Drug Pumping Mechanisms in Canadida albicans
Japanese Journal of Medical Mycology / Nihon Ishinkin Gakkai
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 CDRJ, CDR2 or BENT 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
... to study the pumping function of Cdr 1 and Benr. The C, albicans CDRI and BENT genes were cloned separately into plasmid pVT101-U, to form plasmids pKY1411 and pKN5001 respectively. Plasmids pVT101-U, pKY1011 and pKN5001 were transformed into Saccharomyces cerevisiae SY 1, a sec6-4 mutant with a temperature-sensitive mutation in the secretory pathway. SY 1 cells transformed with pKY 1011 or pKN5041, were more resistant to fluconazole (MICs in both cases 64ug/ml) than SY1 cells (MIC 32ug/ml). In addition, cells transformed with pKY 1011 were more resistant to cycloheximide (MIC 16ug/ml) than SY1 cells (MIC 2ug/ml). Intact secretory vesicles were isolated from SY1 cells expressing Cdr 1 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.