The ‘obligate diploid’ Candida albicans forms mating-competent haploids

Journal name:
Nature
Volume:
494,
Pages:
55–59
Date published:
DOI:
doi:10.1038/nature11865
Received
Accepted
Published online

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.

At a glance

Figures

  1. C. albicans haploid and auto-diploid genotypes.
    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). CEN, centromere; Chr, chromosome; MRS, major repeat sequence. c, Flow cytometry after prolonged propagation of a haploid revealed a mixture of ploidies within a single population (c), whereas single colonies from this haploid (d) show distinct haploid (left) and diploid (right) ploidy. e, Flow cytometry of a homozygous auto-diploid.

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

    a, Representative differential interference contrast microscopy (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, 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; 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 (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. Ade, adenine; NAT, nourseothricin; YPAD, yeast peptone dextrose media with adenine.

  3. Haploid growth in vitro and in vivo.
    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) 48h post-infection.

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

    a, Series of strains constructed from a stable haploid isolate, GZY792 (MTLα, his4), which 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 approach50. 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°C (hyphae) are similar to phenotypes seen for the corresponding diploid null mutants.

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Author information

Affiliations

  1. Department of Genetics, Cell Biology & Development, University of Minnesota, Minneapolis, Minnesota 55455, USA

    • Meleah A. Hickman,
    • Darren Abbey,
    • Benjamin D. Harrison &
    • Judith Berman
  2. Institute of Molecular and Cell Biology, Agency for Science, Technology & Research, Singapore 138673, Singapore

    • Guisheng Zeng,
    • Yan-Ming Wang &
    • Yue Wang
  3. Bowdoin College, Brunswick, Maine 04011, USA

    • Anja Forche
  4. Department of Molecular Microbiology and Immunology, Brown University, Providence, Rhode Island 02912, USA

    • Matthew P. Hirakawa &
    • Richard J. Bennett
  5. Department of Microbiology and Immunology, Taipei Medical University, Taipei, Taiwan

    • Ching-hua Su
  6. Department of Molecular Microbiology and Biotechnology, George Wise Faculty of Life Sciences Tel Aviv University, Ramat Aviv, 69978 Israel

    • Judith Berman

Contributions

M.A.H. performed flow cytometry analysis, SNP/CGH hybridizations, species identification, white-opaque switching and mating assays, and in vitro growth assays. Y.W. and G.Z. designed and analysed auxotrophs, morphogenesis mutants and in vivo growth experiments; G.Z. constructed the mutants; Y.-M.W. collected isolates post-in vivo. A.F. and C.-h.S. initially isolated haploid/homozygous isolates. D.A. developed the flow cytometry analysis and SNP/CGH pipelines. M.P.H. performed virulence and in vivo competition assays. B.D.H. collected and analysed cell and nuclear size data and budding patterns. M.A.H. and J.B. assembled the data and wrote the manuscript with editorial input from A.F., R.J.B. and Y.W.

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The authors declare no competing financial interests.

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