The role of Mycobacterium marinum protein ESAT-6 in promoting granuloma formation

描述（由申请人提供）：分枝杆菌利用几种机制利用宿主免疫系统为自己的利益。而不是被巨噬细胞杀死，通常吞噬和根除病原体的特化免疫细胞，分枝杆菌利用巨噬细胞作为其宿主生态位存活并持续存在。然后，被感染的巨噬细胞招募额外的巨噬细胞和其他免疫细胞到感染灶形成肉芽肿，这是一种有组织的免疫细胞聚集，是结核感染的标志。虽然传统上认为它是一种宿主保护结构，但最近来自海洋分枝杆菌-斑马鱼感染模型的证据表明，分枝杆菌利用正在形成的肉芽肿作为传播感染的手段。它通过分泌细菌因子ESAT-6来诱导上皮细胞中宿主基质金属蛋白酶-9 （MMP9）的表达。MMP9是一个更大的蛋白酶家族的成员，它可以修饰细胞外基质，以及细胞迁移和炎症分子。在分枝杆菌感染期间，MMP9是巨噬细胞募集到发展中的肉芽肿所必需的。本研究将探讨ESAT-6诱导MMP9分泌促进肉芽肿形成的机制。我将首先确定ESAT-6是否对mmp9表达的激活是必要的，或者是否有其他细菌分泌因子起作用。我将利用斑马鱼胚胎的光学透明性来研究ESAT-6是否足以诱导巨噬细胞募集，因为肉芽肿的形成可能需要感染。我假设ESAT-6直接与上皮细胞相互作用诱导mmp9表达，并将在体外使用人类上皮细胞系进行研究。我将识别与ESAT- 6结合并传递促进肉芽肿信号的宿主决定因子。最后，我将采用突变分析来确定ESAT-6的哪些区域是mmp9表达所必需的。本实验将揭示分枝杆菌利用宿主免疫系统和促进发病机制的机制。

DESCRIPTION (provided by applicant): Mycobacteria employ several mechanisms to exploit the host immune system for its own benefit. Rather than being killed by macrophages, specialized immune cells that normally engulf and eradicate pathogens, mycobacteria survive and persist by using macrophages as their host niche. The infected macrophages then recruit additional macrophages and other immune cells to the infection foci to form a granuloma, an organized aggregate of immune cells that is a hallmark of tuberculous infections. Although traditionally thought to be a host-protective structure, recent evidence from the Mycobacterium marinum- zebrafish model of infection has demonstrated that mycobacteria exploit the developing granuloma as a means to disseminate infection. It does so by secreting the bacterial factor ESAT-6 which induces the expression of host matrix metalloproteinase-9 (MMP9) from epithelial cells. MMP9 is a member of a larger family of proteases that modify the extracellular matrix, as well as cell migration and inflammatory molecules. During mycobacterial infections, MMP9 is required for the recruitment of macrophages to the developing granuloma. This proposal will investigate the mechanism by which ESAT-6 induces MMP9 secretion to promote granuloma development. I will begin by determining if ESAT-6 is necessary for the activation of mmp9 expression or if additional bacterial secreted factors play a role. I will take advantage of the optical transparency of zebrafish embryos to investigate if ESAT-6 is sufficient to induce macrophage recruitment, as infection may be required for granuloma formation. I hypothesize that ESAT-6 interacts directly with epithelial cells to induce mmp9 expression and will investigate this in vitro using human epithelial cell lines. I will identify the host determinant that binds to ESAT- 6 and transmits the granuloma-promoting signal. Lastly, I will employ mutational analysis to determine which regions of ESAT-6 are required for mmp9 expression. The experiments outlined in this proposal will shed light on the mechanisms employed by mycobacteria to exploit the host immune system and promote pathogenesis. PUBLIC HEALTH RELEVANCE: Due to the increasing prevalence of multi-drug resistant strains of Mycobacterium tuberculosis, coupled with the HIV pandemic, human tuberculosis disease remains a major global health problem. This proposal will investigate the interactions between host and pathogen factors during mycobacterial infections. These experiments may lead to the discovery and characterization of new host drug targets and reduce the time required to treat drug-resistant tuberculosis infections. )