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许多病原体的关键疾病决定因素。在这个受保护的生态位中，微生物受到各种细胞杀伤机制以及细胞质先天免疫感知的保护。这个隔室的成熟是每个病原体寄生于宿主分泌途径的特定臂的副产物。嗜肺军团菌就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，这些蛋白被认为劫持了宿主细胞内质网（ER）在通往高尔基体的途中产生的分泌囊泡。然而，宿主细胞的地形与该模型不一致，因为细菌经常在远离囊泡运输到高尔基体的位置接触宿主细胞，这表明与外周内质网小管的通信是复制液泡构建的第一步。拟议的研究将验证内质网小管功能的操纵启动液泡形成的模型，并且存在备份策略以确保该隔室的有效构建。工作将集中在细菌控制宿主细胞网状蛋白4 （rtn4）异构体上，这是一组进化上保守的蛋白质，在物理上管化内质网。特别是，研究将分析三种嗜肺乳杆菌Sde家族成员的功能，这些蛋白被注射到宿主细胞中并靶向Rtn4。实验将测试Sde蛋白修饰Rtn4亚型作为复制液泡形成的第一步的模型，并且作为对这种修饰的响应，宿主蛋白聚集成抗洗涤剂结构。为了验证这一假设，我们将分析宿主细胞无法支持这种修饰，以及在修饰位点发生改变的Rtn4突变体，以确定它们对Rtn4动力学的控制是否存在缺陷。此外，将测试一个模型，即Sde蛋白引起Rtn4小种子库的结构变化，导致沿着内质网小管长度的构象变化的连锁反应，使人想起朊病毒的形成。为了解释在没有Rtn4功能的情况下发生的细胞内生长，将进行突变体寻找，以确定与Sde家族平行参与膜运输途径的嗜肺乳杆菌蛋白。在Sde家族缺失或Rtn4被强行从复制液泡中移除的情况下，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。在这样做的过程中，实验将旨在确定操纵内质网小管促进细胞内生长的策略中的薄弱环节。

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.