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

开发体内工具箱来探究曲霉菌孢子的细胞内运输和杀灭

基本信息

批准号：
10569606
负责人：
Emily Rosowski
金额：
$ 18.62万
依托单位：
CLEMSON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-02-09 至 2025-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10569606
关键词：
ATP phosphohydrolase Adrenal Cortex Hormones Animals Antifungal Agents Aspergillus Aspergillus fumigatus Automobile Driving Autophagocytosis Biological Assay C Type Lectin Receptors CRISPR/Cas technology Candidate Disease Gene Cells Cloning Clustered Regularly Interspaced Short Palindromic Repeats Complex Data Development Endosomes Fungal Spores Future Genes Goals Guide RNA Hour Human Image Immune Immune system Immunocompromised Host Immunotherapy In Vitro Individual Infection Infection Control Larva Lectin Receptors Life Macrophage Mediating Methods Modeling Mus Mutate Mutation Mycoses Oxidases Pathogenesis Pathway interactions Patients Persons Phagocytes Phagocytosis Phagosomes Pharmaceutical Preparations Predisposition Process Proteins Reporting Reproduction spores Research Resolution Risk Role Signal Transduction System Testing Toll-like receptors Transplant Recipients Visualization Work Zebrafish combat experimental study extracellular fungus in vivo interest molecular marker neutrophil particle pathogen pathogenic fungus rab GTP-Binding Proteins receptor response trafficking transmission process uptake

项目摘要

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.

项目概要/摘要包括烟曲霉在内的真菌病原体导致超过 200 万人受到危及生命的感染每年全世界有免疫功能低下的人。健康人遇到并成功对抗A. 每天都有烟菌，但我们并不完全了解促进真菌清除的免疫途径。在特别是，巨噬细胞在杀死受感染宿主体内的烟曲霉孢子方面的功能尚不清楚。这拟议研究的总体目标是描绘细胞途径巨噬细胞吞噬、内体运输并杀死烟曲霉孢子。这些实验将在斑马鱼幼体中进行，可以对活体、完整的亚细胞宿主-病原体动态进行成像宿主经历多天的感染。烟曲霉感染的斑马鱼幼虫模型概括了人类患者感染的发病机制。在这个模型中，我们发现只有约 50% 的孢子被杀死巨噬细胞和剩余的孢子可以持续存在于这些细胞内。我们假设孢子被杀死与孢子定位到特定吞噬体区室以及对此的调节相关通过区室定义基因突变进行的定位将改变这种杀伤作用。针对 15 名候选人组成的小组在体外显示与真菌吞噬体相关的基因/蛋白质，我首先建议对每个基因/蛋白质进行荧光标记斑马鱼幼虫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整个感染过程中，以确定孢子运输的隔室。其次，我建议使用 CRISPR 管道系统地突变 15 个候选基因中的每一个，并测试对每个基因都参与巨噬细胞介导的孢子杀死和宿主存活。总而言之，这项研究将确定促进巨噬细胞杀死烟曲霉孢子的细胞内机制并开辟未来调节这些途径以增加真菌杀灭的机会。