Fungal pathogens have a major impact on human health; the lack of effective antifungal therapies, the diversity of species infecting humans, and the emergence of new lineages, species and drug resistance represent major challenges to treatment. This proposal will examine two species causing the largest number of opportunistic invasive mycoses and a third species representing a major cause of endemic dimorphic mycoses. We will compare clinical isolates from each pathogen to determine the genetic basis of highly virulent or antifungal resistant isolates, and to discriminate how isolates that cause invasive infections differ from those of low virulence or that are part of a healthy mycoflora. In the first aim, we will study the genetic basis of pathogenesis of Cryptococcus neoformans, a major cause of infection in immunocompromised individuals, by combining large- scale phenotyping, whole genome sequencing, and transcriptional analysis of natural isolates. We will characterize how natural isolates differ in in vivo growth during pulmonary infection and dissemination to the central nervous system and will use these measures to carry out genome-wide association screens of the pathogen; in parallel, we will measure the growth of the Cryptococcus gene deletion collection of over 4,000 strains. To highlight changes in gene regulation between isolates and pinpoint genes that are highly expressed at different infection stages, we will carry out RNA-Seq of in vivo stages and key cells involved in fungal interactions. We will also examine the microevolution of this pathogen during host infection and compare that to changes observed in an animal model. In the second aim, we will address a major question about the origin of bloodstream Candida infections, which are a major cause of mortality in immunocompromised patients. We will examine the intra-host diversity of Candida isolates between commensal sites in the gut and the skin and isolates from patients with candidemia to trace the origin of these bloodstream infections. We will adapt new enrichment approaches to increase the proportion of Candida reads in metagenomic samples. Lastly, we will test isolates that are more frequently associated with the bloodstream origin to confirm their relative virulence in an animal model. In aim 3, we will examine isolates of Talaromyces (Penicillium) marneffei collected from patients in a recent clinical trial comparing two antifungal treatments. This represents the largest collection of clinical isolates collected for a clinical trial with patient metadata for an endemic mycosis. We will use whole genome and RNA- Seq analyses to define properties associated with drug resistance and aggressive infections with high mortality. While these three aims represent independent projects to examine fungal pathogenesis, each utilizes large-scale ?omics approaches to extend and develop new paradigms for studying and understanding fungal virulence in the context of natural population variation. This work will provide an unprecedented whole-genome view into pathogens associated with clinical samples, and a synthesis of its findings will suggest new approaches for diagnosis and treatment for individual patients and risk factors to monitor prospectively.