Neofunctionalization of a P-type ATPase enables fluconazole efflux in Candida auris

Candida auris is an emerging fungal pathogen known for its high resistance to fluconazole, the most commonly prescribed antifungal. However, the genetic regulators of fluconazole susceptibility remain insufficiently understood in C. auris. Using a pooled screen of piggyBac (PB) transposition mutants, we identify significant enrichment of mitochondrial genes whose inactivation causes reduced fluconazole susceptibility. Genome-wide genetic interaction analysis of a representative mitochondrial gene-deleted mutant, pet309?, implicates that mitochondrial dysfunction reduces fluconazole susceptibility by enhancing Cdr1 efflux activity post-transcriptionally and through the vacuolar calcium pump homolog Cdt1. We further demonstrate that Cdt1, beyond its canonical calcium-pumping function, mediates fluconazole efflux, evidenced by its fluconazole-induced, calcineurin-dependent plasma membrane localization and dependence on ATP hydrolysis. Additionally, Cdt1 accelerates the evolution of fluconazole resistance, and its transcript levels correlate with susceptibility across clinical isolates. Our findings define a novel role for Cdt1 in fluconazole efflux in C. auris. Overall design: mRNA profiles of pet309? and WT cells.