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The role of lipid droplets in immunity to M. tuberculosis infection

脂滴在结核分枝杆菌感染免疫中的作用

基本信息

批准号：
9168274
负责人：
Sarah A Stanley
金额：
$ 19.63万
依托单位：
UNIVERSITY OF CALIFORNIA BERKELEY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-06-01 至 2018-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9168274
关键词：
Anabolism Bacteria Biological Process Biotinylation Carbon Cell Death Cell physiology Cells Cessation of life Characteristics Cholesterol Cytosol Data Development Eicosanoid Production Eicosanoids Gene Targeting Genes Genetic Genome engineering Goals Growth Host Defense Immune Immune response Immunity In Vitro Infection Infection Control Inflammation Interferon Type II Knowledge Lipids Macrophage Activation Maintenance Mammalian Cell Mediating Metabolism Methods Mus Mycobacterium tuberculosis Nutrient Organelles Outcome Pathogenesis Phagosomes Phospholipids Play Production Proteins Proteome Proteomics Rest Role Running Sensitivity and Specificity Signal Transduction Site Source Technology Testing Transformed Cell Line Triglycerides Tuberculosis Vaccine Therapy base cancer cell cell type cytokine genome editing hypoxia inducible factor 1 in vivo killings lipid mediator macrophage monolayer novel pathogen pleiotropism research study response tool trafficking transcription factor

项目摘要

Project Summary Despite the clear importance of IFN-γ for control of M. tuberculosis infection, there are still fundamental gaps in understanding mechanisms of IFN-γ dependent immunity to this globally significant pathogen. An important long-term goal is to understand whether and how IFN-γ induces macrophages to restrict nutrients from the M. tuberculosis containing phagosome. Lipids, including triglycerides and cholesterol, are important nutrient sources for M. tuberculosis during infection. In mammalian cells, lipids are often stored in lipid droplets, cytosolic organelles that consist primarily of triglycerides and cholesterol surrounded by a phospholipid monolayer. Lipid droplets are a characteristic of M. tuberculosis infected macrophages and it is thought that the bacteria exploit lipid droplets as a nutrient rich reservoir. However, lipid droplets serve a variety of biological functions, and can play an important role in host immune responses. Importantly, few if any studies of the role of lipid droplets during M. tuberculosis infection of macrophages have been conducted in the context of IFN-γ activation. The primary hypothesis of this proposal, guided by strong preliminary data, is that in the context of IFN-γ activation, lipid droplets serve to amplify the production of lipid derived eicosaonids that are essential for productive immune responses to infection, and that these lipid droplets are no longer accessible to M. tuberculosis as a nutrient source. To test this hypothesis and clarify the role of lipid droplets during M. tuberculosis infection two specific aims are proposed. 1) Elucidate the lipid droplet proteome in Mtb infected IFN-γ activated macrophages and 2) Develop genetic tools for manipulating LD abundance in macrophages. Lipid droplets are known to have dynamic proteomes that mediate their cellular functions. In the first aim directed and unbiased methods to identify proteins that localize to lipid droplets will be employed to elucidate the lipid droplet proteome. Definition of cell type specific LD proteomes can provide significant clues as to context specific lipid droplet function. To maximize the sensitivity and specificity of proteomic studies, a novel proximity base biotinylation strategy will be employed. In the second aim, Cas9 mediated genome engineering will be used to specifically manipulate the abundance of LDs during infection with M. tuberculosis. The impact of decreasing LD accumulation in IFN-γ activated macrophages on M. tuberculosis restriction, bacterial nutrient acquisition, and host eicosanoid production will be determined. If successful, the proposed experiments will shift the paradigm of the role of lipid droplets in M. tuberculosis pathogenesis and will uncover new mechanisms of IFN-γ dependent immunity. Such knowledge will illuminate our understanding of the basis of successful immune responses to M. tuberculosis, and may inform the development of immune-modulating therapies and vaccines.

项目概要尽管 IFN-γ 对于控制结核分枝杆菌感染具有明显的重要性，但在控制结核分枝杆菌感染方面仍存在根本性差距了解针对这种全球重要病原体的 IFN-γ 依赖性免疫机制。一个重要的长期目标是了解 IFN-γ 是否以及如何诱导巨噬细胞限制来自分枝杆菌的营养物质。含有吞噬体的结核菌。脂质，包括甘油三酯和胆固醇，是重要的营养素感染期间结核分枝杆菌的来源。在哺乳动物细胞中，脂质通常储存在脂滴中，主要由甘油三酯和胆固醇组成，周围有磷脂的胞质细胞器单层。脂滴是结核分枝杆菌感染的巨噬细胞的一个特征，人们认为细菌利用脂滴作为营养丰富的储存库。然而，脂滴具有多种生物功能功能，并在宿主免疫反应中发挥重要作用。重要的是，很少有关于这个角色的研究结核分枝杆菌感染巨噬细胞期间脂滴的变化已在 IFN-γ 的背景下进行激活。该提案的主要假设是在强有力的初步数据的指导下， IFN-γ 激活，脂滴有助于放大脂质衍生的二十碳酸的产生，这对于对感染产生有效的免疫反应，并且这些脂滴不再能够被 M. 结核菌作为营养源。为了检验这一假设并阐明脂滴在 M. 结核病感染提出了两个具体目标。 1) 阐明感染 Mtb 的脂滴蛋白质组 IFN-γ 激活巨噬细胞，2) 开发用于操纵巨噬细胞中 LD 丰度的遗传工具。已知脂滴具有介导其细胞功能的动态蛋白质组。在第一个目标中将采用定向且公正的方法来识别定位于脂滴的蛋白质来阐明脂滴蛋白质组。细胞类型特异性 LD 蛋白质组的定义可以提供重要线索上下文特定的脂滴功能。为了最大限度地提高蛋白质组研究的敏感性和特异性，一种新颖的方法将采用邻近碱基生物素化策略。第二个目标是Cas9介导的基因组工程将用于在结核分枝杆菌感染期间专门控制 LD 的丰度。影响减少 IFN-γ 激活巨噬细胞中 LD 积累对结核分枝杆菌限制、细菌营养的影响采集和宿主类二十烷酸的产生将被确定。如果成功的话，拟议的实验将改变脂滴在结核分枝杆菌发病机制中的作用范式并将揭示新的 IFN-γ依赖性免疫机制。这些知识将阐明我们对基础的理解对结核分枝杆菌的成功免疫反应，并可能为免疫调节的发展提供信息疗法和疫苗。