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

• 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
• 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

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.

项目摘要 尽管IFN-γ在控制结核分枝杆菌感染方面显然很重要，但仍存在基本差距 了解IFN-γ依赖性免疫组织化学对这种全球重要病原体的机制。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M。 结核病含有吞噬体。脂质，包括甘油三酸酯和胆固醇，是重要的营养素 感染过程中结核分枝杆菌的来源。在哺乳动物细胞中，脂质通常存储在脂质液滴中， 胞质细胞器主要由磷脂周围的甘油三酸酯和胆固醇组成 单层。脂质液滴是结核分枝杆菌感染巨噬细胞的特征，人们认为 细菌利用脂质液滴作为营养丰富的储层。但是，脂质液滴提供多种生物学 功能，可以在宿主免疫反应中发挥重要作用。重要的是，很少有关于该角色的研究 在IFN-γ的背景下，已经进行了结核分枝杆菌感染期间的脂质液滴 激活。该提案的主要假设在强大的初步数据的指导下，是在 IFN-γ激活，脂质液滴用于扩增脂质衍生的二十酮的产生，这对于对 生产性免疫反应对感染的反应，并且这些脂质液滴不再可用于M。 结核病作为营养来源。检验这一假设并阐明脂质液滴在M中的作用。 结核病感染提出了两个具体目标。 1）阐明感染MTB中的脂质液滴蛋白质组 IFN-γ激活的巨噬细胞和2）开发遗传工具来操纵巨噬细胞中的LD抽象。 已知脂质液滴具有介导其细胞功能的动态蛋白质组。在第一个目标 识别定位于脂质滴的蛋白质的定向和公正方法将被雇用以阐明 脂质液滴蛋白质组。细胞类型特异性LD蛋白质组的定义可以提供有关的重要线索 上下文特定的脂质液滴功能。为了最大程度地提高蛋白质组学研究的敏感性和特异性 将采用接近碱基生物素化策略。在第二个目标中，CAS9介导的基因组工程 将用于特异性操纵结核分枝杆菌感染期间LDS的抽象。影响 在结核分枝杆菌限制的IFN-γ激活巨噬细胞中降低LD的积累 将确定获取和宿主类固醇生产。如果成功，建议的实验将 改变脂质液滴在结核分枝杆菌发病机理中的作用的范式，并将发现新的 IFN-γ依赖性免疫的机制。这样的知识将阐明我们对 成功对结核分枝杆菌的免疫反应，并可能告知免疫调节的发展 疗法和疫苗。