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Pathogenicity and Medical Microbiology |
-demethylase contribute to azole resistance in Candida albicans
Novartis Research Institute, Brunner Strasse 59, A-1235 Vienna, Austria1
Author for correspondence: Neil S. Ryder. Tel: +43 1 86 634 324. Fax: +43 1 86 634 354. e-mail: neil.ryder{at}pharma.novartis.com
Lanosterol 14
-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 
32 µg ml-1) or dose-dependent resistance (MICs 8–16 µg ml-1) 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.
Keywords: azoles, fluconazole resistance, Candida albicans, lanosterol 14-demethylase
Abbreviations: 14DM, lanosterol 14-demethylase
a Present address: Department of Dermatology, Hôpital Cantonal Universitaire de Genève, 24 Rue Micheli-du-Crest, CH-1211 Geneva 14, Switzerland
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