analysis of ligand-bound structural models revealed a conserved interaction niche, consistent with structural and functional data from major facilitator superfamily (MFS) proteins, particularly DHA1 transporters. Furthermore, we also identified clorgyline as a selective inhibitor of CauMdr1.2, capable of modulating 5-FC susceptibility. Collectively, our findings uncover a DHA1 transporter-mediated mechanism of 5-FC resistance in C. auris, expanding the functional repertoire of such proteins and providing potential new targets for antifungal intervention. The findings further provide a rationale for designing 5-FC-based combination strategies incorporating transporter inhibitors to enhance drug efficacy and counteract efflux-mediated resistance.