Multiple amino acid substitutions in lanosterol 14α-demethylase contribute to azole resistance in Candida albicans

Abstract

Lanosterol 14α-demethylase (14DM) is the target of the azole antifungals, and alteration of the 14DM sequence leading to a decreased affinity of the enzyme for azoles is one of several potential mechanisms for resistance to these drugs in Candida albicans. In order to identify such alterations the authors investigated a collection of 19 C. albicans clinical isolates demonstrating either frank resistance (MICs Z32 µg ml N1 ) or dose-dependent resistance (MICs 8-16 µg ml N1 ) to fluconazole. In cell-free extracts from four isolates, including the Darlington strain ATCC 64124, sensitivity of sterol biosynthesis to inhibition by fluconazole was greatly reduced, suggesting that alterations in the activity or affinity of the 14DM could contribute to resistance. Cloning and sequencing of the 14DM gene from these isolates revealed 12 different alterations (two to four per isolate) leading to changes in the deduced amino acid sequence. Five of these mutations have not previously been reported. To demonstrate that these alterations could affect fungal susceptibility to azoles, the 14DM genes from one sensitive and three resistant C. albicans strains were tagged at the carboxyl terminus with a c-myc epitope and expressed in Saccharomyces cerevisiae under control of the endogenous promoter. Transformants receiving 14DM genes from resistant strains had fluconazole MICs up to 32-fold higher than those of transformants receiving 14DM from a sensitive strain, although Western blot analysis indicated that the level of expressed 14DM was similar in all transformants. Amino acid substitutions in the 14DM gene from the Darlington strain also conferred a strong cross-resistance to ketoconazole. In conclusion, multiple genetic alterations in C. albicans 14DM, including several not previously reported, can affect the affinity of the enzyme for azoles and contribute to resistance of clinical isolates.

Inactivation of Sterol Δ 5,6 -Desaturase Attenuates Virulence in Candida albicans

“…A laboratory-generated homozygous erg3 deletion mutant (DSY1751) along with its heterozygous progenitor (DSY1366) and an engineered revertant (ASC100) were similarly tested (C). isolates with multiple mutations might be expected to occur, it is curious that such isolates appear to be recovered both from patients (3,4,8,15,16,21) and from in vitro experiments (2) more frequently than isolates with homozygous nonsense mutations in ERG3 (6,13). One explanation for this apparent anomaly is that erg3 mutants are at a competitive disadvantage, possibly due to a significant reduction in either growth rate or virulence.…”

Reliability of the Vitek 2 Yeast Susceptibility Test for Detection of In Vitro Resistance to Fluconazole and Voriconazole in Clinical Isolates of Candida albicans and Candida glabrata

“…Among the 11 fluconazole-resistant C. albicans isolates, 7 isolates were found to upregulate CDR and/or MDR1 genes and to exhibit the Erg11p amino acid substitutions shown to cause azole resistance (24,25); for the remaining isolates, two showed CDR1 and CDR2 overexpression and two Erg11p mutations. Interestingly, only upregulated expression of multidrug transporters was noted in all of eight fluconazole-SDD C. albicans isolates (see Table S1 in the supplemental material), emphasizing the concept of the presence of Erg11p mutations, alone or in combination with other mechanisms, as a contributor to azole resistance in C. albicans (10,13).…”

Azole susceptibility and resistance in Candida dubliniensis

“…In C. albicans, mutations that affect the affinity of the target enzyme Erg11p for azole antifungal drugs have been well documented as a drug-resistance mechanism [41][42][43][44][45][46]. In C. dubliniensis, Perea et al [31] have described mutations in the CdERG11 gene which were associated with fluconazole resistance.…”