Multiple amino acid substitutions in lanosterol 14alpha-demethylase contribute to azole resistance in Candida albicans
Details
Serval ID
serval:BIB_08EF0C03E2FA
Type
Article: article from journal or magazin.
Collection
Publications
Institution
Title
Multiple amino acid substitutions in lanosterol 14alpha-demethylase contribute to azole resistance in Candida albicans
Journal
Microbiology
ISSN
1350-0872 (Print)
Publication state
Published
Issued date
10/1999
Volume
145 ( Pt 10)
Pages
2715-25
Notes
Journal Article --- Old month value: Oct
Abstract
Lanosterol 14alpha-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 > or = 32 microg ml(-1)) or dose-dependent resistance (MICs 8-16 microg 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
Amino Acid Sequence
*Amino Acid Substitution
Antifungal Agents/metabolism/*pharmacology
Candida albicans/*drug effects/enzymology/*genetics/isolation &
purification
Cytochrome P-450 Enzyme System/chemistry/*genetics/metabolism
Drug Resistance, Microbial
Drug Resistance, Multiple
Fluconazole/metabolism/pharmacology
Ketoconazole/metabolism/pharmacology
Molecular Sequence Data
Mutation/genetics
Oxidoreductases/chemistry/*genetics/metabolism
Saccharomyces cerevisiae/genetics/metabolism
Sequence Alignment
Pubmed
Web of science
Create date
25/01/2008 16:32
Last modification date
20/08/2019 12:31