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. 2009 Apr;53(4):1344-52.
doi: 10.1128/AAC.00926-08. Epub 2009 Feb 17.

Relative contributions of the Candida albicans ABC transporters Cdr1p and Cdr2p to clinical azole resistance

Sarah Tsao  1 , Fariba Rahkhoodaee, Martine Raymond
Affiliations

Relative contributions of the Candida albicans ABC transporters Cdr1p and Cdr2p to clinical azole resistance

Sarah Tsao et al. Antimicrob Agents Chemother. 2009 Apr.

Abstract

Candida albicans frequently develops resistance to treatment with azole drugs due to the acquisition of gain-of-function mutations in the transcription factor Tac1p. Tac1p hyperactivation in azole-resistant isolates results in the constitutive overexpression of several genes, including CDR1 and CDR2, which encode two homologous transporters of the ATP-binding cassette family. Functional studies of Cdr1p and Cdr2p have been carried out so far by heterologous expression in the budding yeast Saccharomyces cerevisiae and by gene deletion or overexpression in azole-sensitive C. albicans strains in which CDR1 expression is low and CDR2 expression is undetectable. Thus, the direct demonstration that CDR1 and CDR2 overexpression causes azole resistance in clinical strains is still lacking, as is our knowledge of the relative contribution of each transporter to clinical azole resistance. In the present study, we used the SAT1 flipper system to delete the CDR1 and CDR2 genes from clinical isolate 5674. This strain is resistant to several azole derivatives due to a strong hyperactive mutation in Tac1p and expresses high levels of Cdr1p and Cdr2p. We found that deleting CDR1 had a major effect, reducing resistance to fluconazole (FLC), ketoconazole (KTC), and itraconazole (ITC) by 6-, 4-, and 8-fold, respectively. Deleting CDR2 had a much weaker effect, reducing FLC or KTC resistance by 1.5-fold, and had no effect on ITC resistance. These results demonstrate that Cdr1p is a major determinant of azole resistance in strain 5674 and potentially in other clinical strains overexpressing Cdr1p and Cdr2p, while Cdr2p plays a more minor role.

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Figures

FIG. 1.
FIG. 1.
Expression of CDR1 and CDR2 in strains SC5314, 5457, and 5674 and in 5674 mutant derivatives. (A) Northern blot analysis. Total RNA extracts were prepared from the strains indicated at the top and analyzed by Northern blotting with gene-specific probes for CDR1 (top) or CDR2 (middle). rRNAs are shown as loading controls (bottom). (B) Western blot analysis. For the immunodetection of Cdr1p and Cdr2p, total membrane protein extracts were prepared from the strains and analyzed by Western blotting with the anti-Cdr1p, anti-Cdr2p, and anti-Cdrp antibodies. A Coomassie-stained gel of the protein extracts is shown at the bottom, with the positions of the molecular mass standards and the predicted positions of Cdr1p and Cdr2p indicated on the left and right, respectively. The values on the left are molecular sizes in kilodaltons.
FIG. 2.
FIG. 2.
Profiles of resistance of strains 5457 and 5674 and strain 5674-derived tac1 and cdr mutants to azole drugs and different compounds with antifungal activity, as determined by microtiter plate liquid assays. (A) FLC resistance. Cells were incubated for 48 h at 30°C in liquid YPD medium with the indicated concentrations of FLC. The data are presented as the relative growth of cells in FLC-containing medium compared to the growth of the same strain in FLC-free medium, which was set at 100%. The data are the mean of three independent experiments performed in duplicate. (B) KTC resistance. (C) ITC resistance. (D) FPZ resistance. (E) R6G resistance. (F) AMB resistance. For panels B to F, the experiments were performed as described for panel A.
FIG. 3.
FIG. 3.
Phenotypic analysis of the CDR2 revertant. The FLC susceptibilities of strains 5457, 5674, STY19, STY31, and STY47 were analyzed by spot (A) and MIC (B) assays as described in the legends to Fig. 2 and 4.
FIG. 4.
FIG. 4.
Profiles of resistance of strains 5457 and 5674 and strain 5674-derived tac1 and cdr mutants to azole drugs and different compounds with antifungal activity, as determined by spot assays. Serial 10-fold dilutions of cells, starting at an OD600 of 0.1, were spotted onto YPD plates containing FLC, KTC, ITC, FPZ, R6G, or AMB at the indicated concentrations (micrograms per milliliter) or no drug (CTL). The plates were incubated for 48 h at 30°C.
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