Mechanisms of Innate Immune Evasion by Mycobacterium Tuberculosis

总结 本项目的目的是了解结核分枝杆菌（Mtb）的两种毒力因子CpsA 和Phthiocerol dimycerosate（PDIM），通过破坏经典和非经典免疫力 自噬结核分枝杆菌是结核病（TB）的病原体，结核病是全世界由细菌引起的死亡的主要原因。 感染结核分枝杆菌的主要细胞生态位是巨噬细胞和嗜中性粒细胞， 清除感染结核分枝杆菌如何在先天免疫反应中存活以建立感染尚不清楚。在 在前一个项目期间，我们发现输出蛋白CpsA对Mtb毒力至关重要。 我们发现CpsA抑制吞噬体募集NADPH氧化酶。NADPH氧化酶使 活性氧（ROS）是先天性免疫反应的重要介质。除了直接 抗微生物活性，ROS是称为LC 3相关吞噬作用的溶酶体运输途径所必需的 (LAP)，一种非经典的自噬形式。因此，通过抑制ROS，CpsA也抑制ROS。我们展示了 巨噬细胞和小鼠中，CpsA保护Mtb免受NADPH氧化酶和NADPH氧化酶的影响。有趣的是，CpsA在物理上 NDP 52和TAX 1BP 1相互作用，自噬衔接子以经典的自噬（xenophagy）形式起作用， 这表明CpsA也可能损害嗜异性。此外，我们发现Mtb毒力脂质PDIM也 抑制NADPH氧化酶。以前的研究提出了PDIM的一系列作用，并表明它可以保护 结核分枝杆菌从一个定义不清的先天杀伤机制。我们的数据表明，一种未被重视的毒力特性 PDIM的作用是阻断NADPH氧化酶和NADPH氧化酶。因此，我们假设CpsA抑制了异嗜性， 与PDIM协同作用以抑制NADPH氧化酶和NADPH。此外，我们建议， 结核分枝杆菌的感染依赖于它逃避这些在骨髓细胞中被招募到肺部的先天防御的能力 在最初的感染。在这里，我们将定义CpsA如何抑制NADPH氧化酶，并调查它是否也 通过阻断NDP 52和TAX 1BP 1功能来削弱异嗜性。我们将确定PDIM是否也会损害 招募NADPH氧化酶的分枝杆菌吞噬体和评估的贡献，PDIM发挥 在体内破坏NADPH氧化酶和NADPH氧化酶。为了确定CpsA是否促进了 感染的建立，我们将采用超低剂量感染小鼠模型。使用条件敲除 (cKO)小鼠，我们将确定在哪些细胞CpsA功能，以抑制NADPH氧化酶和NADPH在急性 慢性感染。我们的研究结果将为结核分枝杆菌的两个关键毒力因子 合作破坏豁免权我们的研究将揭示杆菌的细胞类型特异性毒力策略， 描述参与体内细胞增殖的细胞类型，并定义控制细胞增殖的宿主-病原体相互作用。 建立感染。我们的研究将为免疫逃避提供详细的机制见解， 世界上最可怕的病原体之一的战略。通过阐明两个关键的作用机制 我们将推进预防和治疗结核病的创新方法。

SUMMARY The goal of this project is to understand how two virulence factors from Mycobacterium tuberculosis (Mtb), CpsA and phthiocerol dimycocerosate (PDIM), impair immunity by undermining both classical and non-classical autophagy. Mtb is the causative agent of tuberculosis (TB), the leading cause of death worldwide from a bacterial infection. The main cellular niche for Mtb is macrophages and neutrophils, the very immune cells that are meant to clear infection. How Mtb survives the innate immune response to establish infection is not well understood. In the previous project period, we discovered that an exported protein, CpsA, is critically important for Mtb virulence. We showed that CpsA inhibits phagosomal recruitment of the NADPH oxidase. The NADPH oxidase makes reactive oxygen species (ROS), an important mediator of the innate immune response. In addition to its direct antimicrobial activity, ROS is required for a lysosomal trafficking pathway called LC3-associated phagocytosis (LAP), a non-classical form of autophagy. Thus, by inhibiting ROS, CpsA also inhibits LAP. We showed both in macrophages and mice that CpsA protects Mtb from the NADPH oxidase and LAP. Interestingly, CpsA physically interacts NDP52 and TAX1BP1, autophagy adaptors that function in a form of classical autophagy (xenophagy), suggesting that CpsA may also impair xenophagy. Moreover, we found that the Mtb virulence lipid, PDIM, also inhibits the NADPH oxidase. Previous studies proposed an array of roles for PDIM and showed that it protects Mtb from a poorly defined innate killing mechanism. Our data suggest that an unappreciated virulence property of PDIM is blocking the NADPH oxidase and LAP. Thus, we hypothesize that CpsA inhibits xenophagy and works in concert with PDIM to inhibit the NADPH oxidase and LAP. Further, we propose that the infectious dose of Mtb depends upon its ability to evade these innate defenses in myeloid cells that are recruited to the lungs during initial infection. Here, we will define how CpsA inhibits the NADPH oxidase and investigate whether it also impairs xenophagy by blocking NDP52 and TAX1BP1 function. We will determine whether PDIM also impairs recruitment of the NADPH oxidase to mycobacterial phagosomes and evaluate the contribution that PDIM plays towards subverting the NADPH oxidase and LAP in vivo. To determine whether CpsA promotes the establishment of infection, we will use an ultra-low dose infection model in mice. Using conditional knockout (cKO) mice, we will determine in which cells CpsA functions to inhibit the NADPH oxidase and LAP during acute and chronic infection. Our findings will provide mechanistic insight into how two key virulence factors in Mtb collaborate to undermine immunity. Our studies will reveal a cell type-specific virulence strategy of the bacilli, delineate the cell types that participate in LAP in vivo, and define host-pathogen interactions that govern the establishment of infection. Our studies will provide detailed mechanistic insight into the immune evasion strategies of one of the world's most formidable pathogens. By elucidating the mechanism of action of two crucial virulence factors in Mtb, we will advance innovative approaches to prevent and treat TB.