Haploidy, diploidy and evolution of antifungal drug resistance in Saccharomyces cerevisiae

Abstract

We tested the hypothesis that the time course of the evolution of antifungal drug resistance depends on the ploidy of the fungus. The experiments were designed to measure the initial response to the selection imposed by the antifungal drug fluconazole up to and including the fixation of the first resistance mutation in populations of Saccharomyces cerevisiae. Under conditions of low drug concentration, mutations in the genes PDR1 and PDR3, which regulate the ABC transporters implicated in resistance to fluconazole, are favored. In this environment, diploid populations of defined size consistently became fixed for a resistance mutation sooner than haploid populations. Experiments manipulating population sizes showed that this advantage of diploids was due to increased mutation availability relative to that of haploids; in effect, diploids have twice the number of mutational targets as haploids and hence have a reduced waiting time for mutations to occur. Under conditions of high drug concentration, recessive mutations in ERG3, which result in resistance through altered sterol synthesis, are favored. In this environment, haploids consistently achieved resistance much sooner than diploids. When 29 haploid and 29 diploid populations were evolved for 100 generations in low drug concentration, the mutations fixed in diploid populations were all dominant, while the mutations fixed in haploid populations were either recessive (16 populations) or dominant (13 populations). Further, the spectrum of the 53 nonsynonymous mutations identified at the sequence level was different between haploids and diploids. These results fit existing theory on the relative abilities of haploids and diploids to adapt and suggest that the ploidy of the fungal pathogen has a strong impact on the evolution of fluconazole resistance.

Figure 1.—

Evolution of resistance to FLC in 50-ml cultures of 0.5× YPD inoculated with 5 × 10⁴ cells. Circles represent the mean OD₆₀₀ of four haploid populations, two replicates of MATa and two of MATα. Squares represent the mean OD₆₀₀ of six diploid populations, two replicates of MATa/α, two of MATα/α, and two of MATa/a. The measure of dispersion at each time point was standard error. On the right, MIC of FLC and expression of PDR5 and ERG11 were measured for three single-colony isolates of each population. For MIC, black, 16 mg/ml; dull red, 64 mg/ml; and bright red, 256 μg/ml. For gene expression, black, not overexpressed; and red, significantly overexpressed [see supplementary data appendix (http://www.genetics.org/supplemental/) for original values]. Each rectangle in the grids is divided into three squares, one for each single-colony isolate tested.

Figure 2.—

Diploid advantage at 32 mg/ml FLC with respect to the waiting time for FLC-resistance mutations. As in Figure 1, circles show haploid populations and squares show diploid populations. Top, diploid populations were started with 2.5 × 10⁴ cells and the haploids with 5 × 10⁴ cells. Bottom, diploid populations were started with 5 × 10⁴ cells and the haploids with 10⁵ cells.

Figure 3.—

Waiting time and the copy number of PDR1 and PDR3. Squares, mean OD₆₀₀ for six populations that were PDR1/PDR1 PDR3/PDR3. Circles, mean OD₆₀₀ for six populations, that were PDR1/PDR1Δ PDR3/PDR3Δ. Diamonds, mean OD₆₀₀ for six populations that were PDR1Δ/PDR1Δ PDR3Δ/PDR3Δ. Each diploid genotype was constructed twice, from different matings, and was used to found three replicate populations each.

Figure 4.—

Distribution of nonsynonymous mutations in PDR1 and PDR2 in haploid and diploid populations evolved for 100 generations in 16 mg/ml FLC.