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

• 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
• 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

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.

项目总结/文摘