Gain-of-Function Mutations in the Transcription Factor MRR1 Are Responsible for Overexpression of the MDR1 Efflux Pump in Fluconazole-Resistant Candida dubliniensis Strains

CdMRR1 complements the defect in inducible MDR1 expression of a C. albicans mrr1Δ mutant. (A) Structure of the cassette that was used to express CdMRR1 or CaMRR1 (white arrow) from the ADH1 promoter (P_ADH1, bent arrow) after integration into the C. albicans genome with the help of the caSAT1 marker (gray arrow). T_ACT1, transcription termination sequence of the ACT1 gene (filled circle). (B) Fluorescence of C. albicans strains carrying a P_MDR1-GFP reporter fusion in an mrr1Δ background and expressing either CaMRR1 or CdMRR1 from the ADH1 promoter. Two independent transformants of parental strain CAG48MRR1M4B (mrr1Δ) were used in each case. Strain SC5314 (control), which does not contain the GFP gene, was included to control for background fluorescence. The strains were grown in the presence (+) or the absence (−) of benomyl, and the mean fluorescence of the cells was determined by flow cytometry, as detailed in Materials and Methods.