Control of Endoplasmic Reticulum Tubule Formation by Legionella pneumophila

描述（由申请人提供）：宿主细胞内膜结合复制液泡的构建是多种病原体的关键疾病决定因素。在这个受保护的生态位中，微生物受到保护，免受各种细胞杀伤机制以及细胞质先天免疫感应的影响。该区室的成熟是寄生于宿主分泌途径的特定臂的每种病原体的副产品。嗜肺军团菌是一种使用这种策略的细菌，在肺炎疾病期间在巨噬细胞内的液泡中生长。一组军团菌蛋白促进了液泡的构建，这些蛋白被认为会劫持从宿主细胞内质网（ER）发出的分泌小泡，并在通往高尔基体的途中。然而，宿主细胞的拓扑结构与该模型不一致，因为细菌经常在远离囊泡运输到高尔基体的位点接触宿主细胞，认为与外周内质网小管的通信是复制液泡构建的第一步。拟议的研究将测试操纵内质网小管功能启动液泡形成的模型，以及存在后备策略以确保该室的有效构建。工作重点是宿主细胞网状蛋白 4 (Rtn 4) 同工型的细菌控制，这是一组进化上保守的蛋白质，在物理上管状内质网。特别是，研究将分析三个嗜肺军团菌 Sde 家族成员的功能，这些成员是注射到宿主细胞中并靶向 Rtn4 的蛋白质。将进行实验来测试 Sde 蛋白修饰 Rtn4 同工型作为复制液泡形成的第一步的模型，并且响应于这种修饰，宿主蛋白聚集成去污剂抗性结构。为了实现这一假设，将分析预计无法支持这种修饰的宿主细胞以及在修饰位点发生改变的 Rtn4 突变体，以确定它们是否在控制 Rtn4 动力学方面存在缺陷。此外，还将测试一个模型，证明 Sde 蛋白会引起 Rtn4 小种子池的结构变化，从而导致沿 ER 小管长度的构象变化的连锁反应，让人想起朊病毒的形成。为了解释在缺乏 Rtn4 功能的情况下发生的细胞内生长，将进行突变体搜寻，以鉴定与 Sde 家族平行参与膜运输途径的嗜肺军团菌蛋白。在 Sde 家族缺失或 Rtn4 从复制液泡中强行去除的情况下，这些蛋白质丢失的后果将通过确定隔室是否分解或进入宿主细胞中的抗菌细胞器来评估。在此过程中，实验将旨在确定操纵 ER 小管促进细胞内生长策略中的薄弱环节， 着眼于开发针对这一过程的抗菌药物。

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.