Modulation of host Apoptotic pathways by Legionella pneumophila

描述(申请人提供)：我们的目标是了解细胞内病原体嗜肺军团菌如何操纵宿主细胞功能以促进其增殖，从而为设计治疗传染病的新治疗策略提供知识。嗜肺性乳杆菌的细胞内生长需要Dot/LCM蛋白分泌系统，该系统将细菌效应物转移到感染细胞中。这些效应器被认为调节宿主细胞的功能，以建立支持细菌复制的利基环境。我们的具体假设是，在感染过程中，Dot/LCM系统的底物SIDF积极地阻止受感染的细胞发生凋亡。这一假说基于四个观察结果：1)嗜肺乳杆菌感染的允许型巨噬细胞开始发生凋亡，但细胞并未发生广泛的凋亡；2)感染SIDF突变体的巨噬细胞比感染野生型细菌的巨噬细胞更容易发生凋亡；3)SIDF与两种促凋亡蛋白BNIP3和Bcl-Rambo相互作用；4)表达SIDF的细胞对这些促死亡蛋白或化学制剂诱导的凋亡具有抵抗力。我们的具体目标是：1.对SIDF突变体的感染表型进行彻底的分析。我们将研究(I)SIDF突变体是否在获取内质网标志物方面存在缺陷，(Ii)SIDF是否是嗜肺乳杆菌在其他类型宿主细胞内生长所必需的，以及(Iii)突变体感染的原代巨噬细胞的凋亡反应，(Iv)缺失BclRambo对细菌细胞内生长的影响。2.分析SIDF与促凋亡蛋白BNIP3的相互作用及其对嗜肺乳杆菌胞内生长的影响。我们将研究(I)SIDF结合对促凋亡蛋白BNI PS介导的细胞凋亡的影响，(Ii)BNI PS介导的细胞凋亡如何影响嗜肺乳杆菌的细胞内生长，以及(Iii)SIDF如何干扰BNIP3的活性的机制。3.鉴定嗜肺乳杆菌效应子(S)与SIDF协同调节宿主功能。我们将确定(I)与BNIP3相互作用的非SIDF效应器，(Ii)在SIDF缺失突变背景下细胞内生长所必需的蛋白质，以及(Iii)与SIDF特异性相互作用形成多组分效应器复合体的蛋白质。

DESCRIPTION (provided by applicant): Our goal is to understand how the intracellular pathogen Legionella pneumophila manipulates host cellular functions to facilitate its multiplication, thus to provide knowledge for designing novel therapeutic strategies for treating infectious diseases. Intracellular growth of L. pneumophila requires the Dot/lcm protein secretion system that translocates bacterial effectors into infected cells. These effectors are believed to modulate host cellular functions for the establishment of a niche that supports bacterial replication. Our specific hypothesis is that during infection, SidF, a substrate of the Dot/lcm system actively prevents infected cells from undergoing apoptosis. This hypothesis is based on four observations: 1) Apoptosis is initiated in permissive macrophages infected by L. pneumophila but the cells are not extensively apoptotic; 2) Macrophages infected by a sidF mutant are more apoptotic than those infected by wild-type bacterium; 3) SidF interacts with two pro-apoptotic proteins, BNIP3 and Bcl-rambo; 4) Cells expressing SidF are resistant to apoptosis induced by these pro-death proteins or chemical agents. Our specific aims are to: 1. Perform a thorough analysis of infection phenotypes of a sidF mutant. We will examine (i) whether the sidF mutant is defective in acquiring endoplasmic reticulum markers, (ii) whether sidF is required for intracellular growth of L. pneumophila in other types of host cells and (iii) apoptotic response of primary macrophages infected by the mutant, (iv) how depletion of Bcl-rambo affects bacterial intracellular growth. 2. Analyze the interactions between SidF and the pro-apoptotic protein BNIP3 and how such interactions affect L. pneumophila intracellular growth. We will examine (i) the effect of SidF binding on the pro- apoptotic protein BNI PS-mediated apoptosis, (ii) how BNI PS-mediated apoptosis affects intracellular growth of L. pneumophila and (iii) the mechanisms of how SidF interferes with the activity of BNIP3. 3. Identify L. pneumophila effector(s) that function synergistically with SidF in modulating host functions. We will identify (i) non-SidF effectors that interact with BNIP3, (ii) proteins necessary for intracellular growth in the sidF deletion mutant background and (iii) proteins that specifically interact with SidF to form a multi- component effector complex.