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

项目总结/摘要 包括烟曲霉菌在内的真菌病原体导致200多万人受到危及生命的感染 免疫力低下的人。健康的人遇到并成功地战胜了A。 我们每天都在研究烟曲霉，但我们并不完全了解促进真菌清除的免疫途径。在 特别是巨噬细胞对A.感染宿主体内的烟曲霉孢子尚不清楚。的 这项研究的首要目标是描绘细胞通路， 巨噬细胞吞噬、内体运输和杀死A.烟曲霉孢子这些实验将 在斑马鱼幼体中进行，这允许在活的、完整的、 在一个多天的感染宿主。A.烟曲霉感染重演了 人类患者感染的发病机制。在这个模型中，我们发现只有~50%的孢子被杀死， 巨噬细胞和剩余的孢子可以在这些细胞内存活。我们假设杀死孢子 与孢子定位于特定的吞噬体隔室相关， 通过区室限定基因的突变进行定位将改变这种杀伤。针对15名候选人的小组 在体外与真菌吞噬体相关的基因/蛋白质，我首先提出荧光标记每个 斑马鱼幼鱼巨噬细胞中的蛋白质，并使用实时成像来量化与孢子的共定位 在整个感染过程中，以确定孢子被运输的隔室。第二，我建议 使用CRISPR管道系统地突变15个候选基因中的每一个，并测试 每个基因在巨噬细胞介导的孢子杀伤和宿主存活中的作用。总之，这项研究将确定 促进A.烟曲霉孢子通过巨噬细胞和开辟未来 调节这些途径以增加真菌杀灭的机会。

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.