The mechanism by which the Darlington strain of Candida albicans is resistant to azole antifungal agents was investigated. This strain produces fecosterol, not ergosterol, suggesting a defect in 5,6 desaturase, an enzymatic activity coded for by the ERG3 gene. We cloned one copy of ERG3 from the Darlington strain and found that the other ERG3 copy differs in two restriction sites. We are currently attempting to clone the other copy. A GAL1 mutant of Darlington created by A. Geber using 2-deoxgalactose was transformed with GAL1 in tandem with ERG3 cloned from a wild type strain. The transformant had increased ERG3 transcript but no change in azole susceptibility. If sterol anaylsis shows ERG3 complementation, then it will be unlikely that a defect in ERG3 accounts for azole resistance in this strain. As another approach, we are attempting to complement a Candida glabrata ERG3 deletant and a Saccharomyces cerevisiae ERG3 using the Darlington ERG3. Isolates of Candida glabrata from an AIDS patient~s mouth showed increasing fluconazole resistance during therapy. The resistant and susceptible isolates seemed to be the same strain on restriction fragment length polymorphism (RFLP) and rapid amplification of polymorphic DNA (RAPD) analysis. Radiolabeled fluconazole efflux was increased in the resistant strains. This efflux was energy dependent, in that it could be blocked by carbonyl cyanide m-chlorophenylhydrazone. No alteration of membrane sterols were found in the resistant strains, indicating that increased drug efflux accounted at least in part for the fluconazole resistance.
