Control of Endoplasmic Reticulum Tubule Formation by Legionella pneumophila

嗜肺军团菌对内质网小管形成的控制

基本信息

批准号：
9277999
负责人：
Ralph R. Isberg
金额：
$ 59.19万
依托单位：
TUFTS UNIVERSITY BOSTON
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-06-01 至 2019-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9277999
关键词：
Aerosols Alveolar Macrophages Amoeba genus Back Bacteria Bacterial Model Bacterial Proteins Biological Assay Cell Compartmentation Cells Cessation of life Communication Defect Detergents Development Disease Distant Endoplasmic Reticulum Ensure Event Exposure to Eye Family Family member Genetic screening method Goals Golgi Apparatus Growth Immune Individual Injectable Legionella Legionella pneumophila Length Light Link Mammalian Cell Membrane Microbe Modeling Modification Molecular Molecular Conformation Monoubiquitination Mutate Mutation Parasites Pathway interactions Peripheral Play Post-Translational Protein Processing Prions Process Protein Isoforms Proteins RNA Interference RTN4 gene Reaction Recruitment Activity Regulation Resistance Role Route Secretory Cell Secretory Vesicles Seeds Set protein Site Source Streptococcus pneumoniae Structure System Testing Tubular formation Ubiquitination Vacuole Vesicle Water Work antimicrobial arm chromophore conformational alteration contaminated water experimental study killings macrophage member microbial microorganism monomer mutant pathogen premature prevent protein aggregate public health relevance response trafficking

项目摘要

DESCRIPTION (provided by applicant): Construction of membrane-bound replication vacuoles within host cells is a critical disease determinant in a wide swath of pathogens. Within this protected niche, microorganisms are protected from a variety of cellular killing mechanisms as well as cytoplasmic innate immune sensing. Maturation of this compartment is a byproduct of each pathogen parasitizing a specific arm of the host secretory pathway. Legionella pneumophila is one bacterium that uses this strategy, growing in a vacuole within macrophages during pneumonic disease. Construction of the vacuole is promoted by a group of Legionella proteins, which are thought to hijack secretory vesicles emanating from the host cell endoplasmic reticulum (ER) en route to the Golgi. The topography of the host cell, however, is inconsistent with this model, as bacteria often contact host cells at sites distant from vesicle trafficking to the Golgi, arguing that communication with peripheral ER tubules is the first step i replication vacuole construction. The proposed studies will test the model that manipulation of ER tubule function initiates vacuole formation, and that back-up strategies exist to ensure efficient construction of this compartment. Work will focus on bacterial control of host cell reticulon 4 (Rtn 4) isoforms, an evolutionarily conserved set of proteins that physically tubulate ER. In particular, studies will analyze the function of three L. pneumophila Sde family members, proteins that are injected into host cells and target Rtn4. Experiments will be performed to test the model that Sde proteins modify Rtn4 isoforms as a first step in replication vacuole formation and that, in response to this modification, the host protein aggregates into a detergent resistant structure. To pursue this hypothesis, host cells predicted to be unable to support this modification, and Rtn4 mutants altered in the modification site, will be analyzed to determine if they are defective for control of Rtn4 dynamics. In addition, a model will be tested that Sde proteins cause a structural change in a small seed pool of Rtn4 that results in a chain reaction of conformational changes along the length of the ER tubule, reminiscent of prion formation. To account for intracellular growth that occurs in the absence of Rtn4 function, a mutant hunt will be performed to identify L. pneumophila proteins that participate in membrane trafficking pathways in parallel to the Sde family. The consequence of loss of these proteins under conditions in which either the Sde family is missing, or when Rtn4 is forcibly removed from the replication vacuole will be evaluated by determining if the compartment decomposes or routes into an antimicrobial organell in the host cell. In so doing, experiments will be directed toward identifying a weak link in the strategy of manipulating ER tubules to promote intracellular growth, with an eye toward developing antimicrobials that target this process.

描述（由申请人提供）：宿主细胞内膜结合的复制液泡的构建是广泛的病原体中的关键疾病决定因素。在这个受保护的小众市场中，微生物受到各种细胞杀戮机制以及细胞质先天免疫感应的保护。该室的成熟是每种病原体的副产品，使宿主分泌途径的特定臂寄生。肺炎军团菌是一种使用该策略的细菌，在肺炎疾病期间在巨噬细胞中生长在液泡中。液泡的结构是由一组军团菌蛋白促进的，这些军团菌蛋白被认为是劫持从宿主细胞内质网（ER）散发出的分泌囊泡（ER）。然而，宿主细胞的地形与该模型不一致，因为细菌经常接触距离囊泡运输的部位与高尔基体接触宿主细胞，认为与外围ER小管的通信是I Replication acepration actrongution I的第一步。拟议的研究将测试该模型，即操纵ER小管功能会启动液泡形成，并存在备用策略以确保该隔室的有效构造。工作将重点放在宿主细胞网状4（RTN 4）同工型的细菌控制上，这是一组物理性ER的进化保守的蛋白质。特别是，研究将分析三种肺炎李斯特拉乳杆菌SDE家族成员的功能，这些蛋白质被注入宿主细胞并靶向RTN4。将进行实验，以测试SDE蛋白将RTN4同工型修改为复制液泡形成的第一步，并响应于这种修饰，宿主蛋白聚集成耐洗涤剂的结构。为了提出这一假设，将分析宿主细胞无法支持这种修饰，并且将在修饰位点发生变化的RTN4突变体，以确定它们是否有缺陷以控制RTN4动力学。此外，将测试一个模型，即SDE蛋白会在RTN4的小种子池中导致结构变化，从而导致沿着ER小管长度的构象变化的链反应，从而让人联想到prion虫的形成。为了说明在没有RTN4功能的情况下发生的细胞内生长，将进行突变的狩猎，以识别与SDE家族并行参与膜运输途径的肺炎乳杆菌蛋白。在SDE家族缺失或将RTN4从复制液泡中删除的条件下，这些蛋白质丢失的结果将通过确定该腔室分解或路由分解或路由到宿主细胞中的抗微生物细胞器中来评估。这样一来，实验将用于确定操纵ER小管以促进细胞内生长的策略中的薄弱环节，着眼于开发针对此过程的抗菌剂。