Department of Medical Specialties, Section of Infectious Diseases, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd, Box 47, Houston, TX 77030, USA
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Abstract |
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Introduction |
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Saccharomyces cerevisiae, a genetically tractable fungus closely related to Candida albicans, is an attractive experimental system for studying azole resistance.1,3,4 A well-characterized mechanism of azole resistance in S. cerevisiae is conferred by loss-of-function mutations in sterol 5,6 desaturase which is encoded by the ERG3 gene.24 Instead of accumulating the toxic 14
-methyl-3,6-diol under azole treatment, erg3 mutants accumulate the less toxic 14
-methylfecosterol.2 Another mechanism of resistance and hypersensitivity to azoles is mediated by the pleiotropic drug resistance-5 gene (PDR5), an ATP binding cassette (ABC) transporter, as a result of increased efflux of the drug.1,5 Finally, loss of function of the CPR1 gene, which codes for the NADPH-dependent cytochrome P-450 oxidoreductase, and of YMR034c, which codes for a putative sterol transporter, results in azole hypersensitivity.3,4
In this study, we provide genetic evidence that Pdr5p-mediated resistance to fluconazole is altered in the background of mutations that affect ergosterol homeostasis and suggest a model in which Pdr5p functions in the maintenance of ergosterol homeostasis by extrusion or transport of sterol intermediates.
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Materials and methods |
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Preparation of the yeast growth medium, synthetic complete medium (SC) and standard techniques for the manipulation of yeast have been described.6 All work was done in the 1278b genetic background. The Tn3::LEU2::lacZ fusion library was used as a disruption mutagen and one fluconazole-resistant mutant with a disruption of ERG3 and three fluconazole-hypersensitive mutants with disruptions of the PDR5, CPR1 and YMR034c genes, respectively, were isolated.3 The yeast strains used in this study are described in the Table
. Fluconazole was a gift from Pfizer, Inc. (New York, NY, USA).
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Two assays were used to test independently for sensitivity to fluconazole. In the first assay, the growth of each yeast strain streaked out to form single colonies was examined on SC medium plates containing various concentrations of fluconazole. In the second assay, yeast growth was examined by spreading approximately 105 cells in late logarithmic growth phase on SC medium and placing on this a paper disc containing 500 µg of the drug. In both assays growth was aerobic at 30°C for 3 days.
Screen for dominant resistance to fluconazole
Approximately 108 cells of the diploid strain L5803 were plated on selective SC plates containing leucine and fluconazole at 256 mg/L and incubated for 7 days at 30°C. Genetic analysis showed that 22 of the 30 resistant mutants had dominant mutations in a single gene, which we called the Fluconazole Dominant Resistance-1 gene (FDR-1).
Molecular biological and biochemical methods
Cloning of the FDR-1 gene.
Genomic DNA from FDR-1 was partially digested with MboI. A genomic DNA library was constructed on the URA3-based vector PRS 316.6 Wild-type yeast (strain 10560-14C; Table) was transformed with the FDR-1 genomic DNA library and plated on SC plates containing uracil. The transformation plates were then replicated to SC plates containing uracil and fluconazole at 128 mg/L to select for fluconazole-resistant colonies among the transformants. Resistant colonies were purified and screened to determine which had a URA3 plasmid-dependent fluconazole resistance phenotype. The plasmid-dependent fluconazole resistance phenotype was evaluated by the identification of Ura+ non-resistant candidates by both 5-fluoro-orotic acid (5-FOA) medium selection and the passive loss of URA3 plasmid by streaking in yeast peptone dextrose (YPD) medium. The same wild-type strain was then retransformed with DNA from the candidate clones. Ura+ fluconazole-resistant transformants were deemed true positives, and DNA was sequenced from their corresponding plasmids.
The ß-galactosidase assays were performed in triplicate on extracts of exponential-phase cultures in SC + fluconazole at 8 mg/L as described previously.6
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Results and discussion |
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The FDR-1 allele was mapped to chromosome VII and was centromere linked. Linkage analysis with a URA3-marked pdr1 mutant (constructed by transforming yeast with a pdr1 plasmid p
B6.5)7 showed that FDR-1 was an allele of PDR1, which we called PDR1-100. Dominant point mutations of PDR1, a transcription factor, resulting in pleiotropic drug resistance through overexpression of PDR5 mRNA have been described.5,7 The diploid strain DC188 had an eight-fold increase in ß-galactosidase levels in SC medium and a 16-fold increase at low concentrations (8 mg/L) of fluconazole compared with the diploid strain DC13, by quantitative ß-galactosidase assay. Finally, the cloning of the FDR-1 library revealed one transformant that was highly resistant to fluconazole. Sequencing of the 12 kb genomic clone in the corresponding plasmid revealed the PDR1 locus.
An unexpected genetic interaction was discovered between PDR1-100 and erg3, both of which were resistant to fluconazole as single mutants: the double mutant erg3 PDR1-100 was sensitive to fluconazole (Figure). In contrast, the erg3 pdr5 double mutant was as resistant as erg3 (Figure, b
). In addition, the resistance of PDR1-100 was also decreased in the ymr034c and cpr1 backgrounds (Figure, b
). Loss of function of CPR1 has been shown to influence ergosterol levels.8 In addition, YMR034c, which encodes a putative sterol transporter, could also affect ergosterol homeostasis.3
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Our work has potential therapeutic implications. If azoles compete with endogenous sterols for Pdr5p, strategies for reversing efflux-mediated resistance to these drugs by inhibiting other components of the ergosterol pathway (such as Erg3p), in addition to Erg11p, will be useful. Alternatively, if non-ergosterol sterols affect lipid fluidity and subsequently the activity of Pdr5p, this model will affect drug design. For example, alternative means of azole delivery such as liposomes could theoretically overcome Pdr5p-mediated resistance. Since transporter-mediated resistance to azoles appears to be the most prevalent in vivo,1 further efforts to elucidate the modulators of such resistance could lead to new therapeutic strategies.
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Acknowledgments |
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Notes |
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References |
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Received 30 June 1999; returned 30 November 1999; revised 4 January 2000; accepted 1 March 2000