Molecular mechanisms supporting bacterial survival within immune cells

项目总结 革兰氏阴性菌的抗击难度越来越大，因为现有的抗生素很难达到 它们的细胞内靶点和面孔被外排泵消除。这个问题对细菌来说尤其紧迫。 从寄主的质膜上形成一个允许复制的液泡。由多个 传统的抗生素无法接触到细胞膜、细胞内的病原体。根本性的差距 坚持目前对细菌病原体如何颠覆宿主膜转运过程的理解 而这种差距的持续存在阻碍了我们对细菌病原体用来 协调毒力策略。我们的长期目标是通过系统地揭开主持人的面纱来解决这一差距 嗜肺军团菌感染人肺巨噬细胞的关键途径 一种被称为军团病的严重肺炎的病原体。军团菌感染肺部 巨噬细胞和通过建立和居住在膜结合的隔室中来抵抗降解 被称为含有军团菌的液泡。最初来自宿主细胞的质膜，这是 在感染过程中，液泡膜发生了显著的重塑。为了做到这一点，细菌立即开始 将大量的(效应物)蛋白质直接移位到宿主细胞质中。主体膜运输 网络是嗜肺性乳杆菌效应蛋白的主要靶点。特别是，小泡在 内质网和高尔基体早期被含军团菌的液泡隔离。 感染，而与可降解的溶酶体融合则被阻止。这些观察结果支持这项工作 病原体协调其与宿主的分子相互作用以刺激或抑制融合的模型 寄主囊泡和它的液泡。描述分泌效应器的时空分布是一个关键步骤。 了解嗜肺乳杆菌是如何与宿主细胞相互作用以确保自身生存的。整体而言 目的是研究嗜肺乳杆菌效应蛋白的时空定位。 人巨噬细胞感染和确定嗜肺乳杆菌效应物与宿主的相互作用 磷脂酰肌醇脂类靶向膜室。我们建议：(1)双管齐下 基于化学生物学直接追踪嗜肺乳杆菌效应物在感染者体内的定位 巨噬细胞，以及(2)鉴定嗜肺乳杆菌效应器之间的蛋白质-脂质界面 我们用X射线结晶学进行了初步筛选。这项拟议的研究具有重要意义，因为它 定位于促进我们对细菌病原体如何操纵宿主膜运输的理解 促进细菌细胞内生存的途径。一个重要的间接结果是，这些研究 可能为干预嗜肺性乳杆菌感染和相关疾病提供新的分子靶点。

PROJECT SUMMARY Gram-negative bacteria are increasingly challenging to combat because existing antibiotics struggle to reach their intracellular targets and face elimination by efflux pumps. This issue is particularly pressing for bacteria that establish a replication-permissive vacuole derived from the host's plasma membrane. Shielded by multiple layers of membranes, intracellular pathogens become inaccessible to traditional antibiotics. A fundamental gap persists in the current understanding of how bacterial pathogens subvert host membrane transport processes and continued existence of this gap impedes our understanding of mechanisms that bacterial pathogens use to coordinate virulence strategies. Our long-term goal is to address this gap by systematically unveiling the host pathways critical for infection of human lung macrophages by the bacterial pathogen Legionella pneumophila, the causative agent of a severe pneumonia known as Legionnaires' disease. Legionella infects lung macrophages and resists degradation by establishing and residing within a membrane-bound compartment known as the Legionella-containing vacuole. Initially derived from the host cell's plasma membrane, this vacuolar membrane is dramatically remodeled during infection. To do so, the bacterium immediately begins translocating a large number of (effector) proteins directly into the host cytosol. The host membrane trafficking network is a major target of L. pneumophila effector proteins. In particular, vesicles traveling between the endoplasmic reticulum and the Golgi are sequestered by the Legionella-containing vacuole early during infection, whereas fusion with degradative lysosomes is prevented. These observations support the working model that the pathogen orchestrates its molecular interactions with the host to stimulate or inhibit fusion of host vesicles with its vacuole. Delineating the spatiotemporal distribution of secreted effectors is a critical step to understanding how L. pneumophila interacts with the host cell to ensure its own survival. The overall objective is to examine the spatiotemporal localization of L. pneumophila effector proteins in the context of human macrophage infection and to determine how L. pneumophila effectors interact with host phosphoinositide lipids to target membrane compartments. We propose: (1) to use a dual pronged approach based on chemical biology to directly track localization of L. pneumophila effectors in infected human macrophages, and (2) to characterize the protein-lipid interface between L. pneumophila effectors identified in our preliminary screen using X-ray crystallography. The proposed research is significant because it is positioned to advance our understanding of how bacterial pathogens manipulate host membrane transport pathways to promote intracellular survival of bacteria. A significant collateral outcome is that these studies could suggest new molecular targets for intervention in L. pneumophila infections and related conditions.