Global analysis of mutations driving microevolution of a heterozygous diploid fungal pathogen

Abstract

Candida albicans is a heterozygous diploid yeast that is a commensal of the human gastrointestinal tract and a prevalent opportunistic pathogen. Here, whole-genome sequencing was performed on multiple C. albicans isolates passaged both in vitro and in vivo to characterize the complete spectrum of mutations arising in laboratory culture and in the mammalian host. We establish that, independent of culture niche, microevolution is primarily driven by de novo base substitutions and frequent short-tract loss-of-heterozygosity events. An average base-substitution rate of ∼1.2 × 10^-10 per base pair per generation was observed in vitro, with higher rates inferred during host infection. Large-scale chromosomal changes were relatively rare, although chromosome 7 trisomies frequently emerged during passaging in a gastrointestinal model and was associated with increased fitness for this niche. Multiple chromosomal features impacted mutational patterns, with mutation rates elevated in repetitive regions, subtelomeric regions, and in gene families encoding cell surface proteins involved in host adhesion. Strikingly, de novo mutation rates were more than 800-fold higher in regions immediately adjacent to emergent loss-of-heterozygosity tracts, indicative of recombination-induced mutagenesis. Furthermore, genomes showed biased patterns of mutations suggestive of extensive purifying selection during passaging. These results reveal how both cell-intrinsic and cell-extrinsic factors influence C. albicans microevolution, and provide a quantitative picture of genome dynamics in this heterozygous diploid species.

Keywords: Candida albicans; LOH; aneuploidy; diploid species; microevolution.

Fig. 1.

Microevolution of C. albicans genomes. (A) Schematic of in vitro and in vivo microevolution experiments. (B) Distribution of SNPs and indels, (C) intergenic and coding mutations, (D) synonymous and nonsynonymous mutations, and (E) transitions and transversions. Data are averaged across all microevolution experiments. Note that panels indicate mutations resulting from either GOH or LOH events.

Fig. 2.

Selection shapes microevolution of C. albicans genomes. (A) Frequency of observed and expected GOH mutations in intergenic regions (these regions represent 36.2% of the C. albicans genome). GOH SNP mutations represent de novo base substitutions. (B) Observed and expected fractions of synonymous GOH SNPs in coding regions during microevolution experiments (∼25% of base substitutions are expected to be synonymous if random events). (C) Selection coefficients for nonsynonymous GOH SNPs calculated based on the number of observed vs. expected nonsynonymous substitutions. Expected nonsynonymous substitutions were estimated for each microevolution experiment based on observed synonymous substitutions (■) or observed intergenic substitutions (●). Only isolates for which both nonsynonymous and synonymous/intergenic substitutions were observed were included in this analysis. For each panel, data points represent independently evolved isolates and asterisks indicate significant differences (t test, *P < 0.05).

Fig. 3.

Mutation rates in C. albicans and the impact of chromosomal features on mutation rates. (A) GOH and LOH mutation rates during in vitro growth. Rates include both SNP and indel events. (B) Fluctuations in genome heterozygosity during microevolution relative to starting heterozygosity levels (red line). Significant decreases in heterozygosity are only observed for two isolates that underwent LLOH events (red symbols). (C and D) GOH (C) and LOH (D) mutation rates in specific regions relative to whole-genome rates. These include heterozygous (HET) regions, repeat regions (MRS and LTR), Chr END regions (final 10 kb of each chromosome arm), centromeres, genes encoding ALS and GPI-linked proteins, and TLO genes. Mutations include both changes in SNPs and indel events. Data points represent independently evolved isolates, and asterisks indicate significant differences relative to whole-genome mutation frequencies (t test, *P < 0.05).

Fig. 4.

Microevolution is punctuated by frequent SLOH events. (A) Schematic of different types of LOH events, including mLOH (loss of single heterozygous positions), SLOH (<10 kb and involve loss of two or more heterozygous positions), and LLOH (>10 kb and affect hundreds of heterozygous positions). (B) Distribution of LOH events showing the L_min, L_avg, and L_max size for each LOH event. (C and D) L_min size distribution of LOH events, including mLOH, SLOH, and LLOH (shown in red), for each lineage (C) and each niche (D). For B–D, data points represent independent LOH events.

Fig. 5.

Relationship between LOH location and different genomic regions. (A) Chromosomal location of all LOH events (using L_min) with triangles marking the start (red) and end (blue) of each event. Location of centromeres (CEN) and MRS regions are shown. (B) Proximity of LOH events to the closest genomic feature, including MRS regions, telomeres (Chr ENDs or TLO genes), and centromeres. Each LOH event is uniquely mapped to the closest of these features on the same chromosome arm. Distances equal to 0 indicate an LOH start site inside the respective genomic region. (C) GOH rates (including SNPs and indels) in the duplicated LLOH region and in the rest of the genome in the P78048 GI SD B isolate, relative to whole genome GOH rates. Only GOH SNPs (base substitutions) were observed in the duplicated LLOH region. (D) Number of GOH events (SNPs and indels per 25 bp) observed within 500 bp of LOH tracts in microevolved isolates. (E) GOH rates (including SNPs and indels) in regions adjacent to LOH tracts (within 500 bp) and in the rest of the genome, relative to whole genome GOH rates. Data points represent independently evolved isolates, asterisks indicate significant differences relative to whole genome mutation frequencies (t test, *P < 0.05).

Fig. 6.

Chr 7 trisomy is associated with a fitness advantage for colonization of the GI tract. (A) Competition experiments comparing the relative fitness of evolved (Chr 7 3X) and parental isolates (Chr 7 2X) in the GI SD colonization model. Evolved isolates represent strains that had become trisomic for Chr 7 following passage in the GI SD model. C. albicans cells were recovered from mouse fecal pellets and the relative levels of evolved and parental isolates determined. (B) Trisomic isolates were passaged in vitro to induce loss of the trisomic Chr 7 and resulting isolates were genotyped using KASP. (C) Competition experiments comparing the fitness of GI SD evolved isolates (Chr 7 3X) and in vitro passaged derivatives that were disomic for Chr 7 (2X). Strains were competed in the GI SD colonization model, cells recovered from mouse fecal pellets, and the relative levels of evolved and passaged isolates determined. Error bars represent ±SD; four mice were used for each competition.

Fig. 7.

Schematic illustrating the pattern of mutational events across the C. albicans genome. Figure highlights how certain chromosomal features are associated with elevated mutation rates.