Gene Expression and evolution of antifungal drug resistance

We examined the gene expression changes resulting from the evolution of resistance in experimental populations of the yeast Saccharomyces cerevisiae subjected to two antifungal drugs, fluconazole (FLC) and amphotericin B (AmB). Fluconazole resistance may involve increased efflux or changes in sterol metabolism, while AmB resistance generally involves changes in sterol metabolism; for all of these types of resistance, the gene expression changes are extensive. The goal of these experiments was to test whether failure of gene expression changes all downstream of the original mutation for drug resistance would affect the ability of a mutant cell to evolve and/or to support a drug-resistant phenotype. In this study, replicate populations of Saccharomyces cerevisiae were evolved in increasing concentrations of AmB over hundreds of generations to yield resistant types among which there were permanent changes in gene-expression. Evolution experiments were started with 100 μL of an overnight culture that was derived from a single colony inoculated into 10 ml of YPD containing 0.25 μg/ml AmB. Five different populations were evolved in the presence of AmB in two different regimens, long and short. The rational for choosing different evolution regimens was to increase the chance of recruiting different mechanisms of AmB resistance among all of the replicates. In the long regimen, three populations started at 0.25 μg/ml AmB and this concentration was doubled every 100 generations for a total of 1100 generations and a final AmB concentration of 125 μg/ml (sce2468, sce2469, sce2470). In the short regimen, two populations were also started at 0.25 μg/ml AmB, but were given as much time as necessary to grow to high density, usually two to four days. The populations were then transferred a second time into the same AmB concentration. At the next transfer, the AmB concentration was doubled for another two growth cycles, and so on, until the populations had been through two transfers in 128 μg/ml AmB (sce2539 and sce2542).