Gene expression and evolution of antifungal drug resistance

Abstract

Permanent changes in gene expression result from certain forms of antifungal resistance. In this study, we asked whether any changes in gene expression are required for the evolution of a drug-resistant phenotype in populations. We examined the changes in gene expression resulting from the evolution of resistance in experimental populations of the yeast Saccharomyces cerevisiae with two antifungal drugs, fluconazole (FLC) in a previous study and amphotericin B (AmB) in this study, in which five populations were subjected to increasing concentrations of AmB, from 0.25 to 128 microg/ml in twofold increments. Six genes, YGR035C, YOR1, ICT1, GRE2, PDR16, and YPLO88W, were consistently overexpressed with resistance to AmB reported here and with resistance to FLC involving a mechanism of increased efflux reported previously. We then asked if the deletion of these genes impaired the ability of populations to evolve resistance to FLC over 108 generations of asexual reproduction in 32 and 128 microg/ml FLC, the same conditions under which FLC-resistant types evolved originally. For each of three deletion strains, YOR1, ICT1, and PDR16 strains, extinctions occurred in one of two replicate populations growing in 128 microg/ml FLC. Each of these three deletion strains was mixed 1:1 with a marked version of the wild type to measure the relative ability of the deletion strain to adapt over 108 generations. In these assays, only the PDR16 deletion strain consistently became extinct both at 32 and at 128 microg/ml FLC. The deletion of PDR16 reduces the capacity of a population to evolve to resistance to FLC.

FIG. 1.

Evolution of resistance measured as MIC₅₀ in three replicate yeast populations over 1,100 generations, during which the concentration of AmB was doubled every 100 generations. Circles, AmB concentration in the medium; diamonds, triangles, and squares, MICs of the three evolving populations, respectively. MICs of mass cultures always equaled those of single-colony isolates.

FIG. 2.

Evolution of resistance measured as MIC₅₀ in two replicate populations over 57 transfers, during which the concentration of AmB was doubled every other transfer. Circles, AmB concentration in the medium; diamonds, triangles, and squares, MICs of the two evolving populations, respectively. MICs of mass cultures always equaled those of single-colony isolates.

FIG. 3.

Gene expression in the five evolved populations resistant to AmB measured in the absence of the drug. Replicate bar graphs facing upwards represent increased levels of expression relative to that of the ancestor. Graphs with bars facing downward represent decreased expression. Only changes >1.5-fold up or down in all five populations are shown. Genes are listed in the same order as they occur on the chromosomes.

FIG. 4.

Evolution of FLC resistance in deletion strains for the six genes overexpressed in the AmB resistance strains from this study and in the three FLC-resistant strains reported previously by Anderson et al. (3) evolved through gradual increases in the FLC concentration. Diamonds, 0 μg/ml FLC; circles, 32 μg/ml FLC; x's, 128 μg/ml FLC.