Innate Immune Responses Triggered by M. Tuberculosis

该提案是P01更新中的项目2，题为“细胞内病原体和先天免疫”。M. 结核病是一种重要的人类病原体，在世界各地造成严重的发病率和死亡率。 前一项提案的目标是确定结核分枝杆菌引发和 操纵其主要宿主细胞--巨噬细胞的宿主反应。我们有一个惊人的发现，那就是M。 结核是一种吞噬病原体，它激活宿主的胞浆监视途径(CSP)，这是一种先天的 感受细胞质中细菌分子的信号通路。我们对寄主和细菌感兴趣 CSP激活所需的因子和阐明该途径在结核分枝杆菌中的功能作用 发病机制。我们已经证明ESX-1分泌途径和细胞壁脂PDIM对于 扰乱巨噬细胞的吞噬体膜，允许激活胞浆DNA受体。 出乎意料的是，胞质通道也将细菌定位于自噬途径。我们假设 细胞内病原体扰乱细胞内膜以促进毒力，但CSP的激活 允许宿主细胞区分和建立对病原体的不同免疫反应 非病原体。在目标1中，我们建议阐明结核分枝杆菌获得 并激活CSP，基于我们的证据，即单一ESX-1底物ESAT-6， 具有通透吞噬体膜的功能。我们建议确定DNA的性质，即 被宿主识别，测试负责CSP激活的假定宿主受体的作用，并探测 胞质信号在结核分枝杆菌感染中的作用。在目标2中，我们将研究胞浆通道如何导致 将结核分枝杆菌定位于自噬途径。特别是，我们将测试泛素化的作用 以及泛素结合适配器在感染期间将自噬小泡靶向于结核分枝杆菌。在 最终目标，我们将与Portnoy(项目1)和Vance(项目3)小组广泛合作进行筛选 通过在巨噬细胞中进行筛选来调节结核分枝杆菌的生长和宿主的先天反应 从ENU致突变小鼠中分离，并在巨噬细胞中进行RNAi筛选。

This proposal is Project 2 within a P01 renewal, entitled "Intracellular pathogens and innate immunity". M. tuberculosis is an important human pathogen that causes severe morbidity and mortality around the world. The goal of the previous proposal was to identify the mechanisms by which M. tuberculosis triggers and manipulates host responses of its primary host cell, the macrophage. We made the striking discovery that M. tuberculosis, a phagosomal pathogen, activates the host cytosolic surveillance pathway (CSP), an innate signaling pathway that senses bacterial molecules in the cytoplasm. We are interested in host and bacterial factors required for CSP activation, and in elucidating the functional role of the pathway in M. tuberculosis pathogenesis. We have shown that the ESX-1 secretion pathway and the cell wall lipid PDIM are critical for perturbing phagosomal membranes of macrophages, allowing activation of cytosolic DNA receptors. Unexpectedly, cytosolic access also targets bacteria to the autophagy pathway. We hypothesize that intracellular pathogens perturb intracellular membranes to promote virulence, but activation of the CSP allows host cells to discriminate and mount qualitatively different immune responses to pathogens versus non-pathogens. In Aim 1, we propose to elucidate the mechanism by which M. tuberculosis gains access to the cytosol and activates the CSP, building upon our evidence that a single ESX-1 substrate, ESAT-6, functions to permeabilize the phagosomal membrane. We propose to identify the nature of the DNA that is recognized by the host, test the role of putative host receptors responsible for CSP activation, and probe the role of cytosolic signaling in M. tuberculosis infection. In Aim 2, we will examine how cytosolic access leads to targeting of M. tuberculosis to the autophagy pathway. In particular, we will test the role of ubiquitination and ubiquitin-binding adapters in targeting of autophagic vesicles to M. tuberculosis during infection. In the final Aim, We will collaborate extensively with the Portnoy (Project 1) and Vance (Project 3) groups to screen for modulators of M. tuberculosis growth and host innate responses by carrying out a screen in macrophages isolated from ENU mutagenized mice, and by performing an RNAi screen in macrophages.