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

项目摘要/摘要 包括烟曲霉在内的真菌病原体导致200多万人受到危及生命的感染 全世界每年都有免疫受损的人。健康的人遇到并成功地对抗了A。 但我们并不完全了解促进真菌清除的免疫途径。在……里面 特别是，巨噬细胞在感染宿主体内杀死烟曲霉孢子的功能尚不清楚。这个 这项拟议研究的首要目标是描绘细胞通路，通过这些通路 巨噬细胞吞噬、内吞运输和杀死烟曲霉孢子。这些实验将 在斑马鱼幼体中进行，这允许成像活的完整的亚细胞宿主-病原体动态 宿主贯穿多天的感染过程。一种感染烟曲霉菌的斑马鱼幼虫模型 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留在这些细胞内。我们假设杀灭孢子 与孢子对特定吞噬体室定位以及对此的调节有关 隔室定义基因的突变定位将改变这种杀戮。瞄准15名候选人组成的小组 在体外与真菌吞噬小体相关的基因/蛋白，我首先建议对每个基因/蛋白进行荧光标记 斑马鱼幼体巨噬细胞中的蛋白质和利用活体成像定量与孢子共定位 整个感染过程中，以确定孢子通过哪些隔间进行运输。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 每个基因都在巨噬细胞介导的孢子杀灭和宿主存活中起作用。总而言之，这项研究将确定 促进巨噬细胞杀灭烟曲霉孢子的胞内机制及前景 有机会调节这些途径，以增加对真菌的杀伤力。

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.