The 'obligate diploid' Candida albicans forms mating-competent haploids

Abstract

Candida albicans, the most prevalent human fungal pathogen, is considered to be an obligate diploid that carries recessive lethal mutations throughout the genome. Here we demonstrate that C. albicans has a viable haploid state that can be derived from diploid cells under in vitro and in vivo conditions, and that seems to arise through a concerted chromosome loss mechanism. Haploids undergo morphogenetic changes like those of diploids, including the yeast-hyphal transition, chlamydospore formation and a white-opaque switch that facilitates mating. Haploid opaque cells of opposite mating type mate efficiently to regenerate the diploid form, restoring heterozygosity and fitness. Homozygous diploids arise spontaneously by auto-diploidization, and both haploids and auto-diploids show a similar reduction in fitness, in vitro and in vivo, relative to heterozygous diploids, indicating that homozygous cell types are transient in mixed populations. Finally, we constructed stable haploid strains with multiple auxotrophies that will facilitate molecular and genetic analyses of this important pathogen.

Figure 1. C. albicans haploid and auto-diploid genotypes

a) Flow cytometry analysis of DNA content of haploid strains (red) compared to diploid (SC5314, green) and tetraploid (RBY18, blue) control strains. Thin lines, raw data; bold lines, ‘best fit’ data as described in the Methods Summary. b) SNP/CGH array analysis of indicated strains (left) showing copy number (log2 ratio, black) and SNP allele information (Grey, heterozygous; magenta, allele ‘a’; cyan, allele ‘b’; white, no SNP data.) c) Flow cytometry after prolonged propagation of a haploid revealed a mixture of ploidies within a single population (c), while single colonies from this haploid (d) exhibit distinct haploid (left) and diploid (right) ploidy. e) Flow cytometry of a homozygous auto-diploid.

Figure 2. Morphology and mating competency of haploid C. albicans

a) Representative DIC images of haploid, diploid, and tetraploid cells overlaid with fluorescence images of their nuclei. b) Calcofluor white staining revealed primarily cells with the axial budding pattern, 15% with a bipolar budding pattern (n = 72) and 3% that were difficult to resolve. White arrows, previous bud scars; black arrow, newest bud. Scale bars represent 5μm. c) Haploids form true hyphae, pseudohyphae, and chlamydospores in serum, RPMI, and corn meal agar media, respectively. d) White-opaque switching detected as pink colony sectoring (top) and by microscopy of cells from white and pink/opaque sectors. Diploid, MTLa/MTLα1Δα2Δ (YJB12234), Haploid I (MTLa), and Haploid IV (MTLα). e) Mating between haploid cells. ‘Parents’: Haploid I (MTLa NAT1 ade2Δ), Haploid II (MTLa ADE2), Haploids III and IV (MTLα ADE2) showing growth on media indicated. ‘Crossed with Haploid I’: opaque (Op) or white cells (Wh) cells from Haploid I were mixed with opaque and/or white cells from Haploids II, III or IV and plated to medium selective for mating products.

Figure 3. Haploid growth in vitro and in vivo

a) Growth (doubling times in YPAD) of control diploid, haploids (pink), their auto-diploid derivatives (green), and heterozygous, mating products I × III and I × IV (purple). Error bars reflect one standard deviation from the mean. * p < 0.01, ** p < 0.001, Student’s t-test. b) Survival of mice (tail vein systemic candidiasis model) following inoculation with Haploid II (pink) or its diploid progenitor, YJB12419 (grey). c) Recovery of colony forming units (CFUs) from mouse kidneys (three mice per yeast strain) 48 hours post-infection.

Figure 4. Auxotrophic haploid strains enable one-step gene deletions

a) Series of strains constructed from a stable haploid isolate, GZY792 (MTLα, his4) was isolated after propagation for 30 passages, screening for ploidy by flow cytometry and selection of isolates that were consistently haploid. GZY803 (ura3Δ) was constructed by disruption of URA3 with HIS4. Other auxotrophies were generated by the URA-flipper approach. b) Flow cytometry of these auxotrophic strains. c) Genes disrupted in one-step map to all eight chromosomes. Circles, centromere position. d) Cell morphology phenotypes of haploid mutants grown in minimal media (yeast) or media supplemented with 20% FBS at 37° (hyphae) are similar to phenotypes seen for the corresponding diploid null mutants.