Stress alters rates and types of loss of heterozygosity in Candida albicans

Abstract

Genetic diversity is often generated during adaptation to stress, and in eukaryotes some of this diversity is thought to arise via recombination and reassortment of alleles during meiosis. Candida albicans, the most prevalent pathogen of humans, has no known meiotic cycle, and yet it is a heterozygous diploid that undergoes mitotic recombination during somatic growth. It has been shown that clinical isolates as well as strains passaged once through a mammalian host undergo increased levels of recombination. Here, we tested the hypothesis that stress conditions increase rates of mitotic recombination in C. albicans, which is measured as loss of heterozygosity (LOH) at specific loci. We show that LOH rates are elevated during in vitro exposure to oxidative stress, heat stress, and antifungal drugs. In addition, an increase in stress severity correlated well with increased LOH rates. LOH events can arise through local recombination, through homozygosis of longer tracts of chromosome arms, or by whole-chromosome homozygosis. Chromosome arm homozygosis was most prevalent in cultures grown under conventional lab conditions. Importantly, exposure to different stress conditions affected the levels of different types of LOH events, with oxidative stress causing increased recombination, while fluconazole and high temperature caused increases in events involving whole chromosomes. Thus, C. albicans generates increased amounts and different types of genetic diversity in response to a range of stress conditions, a process that we term "stress-induced LOH" that arises either by elevating rates of recombination and/or by increasing rates of chromosome missegregation.

Importance: Stress-induced mutagenesis fuels the evolution of bacterial pathogens and is mainly driven by genetic changes via mitotic recombination. Little is known about this process in other organisms. Candida albicans, an opportunistic fungal pathogen, causes infections that require adaptation to different host environmental niches. We measured the rates of LOH and the types of LOH events that appeared in the absence and in the presence of physiologically relevant stresses and found that stress causes a significant increase in the rates of LOH and that this increase is proportional to the degree of stress. Furthermore, the types of LOH events that arose differed in a stress-dependent manner, indicating that eukaryotic cells generate increased genetic diversity in response to a range of stress conditions. We propose that this "stress-induced LOH" facilitates the rapid adaptation of C. albicans, which does not undergo meiosis, to changing environments within the host.

FIG 1

Determination of LOH and point mutation rates. (A) Configuration of Chr1 for comparing LOH rates using selection for loss of GAL1 or URA3. One copy of GAL1 was replaced with URA3. Fluctuation analysis was performed using selection on either 2-deoxygalactose (2-DOG) or 5-fluoroorotic acid (5-FOA). (B) Configuration of Chr4 for measuring the rate of point mutations that revert the his4 G929T point mutation at the native HIS4 locus (18). Fluctuation analysis selected for the appearance of rare His⁺ colonies. (C) Positions of URA3 insertions (one per strain) used to measure LOH rates. In addition, the position of ADE2, used for half-sector analysis (strain YJB11848, which is ade2∆/∆ at the native ADE2 locus), is shown. (D) LOH rates for markers on all 16 chromosome arms. The distance between the URA3 marker used and the centromere (CEN) is indicated below.

FIG 2

SNP analysis of LOH types. (A and B) Detection of LOH events using an SNP microarray (A) and using SNP-RFLP analysis (B). (C) Total proportion of each type of LOH event (n = 21 post-fluctuation strains for array analysis [above] and 15 to 24 isolates per marker for 8 different markers yielding n = 173 post-fluctuation analysis strains for SNP-RFLP analysis of the URA3-marked chromosome [below]). (D) Percentages of LOH types from SNP-RFLP analysis at individual Chr arms for the 8 different marked strains tested (n = 15 to 24 for each strain).

FIG 3

Half-sector analysis to distinguish reciprocal versus nonreciprocal LOH events. (A) Configuration of Chr5 in strain YJB11848, which has one copy of ADE2 inserted on the left arm of Chr5 and distal SNP marker 10080A. (B) Examples of half-sectored colonies detected on MIN medium supplemented with uridine, histidine, and adenine. (C and D) Reciprocal (C) and nonreciprocal (D) LOH yield half-sectored colonies in which the phenotype of the white sector is homozygous or heterozygous, respectively. Adapted from the work of Andersen et al. (39).

FIG 4

LOH rate fold changes in stressed cells. The graph shows fold increases in LOH rates in stressed cells for 6 different strains each with URA3 inserted at a different locus (Fig. 1C; see also Table S6 in the supplemental material). The y axis shows fold change in LOH rate (note that the y axis is on a logarithmic scale).

FIG 5

Effect of stress on types and rates of LOH events. (A) Short-tract, long-tract, and whole-Chr events are diagrammed above and framed with a color corresponding to the type: cyan for short-tract, light gray for long-tract, and lavender for whole-Chr events. (B) LOH rates in cells exposed to different concentrations of H₂O₂ (R² = 0.96). (C) Types of LOH events in cells exposed to different H₂O₂ concentrations shift from primarily short-tract events to primarily long-tract events. Color scheme is as in panel A.