Modulation of host Apoptotic pathways by Legionella pneumophila

描述（由申请人提供）：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，从而为设计治疗感染性疾病的新治疗策略提供知识。胞内生长。嗜肺菌需要Dot/lcm蛋白分泌系统，该系统将细菌效应物易位到感染的细胞中。这些效应物被认为调节宿主细胞功能以建立支持细菌复制的小生境。我们的具体假设是，在感染过程中，SidF，Dot/lcm系统的底物，积极防止感染的细胞发生凋亡。这一假说是基于以下四个方面的观察：（1）受L.在一些实施方案中，所述细胞是嗜肺菌，但细胞不广泛地凋亡; 2）被SidF突变体感染的巨噬细胞比被野生型细菌感染的巨噬细胞更凋亡; 3）SidF与两种促凋亡蛋白BNIP 3和Bcl-rambo相互作用; 4）表达SidF的细胞对由这些促死亡蛋白或化学试剂诱导的凋亡具有抗性。我们的具体目标是：1.对一个BMPF突变体的感染表型进行彻底的分析。我们将研究（i）BNF突变体是否在获得内质网标记方面有缺陷，（ii）BNF是否是L. pneumophila在其他类型的宿主细胞和（iii）由突变体感染的原代巨噬细胞的凋亡反应，（iv）如何耗尽Bcl-rambo影响细菌细胞内生长。2.分析SidF和促凋亡蛋白BNIP 3之间的相互作用以及这种相互作用如何影响L。嗜肺菌细胞内生长。我们将研究（i）SidF结合对促凋亡蛋白BNI PS介导的凋亡的影响，（ii）BNI PS介导的凋亡如何影响L. pneumophila和（iii）SidF如何干扰BNIP 3活性的机制。3.识别L.在调节宿主功能中与SidF协同作用的嗜肺菌效应子。我们将鉴定（i）与BNIP 3相互作用的非SidF效应物，（ii）在BNIP 3缺失突变体背景中细胞内生长所必需的蛋白质，和（iii）与SidF特异性相互作用以形成多组分效应物复合物的蛋白质。

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.