ABSTRACT/PROJECT SUMMARY Background: Histoplasma is a pathogenic fungus that causes life-threatening lung infections. About 500,000 people are exposed to Histoplasma each year in the United States, and over 60% of the US population has been exposed to the fungus at some point in their life. We have shown that Histoplasma is composed of at least five different species that vary considerably in the type and magnitude of disease they cause. Broad, long-term objective: The proposed research will help us identify the genes that allow virulence to emerge and spread, as well as develop a panel of isolates that once deep-sequenced can be used by the community of medical mycologists to map any trait of interest in Histoplasma. The objective of this proposal is to discover whether the genes responsible for differences in virulence among isolates are similar across species. Specific aims: Aim 1 of the study proposes to generate genetic reference panels for three species of the human pathogen Histoplasma. We will use this resource to identify alleles involved with virulence differences within and between species. Aim 2 will genetically test the phenotypic effects (i.e., virulence in vitro and in vivo) of the genomic hypotheses produced in Aim1. Aim 3 will study the spread of alleles in clinical samples over a period of 40 years and will integrate the results from Aims 1 and 2, allowing us to determine whether any of the alleles involved in virulence have increased in frequency. Method: This haploid organism is ideal for the laboratory study of fungal pathogens, and it is well-suited for genomic analysis. We will generate genetic reference panels for three different species of Histoplasma with state-of-the-art genomic tools and genome-wide association mapping. We will use this panel to identify the genetic basis of virulence differences within isolates of the same species. Notably, we will generate an online portal to analyze GWAS data, a first in the medical mycology community. Preliminary results show that given the amount of phenotypic variance in virulence, our approach and proposed sample sizes make this project feasible. Validation of candidate virulence genes will be undertaken according to established cell culture and mouse infection assays. Our approach will generate tools and reference panels for the fungal genetics community. Health-relatedness: The disease burden caused by Histoplasma species is substantial in the United States, with a conservative estimate of at least 3.4 cases per 100,000 population. If infectious strains can transmit the ability to cause infection to less harmful strains through gene exchange, the potential future disease burden will grow as global trade, travel and climate change bring new species of the fungus into overlapping geographic regions. The proposed research will identify what loci are involved in the evolution of virulence and will study the influence of natural selection in their evolution in recent timescales. Th...