Systematic Analysis of Morphogenesis, Commensalism, and Virulence in a Leading Human Fungal Pathogen

University Of Toronto, Toronto ON

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Abstract

SUMMARY/ABSTRACT Fungal pathogens pose a devastating threat to human health, infecting billions of people worldwide and causing more than 1.5 million deaths each year. Candida albicans is one of the most pervasive fungal pathogens, killing almost 40% of people suffering from bloodstream infections. Treating these infections is extremely difficult, as fungi are closely related to humans and there are very few drugs that kill the fungus without host toxicity. With the emergence of drug resistance, the development of new therapeutic strategies is now crucial. To address this important clinical need and identify new antifungal drug targets, it is critical to uncover mechanisms that enable C. albicans to cause life-threatening human disease. We are one of the first academic labs to obtain a powerful functional genomics resource that we are uniquely positioned to expand to allow us to test the function of almost every gene in the C. albicans genome. This resource includes a collection of conditional expression strains that covers ~40% of the genome where one allele of a target gene is deleted in the diploid pathogen, and expression of the remaining wild-type allele is governed by the tetracycline-repressible promoter. During the prior funding period we: developed a pipeline to expand the resource to genome scale, optimized a functional genomics platform for massively parallel analysis of fungal virulence traits using next generation sequencing with pooled assays to quantify the relative proportion of each strain, which are uniquely marked with molecular barcodes; optimized high-resolution image analysis of cellular morphology and structures; and developed assays for identifying genes important for commensalism, virulence, and interaction with host immune cells. Our efficient high-throughput analyses established the power of systematic genetic analysis to uncover new biological insights that could not have been predicted based on current paradigms and enabled focused, hypothesis-driven dissection of key mechanisms governing host- pathogen interactions. Our studies will provide the first global analysis of C. albicans morphogenesis, commensalism, and virulence, and will reveal fundamental biological mechanisms that could not be predicted without a systematic genetic approach. Our studies will: 1) complete the collection of tetracycline-repressible conditional expression strains to cover non-essential genes, since genes required for pathogen viability in vitro provide little insight into host adaptation or virulence; 2) identify novel regulators of key virulence traits such as morphogenesis; and 3) identify determinants of C. albicans host adaptation and virulence on a genome scale. Our comprehensive strain resources and compendium of phenotypic profiles will be made available to the community, advancing the field with a publicly accessible database and interpretive machine learning model to maximize insight. This work will provide the most comprehensive functional genomics resource for any fungal pathogen and will reveal genes governing host adaptation, revealing new strategies to cripple fungal pathogens.