Multiple drug resistance is becoming a major problem in the treatment of AIDS patients with oropharyngeal candidosis. Candida albicans strains isolated from candidosis patients who do not respond to fluconazole therapy often show azole drug resistance which usually correlates with the expression of C. albicans CDR1, CDR2 or BENr genes, encoding potential drug efflux pumps. The objective of this study was to develop a yeast secretory vesicle transport assay and use this system to study the pumping function of Cdr1 and Benr. The C. albicans CDR1 and BEN r genes were cloned separately into plasmid pVT101-U, to form plasmids pKY1011 and pKN5001 respectively. Plasmids pVT101-U, pKY1011 and pKN5001 were transformed into Saccharomyces cerevisiae SY1, a sec6-4 mutant with a temperature-sensitive mutation in the secretory pathway. SY1 cells transformed with pKY1011 or pKN5001, were more resistant to fluconazole (MICs in both cases 64 microg/ml) than SY1 cells (MIC 32 microg/ml). In addition, cells transformed with pKY1011 were more resistant to cycloheximide (MIC 16 microg/ml) than SY1 cells (MIC 2 microg/ml). Intact secretory vesicles were isolated from SY1 cells expressing Cdr1 and these vesicles accumulated fluconazole in a time dependent manner. These experiments demonstrated that S. cerevisiae secretory vesicles can be used to examine the mechanism of fluconazole transport by putative C. albicans membrane pumps.

• PMID: 9580031
• DOI full text
• PubMed

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Journal Article | Research Support, Non-U.S. Gov't

Authors

Cannon RD, Fischer FJ, Niimi K, Niimi M, Arisawa M

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