Multidrug Transporters and Alterations in Sterol Biosynthesis Contribute to Azole Antifungal Resistance in Candida parapsilosis

Abstract

While much is known concerning azole resistance in Candida albicans, considerably less is understood about Candida parapsilosis, an emerging species of Candida with clinical relevance. We conducted a comprehensive analysis of azole resistance in a collection of resistant C. parapsilosis clinical isolates in order to determine which genes might play a role in this process within this species. We examined the relative expression of the putative drug transporter genes CDR1 and MDR1 and that of ERG11. In isolates overexpressing these genes, we sequenced the genes encoding their presumed transcriptional regulators, TAC1, MRR1, and UPC2, respectively. We also sequenced the sterol biosynthesis genes ERG3 and ERG11 in these isolates to find mutations that might contribute to this phenotype in this Candida species. Our findings demonstrate that the putative drug transporters Cdr1 and Mdr1 contribute directly to azole resistance and suggest that their overexpression is due to activating mutations in the genes encoding their transcriptional regulators. We also observed that the Y132F substitution in ERG11 is the only substitution occurring exclusively among azole-resistant isolates, and we correlated this with specific changes in sterol biosynthesis. Finally, sterol analysis of these isolates suggests that other changes in sterol biosynthesis may contribute to azole resistance in C. parapsilosis.

FIG 1

Relative fold change in expression of MDR1, CDR1, and ERG11 obtained from qRT-PCR for each resistant isolate of the collection. All experiments include biological and technical replicates in triplicate. Fold expression of the genes was compared to the average of the expression levels of the 4 susceptible isolates within the collection. The dotted line indicates a 2-fold relative change in gene expression. Error bars show standard errors. The isolates are listed in order of increasing MIC to fluconazole.

FIG 2

Fluconazole MICs for isolates in which either Mdr1 (A) or Cdr1(B) have been deleted. Susceptibility testing was performed according to CLSI guidelines, and the 48-h MICs are reported here in micrograms per milliliter. The bars are grouped by a specific isolate, listing the wild-type isolate, the deletion mutant, and the complemented derivative isolate, respectively.

FIG 3

Schematic representation of the ergosterol biosynthesis pathway for Candida species. The solid arrows indicate a single enzymatic step, while the dashed arrows represent multiple enzymatic steps.