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Antimicrobial Agents and Chemotherapy, December 2001, p. 3366-3374, Vol. 45, No. 12
0066-4804/01/$04.00+0   DOI: 10.1128/AAC.45.12.3366-3374.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Functional Expression of Candida albicans Drug Efflux Pump Cdr1p in a Saccharomyces cerevisiae Strain Deficient in Membrane Transporters

Kenjirou Nakamura,1,2 Masakazu Niimi,1,3 Kyoko Niimi,1 Ann R. Holmes,1 Jenine E. Yates,1 Anabelle Decottignies,4 Brian C. Monk,1 Andre Goffeau,4 and Richard D. Cannon1,*

Department of Oral Sciences and Orthodontics, University of Otago, Dunedin, New Zealand1; General Research Institute, Nippon Dental University, Niigata,2 and Department of Bioactive Molecules, National Institute of Infectious Diseases, Tokyo,3 Japan; and Unité de Biochimie Physiologique, Université de Louvain, Louvain, Belgium4

Received 3 January 2001/Returned for modification 20 February 2001/Accepted 27 August 2001

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 [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. 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.

* Corresponding author. Mailing address: Department of Oral Sciences and Orthodontics, University of Otago, P.O. Box 647, Dunedin, New Zealand. Phone: 64-3-479-7081. Fax: 64-3-479-0673. E-mail: richard.cannon{at}stonebow.otago.ac.nz.

Antimicrobial Agents and Chemotherapy, December 2001, p. 3366-3374, Vol. 45, No. 12
0066-4804/01/$04.00+0   DOI: 10.1128/AAC.45.12.3366-3374.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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