ABSTRACT
Analysis of the transport functions of individualCandida albicans plasma membrane drug efflux pumps is hampered by the multitude of endogenous transporters. We have stably expressed C. albicans Cdr1p, the major pump implicated in multiple-drug-resistance phenotypes, from the genomicPDR5 locus in a Saccharomyces cerevisiae mutant (AD1-8u−) from which seven major transporters of the ATP-binding cassette (ABC) family have been deleted. High-level expression of Cdr1p, under the control of the S. cerevisiae PDR5 promoter and driven by S. cerevisiae Pdr1p transcriptional regulator mutation pdr1-3, was demonstrated by increased levels of mRNA transcription, increased levels of nucleoside triphosphatase activity, and immunodetection in plasma membrane fractions. S. cerevisiae AD1-8u− was hypersensitive to azole antifungals (the MICs at which 80% of cells were inhibited [MIC80s] were 0.625 μg/ml for fluconazole, <0.016 μg/ml for ketoconazole, and <0.016 μg/ml for itraconazole), whereas the strain (AD1002) that overexpressed C. albicans Cdr1p was resistant to azoles (MIC80s of fluconazole, ketoconazole, and itraconazole, 30, 0.5, and 4 μg/ml, respectively). Drug resistance correlated with energy-dependent drug efflux. AD1002 demonstrated resistance to a variety of structurally unrelated chemicals which are potential drug pump substrates. The controlled overexpression of C. albicansCdr1p in an S. cerevisiae background deficient in other pumps allows the functional analysis of pumping specificity and mechanisms of a major ABC transporter involved in drug efflux from an important human pathogen.
Efflux‐Pump–Derived Multiple Drug Resistance to Ethambutol Monotherapy inMycobacterium tuberculosisand the Pharmacokinetics and Pharmacodynamics of Ethambutol