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Systematic screens of a Candida albicans homozygous deletion library decouple morphogenetic switching and pathogenicity

Abstract

Candida albicans is the most common cause of serious fungal disease in humans. Creation of isogenic null mutants of this diploid organism, which requires sequential gene targeting, allows dissection of virulence mechanisms. Published analyses of such mutants show a near-perfect correlation between C. albicans pathogenicity and the ability to undergo a yeast-to-hypha morphological switch in vitro. However, most studies have used mutants constructed with a marker that is itself a virulence determinant and therefore complicates their interpretation. Using alternative markers, we created 3,000 homozygous deletion strains affecting 674 genes, or roughly 11% of the C. albicans genome. Screening for infectivity in a mouse model and for morphological switching and cell proliferation in vitro, we identified 115 infectivity-attenuated mutants, of which nearly half demonstrated normal morphological switching and proliferation. Analysis of such mutants revealed that virulence requires the glycolipid glucosylceramide. To our knowledge, this is the first C. albicans small molecule that has been found to be required specifically for virulence.

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Figure 1: C. albicans mutants and screens.
Figure 2: Infectivity screen.
Figure 3: Colony morphology screen.
Figure 4: Proliferation screen and Venn diagram.
Figure 5: Characterization of mutants affecting glucosylceramide biosynthesis.
Figure 6: Virulence analysis of mutants affecting glucosylceramide biosynthesis.

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Acknowledgements

We are extremely grateful to the Stanford Genome Technology Center for releasing the C. albicans genomic sequence before publication, and to the curators of the Candida Genome Database, which has been an invaluable tool in our analysis of the published literature. We are also grateful to many colleagues at the University of California, San Francisco (UCSF), and elsewhere who were generous with advice, protocols and reagents. S. O'Brien constructed our C. albicans bioinformatics database. B. Hromatka assisted with construction of C. albicans mutants. J. Zhe and J. De Risi provided a database of putative unique C. albicans ORFs. M. Fischbach suggested the link between our sphingolipid-associated mutants and the glucosylceramide pathway. E. Chow and J. Cox provided expertise and equipment for lipid extraction. P.E. Bailey and the Agard, Morgan, Shokat and Walter labs at UCSF provided training and access to their mass spectrometer. B. al-Sady and G. Narlikar shared their mass spectrometry plate. H. Madhani wrote software for data analysis. S. Desta prepared hundreds of liters of laboratory media. H. Madhani, J. Cox, L. Connolly and R. Locksley provided helpful comments on the manuscript. This work was supported by US National Institutes of Health grants RO1 A149187 and K08 AI062800, and the UCSF Program for Breakthrough Biomedical Research. S.M.N. received a Howard Hughes Medical Institute Postdoctoral Research Fellowship for Physicians and a Burroughs Wellcome Fund Career Award in the Biomedical Sciences during the course of these studies.

Author information

Authors and Affiliations

Authors

Contributions

S.M.N. constructed mutants, conceived and carried out the majority of experiments, and wrote the manuscript. S.F. assisted with validation of mutants, contributed to the morphogenesis screen and performed the germ tube screen. L.A.K. assisted with mutant construction and validation. V.C. assisted with the morphogenesis screen. A.D.J. provided guidance and participated in writing the manuscript.

Corresponding author

Correspondence to Suzanne M Noble.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1 and 2 and Supplementary Tables 1, 2, 6, 7, 9 and 12 (PDF 996 kb)

Supplementary Table 3

Mutants with abnormal infectivity. (XLS 47 kb)

Supplementary Table 4

Mutants with abnormal morphogenesis (M score >1). (XLS 50 kb)

Supplementary Table 5

Results of the infectivity, morphogenesis, and proliferation screens. (XLS 153 kb)

Supplementary Table 8

Primers used for mutant construction and validation. (XLS 603 kb)

Supplementary Table 10

log2(R/I) ratios of mutants tested in the infectivity screen. (XLS 190 kb)

Supplementary Table 11

log2(R/I) ratios of wild type C. albicans tested in the infectivity screen. (XLS 28 kb)

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Noble, S., French, S., Kohn, L. et al. Systematic screens of a Candida albicans homozygous deletion library decouple morphogenetic switching and pathogenicity. Nat Genet 42, 590–598 (2010). https://doi.org/10.1038/ng.605

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