Abstract/Project Summary Neutropenic patients are at risk for severe infection, and the fungal opportunistic pathogen Aspergillus fumigatus poses a particular risk. While macrophages first respond to and phagocytose Aspergillus spores, on their own these cells do not efficiently kill the fungus in vivo. The overarching goal of the proposed research is to identify novel pathways within in vivo macrophages that can be harnessed to kill Aspergillus spores. The larval zebrafish is an ideal host in which to tackle this problem. Excellent live imaging and genetic tools are available, the immune systems of zebrafish and humans are largely conserved, and an Aspergillus infection model in zebrafish recapitulates human Aspergillosis. I propose to first determine the exact anti- Aspergillus effects of two proteins implicated in susceptibility to Aspergillus: Rac2 and Caspase-1. Rac2 is a pleiotropic regulator of immune cell function and delineation of each of its functions will determine which are implicated in Aspergillus killing. The protease Caspase-1 is the central effector of inflammasome activation, and careful stepwise analysis will determine which of its several targets promote the survival of animals infected with Aspergillus. Concomitantly, I will use an unbiased RNAseq screen for pathways and genes involved in spore killing by sorting “killing” versus “permissive” macrophages from live animals using FACS, identifying gene and pathway candidates for future research. Results from these aims will be applicable to the development of immunotherapies that improve the survival of immunocompromised patients with fungal infections.