Antagonism between two mechanisms of antifungal drug resistance

Abstract

This study tested for interaction between two independently evolved mechanisms of fluconazole resistance in Saccharomyces cerevisiae. One set of strains was from a 400-generation evolution experiment, during which the concentration of fluconazole was increased from 16 to 256 microg/ml in four increments. At 100 generations, populations became fixed for resistance mutations in either of two transcriptional regulators, PDR1 or PDR3. At 400 generations, replicate populations became fixed for another resistance mutation in UNK1, an unmapped gene further increasing resistance. Another genotype used in this study came from a population placed initially in 128 microg/ml of fluconazole; this environment selects for resistance through loss of function at ERG3, resulting in altered sterol metabolism. Mutant strains carrying PDR1(r) or PDR3(r) were crossed with the erg3(r) mutant strain, and the doubly mutant, haploid offspring were identified. The double-mutant strains grew less well than the parent strains at all concentrations of fluconazole tested. In genome-wide assays of gene expression, several ABC transporter genes that were overexpressed in one parent and several ERG genes that were overexpressed in the other parent were also overexpressed in the double mutants. Of the 43 genes that were consistently overexpressed in the PDR1(r) parents at generation 100, however, 31 were not consistently overexpressed in the double mutants. Of these 31 genes, 30 were also not consistently overexpressed after a further 300 generations of evolution in the PDR1(r) parent populations. The two independently evolved mechanisms of fluconazole resistance are strongly antagonistic to one another.

FIG. 1.

Tests for the FLC MIC for the ancestor (diamonds), strains carrying PDR1^r or PDR3^r from generation 100 of evolution experiment 1 (squares), the mutant strain carrying egr3^r (circles), and offspring strains of S. cerevisiae carrying both PDR1^r and erg3^r (triangles).

FIG. 2.

Tests for FLC MIC for the ancestor (diamonds), strains carrying PDR1^r and UNK1^r from generation 400 of evolution experiment 1 (squares), the mutant strain carrying erg3^r (circles), and offspring strains carrying both PDR1^r and erg3^r (triangles), among which segregation of UNK1^r and UNK1^s is expected to occur. In addition, strains carrying the respective PDR1^r mutation at generation 100 (stars) are included for comparison.

FIG. 3.

Uptake and efflux of rhodamine 6G. The protocol used followed that of Kolaczkowski et al. (13); see Materials and Methods for details. The results are from one of two replicate experiments and are representative of the two. RHO was added at time zero. Note that the erg3^r and PDR1^r/erg3^r strains take up RHO faster initially than any of the other strains, before all converge to the same level of uptake. Glucose-mediated efflux occurred in all strains except the PDR5 deletion strain. Cells of all strains maintained viability throughout the experiment.

FIG. 4.

Gene expression among strains from three populations evolving over 400 generations in increasing concentrations of FLC. The systematic gene name appears of the left, and the descriptive gene name appears on the right where available. The functions of genes known to include transport, stress response, and lipid metabolism are marked at the right. The color of boxes represents the constitutive expression of genes, with the scale at the bottom. Included are (a) genes overexpressed (≥1.5-fold) at generations 100 and 400; (b) genes consistently overexpressed in all three populations at generation 100 but not in all three populations at generation 400; (c) genes overexpressed in all three populations at generation 400 but not in any population at generation 100; and (d) genes underexpressed (≤1.5-fold) in all populations, either at generation 100 or generation 400.

FIG. 5.

Gene expression among PDR1^r mutant strains from three populations at generation 100 of evolution experiment 1, the erg3^r mutant strain, and doubly mutant offspring. Genes and gene expression values are denoted as described in the legend to Fig. 4. Included are (a) genes overexpressed (≥1.5-fold) in all three populations at 100 generations, and in all three respective double-mutant offspring, but not in the erg3^r mutant; (b) genes overexpressed in all three populations at 100 generations, in all three respective double-mutant offspring, and in the erg3^r mutant; (c) genes overexpressed in all three populations at 100 generations but not in all three respective double-mutant offspring; (d) genes overexpressed (≥1.5-fold) in the erg3^r mutant strain, and in all three of the double mutants, but not in all three PDR1^r mutants; (e) genes overexpressed (≥1.5-fold) in the erg3^r mutant strain but not in all three of the double mutants; (f) genes overexpressed in the three double mutants but not in erg3^r, nor in all three of the PDR1^r mutants; (g) genes underexpressed (≤1.5-fold) in the erg3^r mutant strain but not in all three of the double mutants; and (f) genes underexpressed either in all three PDR1^r mutants or in all three double mutants.