The azole-resistance phenotype of a Nakaseomyces glabratus clinical strain encoding a wild-type PDR1 allele involves the efflux pumps Aus1 and Pdh1 and Cyb5, an alternative reductase required for ergosterol biosynthesis

ABSTRACT Azole resistance among Candida yeasts is problematic as it increases therapeutic failure, resulting in prolonged hospital stays and worse clinical prognosis. Clinical strains of the N. glabratus species (formerly classified as Candida glabrata ) acquire resistance to azoles mainly by acquiring gain-of-function (GOF) mutations in the regulator Pdr1. However, a small percentage of resistant strains without such modifications have been described. This work focuses on one of such azole-resistant strains encoding a “wild-type” (non-GOF) PDR1 allele, ISTB218. We found that the azole-resistance phenotype in this strain involves: (i) the expression of the multi-drug resistance transporters Pdh1 and Aus1, whose deletion abolished the fluconazole and voriconazole resistance; (ii) an Aus1-mediated increase in plasma membrane fluidity, especially under fluconazole stress; and (iii) the ability to sustain higher pools of ergosterol and lower accumulation of methylated sterols under fluconazole stress. This last trait was linked to the activity of the CYB5 gene, an alternative reductase that is overexpressed in ISTB218 cells and that we found, for the first time, to be required for 14-demethylation of lanosterol and 22-desaturation of episterol. Thus, Cyb5 is added to the panoply of enzymes involved in ergosterol biosynthesis in N. glabratus . Altogether, our results advance knowledge on the biosynthesis of ergosterol and the mechanisms of resistance to azoles independent of Pdr1, thus opening the portfolio of possible therapeutic targets to be used in the advance of new anti- Candida therapies. IMPORTANCE The emergence of resistance to azoles among strains of Nakaseomyces glabratus is a major cause of concern, considering the importance of this yeast in causing human infections. In this work, we disclose a set of genes that mediate the azole-resistance phenotype of a resistant clinical strain, with one of these players (Cyb5) being also implicated in biosynthesis of ergosterol for the first time. With this, we increase a set of possible therapeutic targets that can be used in future studies to develop new antifungals.