Mycobacterium tuberculosis phagosome maturation

描述（申请方提供）：结核分枝杆菌（MTB）通过阻止吞噬体成熟促进其存活。通过靶向宿主蛋白质或途径逆转该过程提供了消除细胞内MTB的潜在未来治疗方法。我们提出了一种新的替代方法，以确定在人类巨噬细胞中的毒性MTB H37 Rv吞噬体成熟途径的关键主机组件。吞噬体成熟是通过从吞噬体募集或消除选择的宿主蛋白质来介导的。因此，介导该过程的宿主蛋白质在成熟吞噬体与未成熟吞噬体上差异表达。虽然常规技术已经鉴定了这些蛋白中的一些，但是我们将通过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的成熟与未成熟毒性MTB H37 Rv吞噬体上差异表达的宿主蛋白。在这些差异表达的宿主蛋白中，特异性抑制MTB吞噬体成熟的那些将通过抑制表达来鉴定。我们还将评估抑制先前鉴定的差异表达的宿主蛋白对人巨噬细胞中MTB H37 Rv吞噬体成熟和活力的影响，因为这些研究中的许多研究仅限于小鼠巨噬细胞中的无毒分枝杆菌菌株。目标1：通过从人单核细胞系THP-1分离的吞噬体的蛋白质组学分析鉴定活的和死的MTB H37 Rv吞噬体中差异表达的宿主蛋白。通过活的和死的MTB H37 Rv吞噬体的蛋白质印迹分析的初步发现。我们先前已经标准化了鼠中MTB吞噬体的分离（Ramachandra等人，J. Exp. 2001（1））和人巨噬细胞（2）。目标2：抑制选定数量的新鉴定的差异表达宿主蛋白的表达，以确定其对THP-1巨噬细胞中MTB H37 Rv吞噬体成熟和活力的影响。我们将另外分析抑制Rab 5、Rab 14和Rab 22 a的表达对MTB H37 Rv吞噬体成熟的影响。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，产生的数据将为未来的研究奠定基础，以确定新的药物靶点，促进MTB吞噬体成熟和杀伤，降低耐药性的风险。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其阻断吞噬体成熟为吞噬溶酶体的能力。靶向宿主蛋白质/途径并促进MTB吞噬体成熟和杀伤的试剂将提供消除细胞内MTB的新方法，并且还使耐药性更难以实现。我们建议使用蛋白质组学作为一种新的，快速的，全面的，公正的和数据驱动的工具，以确定宿主蛋白，可能作为潜在的治疗目标，促进有毒MTB吞噬体成熟和杀伤。

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.