Control of Endoplasmic Reticulum Tubule Formation by Legionella pneumophila

描述（由申请人提供）：在宿主细胞内构建膜结合复制空泡是广泛病原体中的关键疾病决定因素。在这种受保护的生态位内，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。该隔室的成熟是每种病原体寄生于宿主分泌途径的特定臂的副产品。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空泡中生长。一组军团菌蛋白质促进液泡的构建，它们被认为劫持了从宿主细胞内质网（ER）到高尔基体的分泌囊泡。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至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。拟议的研究将测试模型，即操纵内质网小管功能启动空泡形成，并存在备份策略，以确保有效地构建这个隔间。工作将集中在宿主细胞reticulon 4（RTN 4）异构体的细菌控制上，RTN 4是一组进化上保守的蛋白质，物理上使ER微管化。特别是，研究将分析三个L的功能。嗜肺菌Sde家族成员，注射到宿主细胞中并靶向Rtn4的蛋白质。将进行实验以测试Sde蛋白修饰Rtn4同种型作为复制液泡形成的第一步的模型，并且响应于该修饰，宿主蛋白聚集成耐洗涤剂结构。为了实现这一假设，将分析预测不能支持这种修饰的宿主细胞和修饰位点改变的Rtn4突变体，以确定它们是否对Rtn4动力学的控制有缺陷。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ER小管沿着长度的构象变化的连锁反应，使人联想到朊病毒形成。为了解释在没有Rtn4功能的情况下发生的细胞内生长，将进行突变体搜寻以鉴定L.与Sde家族平行参与膜运输途径的嗜肺菌蛋白。在Sde家族缺失或Rtn4被强制从复制液泡中去除的条件下，这些蛋白质丢失的后果将通过确定隔室是否分解或路由到宿主细胞中的抗菌细胞器中来评估。在这样做的过程中，实验将被导向识别操纵内质网小管以促进细胞内生长的策略中的薄弱环节， 着眼于开发针对这一过程的抗菌剂。

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.