Developing an in vivo toolbox to interrogate the intracellular trafficking and killing of Aspergillus spores

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Project Summary/Abstract Fungal pathogens, including Aspergillus fumigatus, cause life-threatening infections in more than 2 million immunocompromised people worldwide per year. Healthy people encounter and successfully combat A. fumigatus every day, but we do not fully understand the immune pathways that promote fungal clearance. In particular, the function of macrophages in killing A. fumigatus spores inside of infected hosts is unclear. The overarching goal of the proposed research is to delineate the cellular pathways through which macrophages phagocytose, endosomally traffick, and kill A. fumigatus spores. These experiments will be performed in larval zebrafish which allow for imaging of subcellular host-pathogen dynamics in live, intact hosts throughout a multi-day infection. A larval zebrafish model of A. fumigatus infection recapitulates the pathogenesis of infections in human patients. In this model we find that only ~50% of spores are killed by macrophages and the remaining spores can persist inside of these cells. We hypothesize that spore killing correlates with localization of spores to specific phagosomal compartments and that modulation of this localization by mutation of compartment-defining genes will alter this killing. Targeting a panel of 15 candidate genes/proteins shown to associate with fungal phagosomes in vitro, I first propose to fluorescently-tag each protein in macrophages in larval zebrafish and use live imaging to quantify colocalization with spores throughout infection to define the compartments through which spores are trafficked. Second, I propose to systematically mutate each of the 15 candidate genes using a CRISPR pipeline and test the requirement for each gene in macrophage-mediated spore killing and host survival. Altogether, this research will identify intracellular mechanisms that promote killing of A. fumigatus spores by macrophages and open up future opportunities to modulate these pathways to increase fungal killing.