title: Functional expression of Candida albicans drug efflux pump Cdr1p in a Saccharomyces cerevisiae strain deficient in membrane transporters.
abstract: Analysis of the transport functions of individual Candida 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 genomic PDR5 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 [MIC(80)s] were 0.625 microg/ml for fluconazole, <0.016 microg/ml for ketoconazole, and <0.016 microg/ml for itraconazole), whereas the strain (AD1002) that overexpressed C. albicans Cdr1p was resistant to azoles (MIC(80)s of fluconazole, ketoconazole, and itraconazole, 30, 0.5, and 4 microg/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. albicans Cdr1p 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.
authors.affiliation: Department of Oral Sciences and Orthodontics, University of Otago, Dunedin, New Zealand.
authors.forename: K
authors.initials: K
authors.lastname: Nakamura
authors.forename: M
authors.initials: M
authors.lastname: Niimi
authors.forename: K
authors.initials: K
authors.lastname: Niimi
authors.forename: A R
authors.initials: AR
authors.lastname: Holmes
authors.forename: J E
authors.initials: JE
authors.lastname: Yates
authors.forename: A
authors.initials: A
authors.lastname: Decottignies
authors.forename: B C
authors.initials: BC
authors.lastname: Monk
authors.forename: A
authors.initials: A
authors.lastname: Goffeau
authors.forename: R D
authors.initials: RD
authors.lastname: Cannon
mesh_terms: D017766:Acid Anhydride Hydrolases / Q000378:metabolism; D000935:Antifungal Agents / Q000378:metabolism; D015152:Blotting, Northern; D015139:Blotting, Southern; D002176:Candida albicans / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics*; D015825:Chromosomes, Fungal / Q000235:genetics; D003470:Culture Media; D004269:DNA, Bacterial / Q000302:isolation & purification; D004352:Drug Resistance, Microbial; D015725:Fluconazole / Q000378:metabolism; D005656:Fungal Proteins; D007120:Immunochemistry; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism*; D008826:Microbial Sensitivity Tests; D043583:Nucleoside-Triphosphatase; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*
chemical_list: D000935:Antifungal Agents; C094693:CDR1 protein, Candida albicans; D002352:Carrier Proteins; D003470:Culture Media; D004269:DNA, Bacterial; D005656:Fungal Proteins; D026901:Membrane Transport Proteins; D015725:Fluconazole; D017766:Acid Anhydride Hydrolases; D043583:Nucleoside-Triphosphatase