Investigating the Role of the GTPase Arl8b in Plasma Membrane Repair of Mtb-Infected Macrophages

 描述(申请人提供)：结核分枝杆菌(Mtb)是一种非常成功的病原体，每年感染860万人，导致130万人死亡。结核分枝杆菌的主要宿主细胞是肺巨噬细胞(M)。在某些情况下，内化的结核分枝杆菌如何保持隔离并最终被杀死，在另一些情况下，如何持续并逃脱免疫系统的识别，目前尚不清楚。我们已经证明，膜修复机制在决定感染结果方面是极其重要的。在Mφ感染M后，膜修复维持Mφ质膜的完整性，这是M Mtb感染M DNA后细胞凋亡的必要步骤，从而杀死病原体。相比之下，毒力结核分枝杆菌会阻止膜修复，而是主动导致坏死，支持感染的传播和逃避免疫控制。在机械上，无毒和强毒结核分枝杆菌均可引起宿主M质膜的微破裂。这些质膜损伤在感染了无毒结核分枝杆菌的M-中被修复，但在强毒结核分枝杆菌的情况下，由于杆菌抑制质膜修复而导致坏死。本项目的目标是描述结核分枝杆菌感染的M-的膜修复，这将导致对保护性宿主反应机制和毒力结核杆菌逃避宿主遏制的关键步骤的进一步了解。膜修复机制的主要组成部分是Ar18b，它是一种小的GTP酶，参与溶菌酶的运动和运输。在目标1中，我们将使用细胞系模型(HeLa细胞)来(A)确定Ar18b的作用，以及(B)确定其他相互作用的效应分子在质膜修复中的作用。在目标2中，我们将测试Ar18b和验证的效应器是否是Mtb感染的M质膜修复所必需的，以及强毒Mtb是否颠覆了它们的功能。所描述的研究针对的是提高宿主抗分枝杆菌反应的治疗干预措施(宿主导向的结核病治疗)。

 DESCRIPTION (provided by applicant): Mycobacterium tuberculosis (Mtb) is an extraordinarily successful pathogen that infects 8.6 million people yearly and causes 1.3 million deaths. The principal host cell of Mtb is the lung macrophage (M). It is not well understood how in some cases the internalized Mtb remain sequestered and are ultimately killed and in other cases persist and escape recognition by the immune system. We have shown that membrane repair mechanisms are extremely important in determining the outcome of infection. Membrane repair maintains the integrity of the Mφ plasma membrane after infection of M with avirulent Mtb, a requisite step for apoptosis of infected Mφ, which kills the pathogen. Virulent Mtb, in contrast, block membrane repair and instead actively induce necrosis, supporting spread of infection and escape from immune control. Mechanistically both avirulent and virulent Mtb induce microdisruptions of the host M plasma membrane. These plasma membrane lesions are repaired in M infected with avirulent Mtb, but in the case of virulent Mtb, necrosis ensues because the bacillus inhibits plasma membrane repair. The goal of this project is the delineation of membrane repair in Mtb-infected M, which will lead to increased understanding of the protective host response mechanism and of the critical steps, by which virulent Mtb escape host containment. A major component of the membrane repair mechanism is Arl8b, a small GTPase involved in lysozyme motility and trafficking. In Aim 1 we will use a cell line model (HeLa cells) to (a) determine the role of Arl8b and (b) identify additional interacting effector molecules in plasma membrane repair. In Aim 2 we will test whether Arl8b and the validated effectors are required for plasma membrane repair of Mtb-infected M and whether virulent Mtb subverts their function. The described studies are directed towards therapeutic interventions to enhance the host anti-mycobacterial response (host-directed tuberculosis therapy).