Genetic and phenotypic intra-species variation in Candida albicans

Abstract

Candida albicans is a commensal fungus of the human gastrointestinal tract and a prevalent opportunistic pathogen. To examine diversity within this species, extensive genomic and phenotypic analyses were performed on 21 clinical C. albicans isolates. Genomic variation was evident in the form of polymorphisms, copy number variations, chromosomal inversions, subtelomeric hypervariation, loss of heterozygosity (LOH), and whole or partial chromosome aneuploidies. All 21 strains were diploid, although karyotypic changes were present in eight of the 21 isolates, with multiple strains being trisomic for Chromosome 4 or Chromosome 7. Aneuploid strains exhibited a general fitness defect relative to euploid strains when grown under replete conditions. All strains were also heterozygous, yet multiple, distinct LOH tracts were present in each isolate. Higher overall levels of genome heterozygosity correlated with faster growth rates, consistent with increased overall fitness. Genes with the highest rates of amino acid substitutions included many cell wall proteins, implicating fast evolving changes in cell adhesion and host interactions. One clinical isolate, P94015, presented several striking properties including a novel cellular phenotype, an inability to filament, drug resistance, and decreased virulence. Several of these properties were shown to be due to a homozygous nonsense mutation in the EFG1 gene. Furthermore, loss of EFG1 function resulted in increased fitness of P94015 in a commensal model of infection. Our analysis therefore reveals intra-species genetic and phenotypic differences in C. albicans and delineates a natural mutation that alters the balance between commensalism and pathogenicity.

Figure 1.

Phylogenetic relationship and clade assignment of studied isolates. The phylogenetic relationship of the strains was inferred based on 112,223 informative SNP positions using maximum parsimony in PAUP*; node labels indicate the support frequency of 1000 bootstrap replicates. Clades were assigned based on multilocus sequence type (MLST) analysis of seven loci or prior work reporting fingerprint clades (FP).

Figure 2.

Phenotypic profiling of C. albicans isolates. (A) Doubling times and biofilm formation. Doubling times were measured in liquid YPD medium at 30°C. Circles represent euploid isolates and triangles represent aneuploid isolates. Biofilm dryweight biomasses ± standard error. Colors represent clade designation as follows: (SA) gray; (I) red; (II) yellow; (III) blue; (E) green. (B) Filamentation of clinical isolates grown on Spider medium for 7 d at 30°C or 37°C. Bar graphs represent filamentation scores ± standard error for strains at 30°C (pink) or 37°C (blue). (C) Assays of clinical isolates grown in the presence of stressors. Tenfold dilutions of cells were plated under regular growth conditions (SCD, 30°C), cell wall stress (SCD + 100 µg/mL calcofluor white, 30°C), oxidative stress (SCD + 2 mM hydrogen peroxide, 30°C), and thermal stress (SCD medium, 42°C).

Figure 3.

Aneuploid regions of clinical isolates. For each sequenced isolate, normalized read depth in 1-kb windows is shown along each chromosome relative to the SC5314 reference. Chromosome position is shown for every 100 kb along the x-axis; average normalized depth on the y-axis is relative to diploid levels. Regions exhibiting partial or full chromosome aneuploidies are noted with the corresponding isolate name. See also Supplemental Figure S8 for chromosome copy number in each strain.

Figure 4.

Loss of heterozygosity in sequenced isolates. For each isolate, the frequency of SNPs, including both heterozygous and homozygous variants relative to SC5314, is plotted for 5-kb windows across the genome (gray bars). Homozygous regions are shaded blue; this includes regions that are homozygous and shared with the reference SC5314 and regions containing a different homozygous haplotype. Additional features shown on the chromosome plot (bottom profile) include centromeres (green circles), major repeat sequences (blue rectangles), and the MTL locus on Chromosome 5 (orange rectangle).

Figure 5.

Pattern of heterozygosity on Chromosome 5 containing the MTL locus. (A) Isolates were typed as either MTL heterozygous (left column) or MTL homozygous (right column). Homozygous regions (shaded blue) of Chromosome 5 were identified from SNP data. The lower axis indicates the position of the MTL locus (orange rectangle) and centromere (green circle) on Chromosome 5, with scale shown in bases. (B) Percentage of Chromosome 5 that is heterozygous in each isolate (fraction of 5-kb windows typed as heterozygous; see Methods). (C) Pattern of SNP heterozygosity immediately flanking the MTL locus. Note that SNPs within the MTL loci were not mapped due to the high sequence divergence of the two loci. MTLa loci are marked by orange triangles and MTLα loci are marked by white triangles. Axis indicates position on Chromosome 5, with scale shown in bases.

Figure 6.

Analysis of native and EFG1-complemented P94015 strains in commensal and pathogenic models of infection. (A,B) Induction of filamentation in SC5314 and P94015 strains in YPD medium supplemented with 10% serum at 37°C. Two independent EFG1-complemented strains are shown: P94015 + EFG1(1) and P94015 + EFG1(2). (C) Survival curves for Galleria mellonella infected with strain P94015 and EFG1-complemented P94015. n = 30 worm per strain, P < 0.01, log-rank Mantel-Cox test. (D) Competition between native P94015 and EFG1-complemented P94015 in a murine model of systemic infection. C. albicans cells were recovered from kidneys after 7 d infection and compared to the inoculum. Data are two independent competition experiments with four mice each. (E) Testing of P94015 or derived strains in a murine model of commensal fitness. C. albicans cells were recovered from fecal pellets each day, and the relative levels of native P94015 or EFG1-complemented P94015 were determined. Error bars, ±SD. n = 3 mice for each competition. (F) Relative levels of P94015 or EFG1-complemented P94015 in gastrointestinal tissues after 2 wk of commensal growth. Error bars, ±SD.