Vacuolar sequestration of azoles: A novel strategy of azole antifungal resistance conserved across pathogenic and non-pathogenic yeast.

Target alteration and over production, drug efflux by overexpression of plasma membrane localized multidrug transporters comprise the conventional mechanisms of azole antifungal resistance. Here, we identify a novel and conserved mechanism of azole resistance not only in the budding yeast, Saccharomyces cerevisiae but also in the pathogenic yeast, Candida albicans We observe that the vacuolar membrane localized, MRP subfamily ABC transporter, of S. cerevisiae , Ybt1, can import azoles into the vacuole. Interestingly, the Ybt1 homologue in C. albicans , Mlt1p, could also complement this function. The underpinnings that the process is energy dependent is evident from the fact that the Mlt1p mutant version, made by converting a critical lysine residue in the "Walker A" motif of NBD1 (required for ATP hydrolysis) to alanine (K710A) wasn't able to transport azoles. Additionally, we have shown that like other eukaryotic MRP subfamily members, deletion of the conserved phenylalanine amino acid at 765 position (F765Δ) results in mislocalization of Mlt1 protein and this mislocalized protein was devoid of the azole resistant attribute. This suggests that the presence of this protein on vacuolar membrane is an important factor in azole resistance. Further, we report the importance of conserved residues, as converting two of the serines (positions 973 and 976) to alanine in the regulatory domain and CKI consensus sequence respectively severely affect the drug resistance. Hence, the present study reveals vacuolar sequestration of azoles by ABC transporter Ybt1 and its homologue Mlt1, as an alternate strategy to circumvent drug toxicity among pathogenic and non-pathogenic yeasts.