Transcriptional Regulation of MDR1 , Encoding a Drug Efflux Determinant, in Fluconazole-Resistant Candida albicans Strains through an Mcm1p Binding Site

Abstract

ABSTRACT Constitutive, high-level transcription of the gene encoding the drug efflux facilitator Mdr1p is commonly observed in laboratory and clinical strains of Candida albicans that are resistant to the antifungal drug fluconazole (FLC). In five independently isolated FLC R laboratory strains, introduction of a wild-type MDR1 promoter fragment fused to the yeast enhanced green fluorescent protein ( yEGFP ) reporter gene resulted in high-level expression of GFP, demonstrating that overexpression of MDR1 is dependent on a trans- acting factor. This study identified a 35-bp MDR1 promoter element, termed the MDRE, that mediates high-level MDR1 transcription. When inserted into a heterologous promoter, the MDRE was sufficient to mediate high-level expression of the yEGFP reporter gene specifically in MDR1 trans -activation strains. The MDRE promoted transcription in an orientation-independent and dosage-dependent manner. Deletion of the MDRE in the full-length promoter did not abolish MDR1 trans- activation, indicating that elements upstream of the MDRE also contribute to transcription of MDR1 in these overexpression strains. Analysis of the MDRE sequence indicated that it contains an Mcm1p binding site very similar in organization to the site seen upstream of the Saccharomyces cerevisiae MFA1 and STE2 genes. Electrophoretic mobility shift analysis demonstrated that both wild-type, FLC-sensitive and MDR1 trans -activated, FLC-resistant strains contain a factor that binds the MDRE. Depletion of Mcm1p, by use of a strain in which MCM1 expression is under the control of a regulated promoter (44), resulted in a loss of MDRE binding activity. Thus, the general transcription factor Mcm1p participates in the regulation of MDR1 expression.