Azole resistance in Candida spp.

Abstract

The emergence of azole-resistant Candida spp. is a significant problem after long-term treatment of recurrent oropharyngeal candidiasis in HIV-infected patients. Several mechanisms can cause this resistance. An important mechanism of azole resistance is reduced intracellular accumulation of the drug. Among the multidrug efflux transporters, ABC transporters and the major facilitator superfamily are reported to cause the resistance. Erg11p, sterol C14α-demethylase, is a target of azole derivatives. It was reported that ERG11 overexpression had only a modest effect on the development of azole resistance. However, mutations in the ERG11 gene can cause the resistance, probably by reducing binding of azole to the target enzyme. We sequenced the ERG11 gene in a high-level azole resistant C. albicans strain, Darlington, and found that two amino acid substitutions, Y132H and I471T, had been encoded in the Darlington ERG11 gene. To assess the significance of these substitutions, we replaced one of the two copies of ERG11 gene in an azole-susceptible strain of C. albicans with a copy of the Darlington ERG11 and this resulted in a modest increase in azole resistance. Furthermore, to estimate the effect of Y132H and I471T individually, ERG11 genes with either or both mutations were expressed in S. cerevisiae. The I471T substitution, not previously described, conferred azole resistance when overexpressed alone and increased this resistance when added to the Y132H substitution. Alterations in the sterol biosynthetic pathway are another resistance mechanism. Inhibition of 14α-demethylase by azole results not only in ergosterol depletion but also in accumulation of methylated sterol 14 α-methylergosta-8, 22 (28)-dien-3 β, 6 α-diol. We deleted the ERG3 gene, which encodes a sterol 5, 6-desaturase, in C. albicans, and the deletion resulted in reduced susceptibility of the mutant to azoles. Sterol analysis revealed that erg3 mutant lost both ergosterol and diol when cultured with fluconazole.