The impact of fungal pathogens on human health is devastating. They infect billions of people worldwide, and kill more than 1.5 million each year. The most vulnerable are people with reduced immune function, such as those with HIV or those undergoing immune suppressing treatments for cancer or organ transplants. One of the most pervasive fungal pathogens is Candida albicans, which kills 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 resource that will allow us to test the function of almost every gene in the C. albicans genome. This resource includes a collection of double barcoded heterozygous mutants covering ~90% of the genome, and a collection of strains covering ~40% of the genome where the expression of the remaining wild-type allele of a gene is governed by the tetracycline-repressible promoter. This resource provides an unprecedented opportunity to identify genes that control key virulence traits such as morphogenesis. It also enables the identification of determinants of commensalism and virulence, and to further elucidate the molecular mechanisms involved. We have optimized a functional genomics platform for massively parallel analysis of fungal virulence traits using next generation sequencing to quantify the relative proportion of each barcoded strain in pooled assays. We have also optimized high- resolution image analysis of cellular morphology and structures, and assays for identifying genes important for commensalism, virulence, and interaction with host immune cells. Our studies will provide the first global analysis of C. albicans morphogenesis, commensalism, and virulence. Our studies will: 1) develop a computational platform to predict C. albicans essential genes, and expand the collection of tetracycline-repressible conditional expression strains to cover most non-essential genes, since genes required for pathogen viability in vitro provide little insight into mechanisms of 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. This work will provide an expanded functional genomics resource to advance the field, and will leverage this resource to elucidate the genes and genetic networks governing morphogenesis and virulence. This will reveal new strategies to cripple fungal pathogens, and drug targets to improve clinical outcome.
The focus of this proposal is to elucidate mechanisms governing morphogenesis, commensalism, and virulence of the fungal pathogen Candida albicans. This eukaryotic pathogen has a profound impact on human health, with a global incidence of Candida bloodstream infections of ~400,000 cases, associated with crude mortality rates of 42% and attributable mortality rates of 27%; C. albicans is the most prevalent Candida species in almost all patient populations. The central hypotheses that govern this proposal are that functional genomic analysis of C. albicans using genome-scale libraries of double barcoded heterozygous gene deletion mutants and tetracycline-repressible conditional expression strains will reveal novel circuitry governing morphogenesis, commensalism, and virulence, and that targeting this circuitry has therapeutic potential for combating life-threatening fungal infections.
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