Mycobacterium tuberculosis phagosome maturation

描述(申请人提供)：结核分枝杆菌(MTB)通过阻止吞噬小体成熟来促进其存活。通过靶向宿主蛋白或途径来逆转这一过程，为未来消除细胞内结核杆菌提供了一种潜在的治疗方法。我们提出了一种新的替代方法来鉴定人巨噬细胞中MTB H37Rv吞噬小体成熟途径的关键宿主成分。吞噬小体的成熟是通过从吞噬小体中招募或消除选定的宿主蛋白来调节的。因此，介导这一过程的宿主蛋白在成熟噬菌体和未成熟噬菌体上有差异表达。虽然常规技术已经鉴定了其中的一些蛋白质，但我们将利用最先进的蛋白质组学技术，系统地鉴定一组在人巨噬细胞中成熟和未成熟的结核分枝杆菌H37Rv噬菌体上差异表达的宿主蛋白。在这些差异表达的宿主蛋白中，那些特异性地抑制结核杆菌吞噬小体成熟的蛋白将通过抑制表达来鉴定。我们还将评估抑制先前识别的、差异表达的宿主蛋白对人巨噬细胞中MTB H37Rv吞噬小体成熟和活性的影响，因为许多这些研究仅限于小鼠巨噬细胞中的无毒分枝杆菌菌株。[段落]目的1：通过对人单核细胞系THP-1的吞噬小体进行蛋白质组学分析，鉴定活的和死亡的MTB H37Rv吞噬小体中差异表达的宿主蛋白。通过活的和死亡的结核分枝杆菌H37Rv噬菌体的Western印迹分析来验证我们的发现。我们以前已经标准化了小鼠中MTB吞噬小体的分离(Ramachandra等人，J.Exp。地中海医院。2001(1))和人巨噬细胞(2)。目的2：抑制一些新发现的、差异表达的宿主蛋白的表达，以确定它们对THP-1巨噬细胞中MTB H37Rv吞噬小体成熟和活性的影响。此外，我们还将分析抑制Rab 5、Rab 14和Rab22a表达对MTB H37Rv吞噬小体成熟的影响。目标1将是第一年的重点。目标2将是第一年和第二年的重点。我们预测，所产生的数据将为未来的研究奠定基础，以寻找新的药物靶点，以促进结核杆菌噬菌体成熟和杀伤，并降低耐药风险。公共卫生相关性：结核分枝杆菌(MTB)每年潜伏感染超过10亿人并导致200万人死亡的能力取决于它阻止吞噬小体成熟进入吞噬小体的能力。靶向宿主蛋白/途径并促进MTB吞噬小体成熟和杀伤的药物将为消除细胞内的MTB提供一种新的途径，同时也使耐药变得更加困难。我们建议使用蛋白质组学作为一种新的、快速的、全面的、无偏见的和数据驱动的工具来识别宿主蛋白，这些宿主蛋白可能通过促进毒力的MTB吞噬小体成熟和杀伤而成为潜在的治疗靶点。

DESCRIPTION (provided by applicant): Mycobacterium tuberculosis (MTB) promotes its survival by preventing phagosome maturation. Reversing this process by targeting host proteins or pathways provides a potential future therapeutic approach to eliminate intracellular MTB. We propose a novel, alternative approach to identify critical host components of the virulent MTB H37Rv phagosome maturation pathway in human macrophages. [PARAGRAPH] Phagosome maturation is mediated by the recruitment or elimination of select host proteins from phagosomes. Therefore, host proteins that mediate this process are differentially expressed on mature versus immature phagosomes. While conventional techniques have identified a few of these proteins, we will systematically identify a set of host proteins that are differentially expressed on mature versus immature virulent MTB H37Rv phagosomes in human macrophages by state-of-the-art proteomic technologies. Of these differentially expressed host proteins, those that specifically inhibit MTB phagosome maturation will be identified through inhibition of expression. We will additionally assess the impact of inhibiting previously identified, differentially expressed host proteins, on MTB H37Rv phagosome maturation and viability in human macrophages, since many of these studies have been limited to avirulent mycobacterial strains in mouse macrophages. [PARAGRAPH] AIM 1: Identify differentially expressed host proteins in live and dead MTB H37Rv phagosomes by proteomic analysis of phagosomes isolated from a human monocyte cell line, THP-1. Validate our findings by Western blot analysis of live and dead MTB H37Rv phagosomes. We have previously standardized the isolation of MTB phagosomes in murine (Ramachandra et al., J. Exp. Med. 2001 (1)) and human macrophages (2). AIM 2: Inhibit expression of a select number of newly identified, differentially expressed host proteins to define their impact on MTB H37Rv phagosome maturation and viability in THP-1 macrophages. We will additionally analyze impact of inhibiting expression of Rab 5, Rab 14 and Rab22a on MTB H37Rv phagosome maturation. [PARAGRAPH] Aim 1 will be the focus of year one. Aim 2 will be the focus of year 1 and 2. We predict that the data generated will lay the foundation for future studies to identify novel drug targets that promote MTB phagosome maturation and killing with a lower risk of drug resistance. PUBLIC HEALTH RELEVANCE: The capacity of Mycobacterium tuberculosis (MTB) to latently infect over one billion people and cause two million fatalities annually rests with its ability to block phagosomal maturation into the phagolysosome. Agents that target host proteins/pathways and promote MTB phagosome maturation and killing will provide a novel way to eliminate intracellular MTB and also make drug resistance more difficult to achieve. We propose to use proteomics as a novel, rapid, comprehensive, unbiased and data-driven tool to identify host proteins that may serve as potential therapeutic targets by promoting virulent MTB phagosome maturation and killing.