yEvo: experimental evolution in high school classrooms selects for novel mutations that impact clotrimazole resistance in Saccharomyces cerevisiae

Abstract

Antifungal resistance in pathogenic fungi is a growing global health concern. Nonpathogenic laboratory strains of Saccharomyces cerevisiae are an important model for studying mechanisms of antifungal resistance that are relevant to understanding the same processes in pathogenic fungi. We have developed a series of laboratory modules in which high school students used experimental evolution to study antifungal resistance by isolating azole-resistant S. cerevisiae mutants and examining the genetic basis of resistance. We have sequenced 99 clones from these experiments and found that all possessed mutations previously shown to impact azole resistance, validating our approach. We additionally found recurrent mutations in an mRNA degradation pathway and an uncharacterized mitochondrial protein (Csf1) that have possible mechanistic connections to azole resistance. The scale of replication in this initiative allowed us to identify candidate epistatic interactions, as evidenced by pairs of mutations that occur in the same clone more frequently than expected by chance (positive epistasis) or less frequently (negative epistasis). We validated one of these pairs, a negative epistatic interaction between gain-of-function mutations in the multidrug resistance transcription factors Pdr1 and Pdr3. This high school-university collaboration can serve as a model for involving members of the broader public in the scientific process to make meaningful discoveries in biomedical research.

Keywords: azole resistance; course-based research experience; experimental evolution; genome sequencing; science education; yeast.

Fig. 1.

Overview of evolution experiment and sequencing. a) Outline of experiment. Yeasts were propagated in increasing concentrations of clotrimazole for several weeks. Clones from these experiments were sequenced to identify mutations that occurred during the experiment. b) Maximum measured clotrimazole tolerance of clones isolated from experiments at different schools and timepoints. c) Number of point mutations in sequenced clones.

Fig. 2.

Copy number variation events and candidate genes. Likely segmental duplications on chromosomes VIII, IV, and XV based on increased coverage in whole-genome sequencing data (Materials and Methods). Regions with increased copy number in at least one sequenced clone are represented with red horizontal lines drawn above their corresponding chromosomes. A number of clones with a given amplification are listed to the right of each red line. Location of candidate genes that could contribute to a fitness benefit is denoted on wild-type chromosome in black.

Fig. 3.

Outline of CSF1 competition experiment. a) Wild-type, synonymous mutant, and nonsynonymous mutant (CSF1^A2913P) were mixed in equal ratios and inoculated into YPD growth media with or without clotrimazole. These populations were propagated for three outgrowths. The frequency of CSF1^A2913P was determined at initial and final timepoints by Sanger sequencing. Representative Sanger sequencing chromatograms are shown. Heterozygous positions are represented with IUPAC codes in sequences below chromatograms (S used when G and C present; M used when A and C present). b) Frequency of CSF1^A2913P allele at beginning and end of competition with (+) or without (−) clotrimazole. Frequencies are averages of three replicates and were quantified by the program QSVAnalyzer. Error bars are one standard deviation in each direction.

Fig. 4.

Analysis of growth rates of PDR1^F749I and PDR3^T949A single and double mutants. Haploid PDR1^F749I and PDR3^T949A evolved strains were crossed and sporulated to generate recombinant haploid spores. Spore genotypes were determined by CAPS markers (Supplementary Table 12 in Supplementary File 1, Materials and Methods). a) Segregants were arrayed in a 96-well plate and grown in 9 µM clotrimazole media at 30°C in a Biotek Synergy H1 plate reader that measured growth of each strain by optical density (Supplementary Table 13 in Supplementary File 1). b) Doubling time of each genotype was calculated by linear fit of logarithmic growth phase for each curve in a (Materials and Methods).

Fig. 5.

Signatures of epistasis involving ERG25. a) Number of clones with genotypes related to the genes ERG25, HAP1, ROX1, ATP1, and ATP2. Frequency is biased toward double mutant groupings in the bottom right of each square (P-values by t-test listed below). Clones were sequenced at multiple timepoints; therefore, not all clones represent independent mutation events (Materials and Methods). b) Lineages within individual replicates were identified by shared mutations in clones from that replicate. Several lineages are made up of clones with ERG25 mutations that later acquire mutations in HAP1, ROX1, ATP1, or ATP2, as evidenced by multiple individuals with the same ERG25 mutation but different mutations in HAP1, ROX1, ATP1, or ATP2. No lineages were detected in which a HAP1, ROX1, ATP1, or ATP2 mutant later acquired an ERG25 mutation. c) Blue lines between ROX1-HAP1 and ATP1-ATP2 are known mechanistic relationships (Hap1 regulates Rox1; Atp1 and Atp2 are in the same complex). Orange lines between HAP1-ATP1 and HAP1-ATP2 are previously identified genetic interactions (van Leeuwen et al. 2016). The six yellow dashed lines are putative novel genetic interactions supported by presented data. d) Doubling time of segregants from backcrossing of strain containing mutations in ERG25 and ATP2 or e) ERG25 and ROX1 (Materials and Methods) grown in a 96-well plate in 4.5 or 9 µM clotrimazole media at 30°C. Doubling time calculated by linear fit of logarithmic growth phase (Supplementary Fig. S3, Materials and Methods). P-value by t-test, *P < 0.05; **P < 0.01; ***P < 0.001.