Activation of the multiple drug resistance gene MDR1 in fluconazole- resistant, clinical Candida albicans strains is caused by mutations in a trans-regulatory factor

Stephanie Wirsching, Sonja Michel, Gerwald Köhler, Joachim Morschhäuser

Research output: Contribution to journalArticle

69 Citations (Scopus)

Abstract

Resistance of Candida albicans against the widely used antifungal agent fluconazole is often due to active drug efflux from the cells. In many fluconazole-resistant C. albicans isolates the reduced intracellular drug accumulation correlates with constitutive strong expression of the MDR1 gene, encoding a membrane transport protein of the major facilitator superfamily that is not detectably expressed in vitro in fluconazole-susceptible isolates. To elucidate the molecular changes responsible for MDR1 activation, two pairs of matched fluconazole-susceptible and resistant isolates in which drug resistance coincided with stable MDR1 activation were analyzed. Sequence analysis of the MDR1 regulatory region did not reveal any promoter mutations in the resistant isolates that might account for the altered expression of the gene. To test for a possible involvement of trans-regulatory factors, a GFP reporter gene was placed under the control of the MDR1 promoter from the fluconazole-susceptible C. albicans strain CAI4, which does not express the MDR1 gene in vitro. This MDR1P-GFP fusion was integrated into the genome of the clinical C. albicans isolates with the help of the dominant selection marker MPA(R) developed for the transformation of C. albicans wild-type strains. Integration was targeted to an ectopic locus such that no recombination between the heterologous and resident MDR1 promoters occurred. The transformants of the two resistant isolates exhibited a fluorescent phenotype, whereas transformants of the corresponding susceptible isolates did not express the GFP gene. These results demonstrate that the MDR1 promoter was activated by a trans-regulatory factor that was mutated in fluconazole-resistant isolates, resulting in deregulated, constitutive MDR1 expression.

Original languageEnglish
Pages (from-to)400-404
Number of pages5
JournalJournal of Bacteriology
Volume182
Issue number2
DOIs
StatePublished - 1 Jan 2000

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Fluconazole
Multiple Drug Resistance
Candida albicans
Mutation
Genes
Gene Expression
Membrane Transport Proteins
Antifungal Agents
Nucleic Acid Regulatory Sequences
Reporter Genes
Drug Resistance
Pharmaceutical Preparations
Genetic Recombination
Sequence Analysis
Genome
Phenotype

Cite this

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title = "Activation of the multiple drug resistance gene MDR1 in fluconazole- resistant, clinical Candida albicans strains is caused by mutations in a trans-regulatory factor",
abstract = "Resistance of Candida albicans against the widely used antifungal agent fluconazole is often due to active drug efflux from the cells. In many fluconazole-resistant C. albicans isolates the reduced intracellular drug accumulation correlates with constitutive strong expression of the MDR1 gene, encoding a membrane transport protein of the major facilitator superfamily that is not detectably expressed in vitro in fluconazole-susceptible isolates. To elucidate the molecular changes responsible for MDR1 activation, two pairs of matched fluconazole-susceptible and resistant isolates in which drug resistance coincided with stable MDR1 activation were analyzed. Sequence analysis of the MDR1 regulatory region did not reveal any promoter mutations in the resistant isolates that might account for the altered expression of the gene. To test for a possible involvement of trans-regulatory factors, a GFP reporter gene was placed under the control of the MDR1 promoter from the fluconazole-susceptible C. albicans strain CAI4, which does not express the MDR1 gene in vitro. This MDR1P-GFP fusion was integrated into the genome of the clinical C. albicans isolates with the help of the dominant selection marker MPA(R) developed for the transformation of C. albicans wild-type strains. Integration was targeted to an ectopic locus such that no recombination between the heterologous and resident MDR1 promoters occurred. The transformants of the two resistant isolates exhibited a fluorescent phenotype, whereas transformants of the corresponding susceptible isolates did not express the GFP gene. These results demonstrate that the MDR1 promoter was activated by a trans-regulatory factor that was mutated in fluconazole-resistant isolates, resulting in deregulated, constitutive MDR1 expression.",
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Activation of the multiple drug resistance gene MDR1 in fluconazole- resistant, clinical Candida albicans strains is caused by mutations in a trans-regulatory factor. / Wirsching, Stephanie; Michel, Sonja; Köhler, Gerwald; Morschhäuser, Joachim.

In: Journal of Bacteriology, Vol. 182, No. 2, 01.01.2000, p. 400-404.

Research output: Contribution to journalArticle

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T1 - Activation of the multiple drug resistance gene MDR1 in fluconazole- resistant, clinical Candida albicans strains is caused by mutations in a trans-regulatory factor

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