The interface between L. pneumophila manipulation of host endoplasmic reticulum and innate immune subterfuge

许多病原体在宿主细胞内生长，通过构建膜结合的复制区室， 增长隔室提供了重要的保护，免受宿主先天免疫细胞质监视 系统和干扰贩运有机体进入降解车厢。隔室生长 是由微生物蛋白驱动的，这些蛋白促进隔室构建， 监视并提供对宿主细胞的调节控制。病原体的这些不同分支 协调是很难理解的。使用这种策略的细菌是嗜肺军团菌， 在肺炎时生长在巨噬细胞内的空泡中。超过300种军团菌蛋白 被转移到宿主细胞中，控制细胞内生活方式的所有方面，包括 复制区室，阻断宿主细胞翻译，并阻止细胞质识别的复制区室， 复制区室该生物体劫持宿主的管状内质网（ER）作为其中一个 复制区室形成的最早步骤，由细菌Sde蛋白促进的事件。 拟议研究将测试两种模型的Sde功能。首先，它将测试Sde蛋白质 控制内质网小管的形成，复制区室的形成，并通过促进免疫回避， 三步路其次，本文将检验宿主翻译启动的操纵对宿主翻译的影响模式， 在协调复制空泡构建和免疫回避中的作用。第一种模式提出， Sde去泛素化酶活性从复制区室上的polyUb释放游离的泛素（Ub）。 这将作为AD核糖基转移酶和磷酸二酯酶结构域的库，以促进Ub 管状ER蛋白Rtn 4和宿主翻译起始因子（eIF）的修饰。在最后一步中， 蛋白质家族用磷酸核糖掩蔽复制区室以防止被宿主识别 自噬途径该模型将通过操纵细胞中的polyUb池，进行电子扫描， 显微镜对在这一途径中紊乱的突变体，并重建细胞中的管状ER重排- 自由系统为了确定靶向eIF蛋白在这些过程中的作用，将靶向 使用纯化组分进行验证，并确定受管制的起始步骤。此外还 确定eIF蛋白是否失活以从粗糙ER去除核糖体并允许eIF蛋白与粗糙ER紧密对接。 如果eIF操作使宿主翻译反应偏向于复制区， 有利于细菌复制。广泛的初步研究支持拟议的实验，提供了一个 为这项工作打下坚实的基础。在这些研究中，目的是确定一个薄弱环节， 操纵内质网小管以促进细胞内生长的微生物策略，着眼于 开发针对这一过程的抗菌剂。

Many pathogens grow within host cells by building membrane-bound replication compartments essential for growth. The compartments provide important protection from host innate immune cytoplasmic surveillance systems and interfere with trafficking organisms into degradative compartments. Growth in the compartment is driven by microbial proteins which promote compartment construction, protection against innate immune surveillance and provide regulatory control over the host cell. How these different arms of the pathogen are coordinated is poorly understood. A bacterium that uses this strategy is Legionella pneumophila, which grows in a vacuole within macrophages during pneumonic disease. A group of over 300 Legionella proteins are translocated into host cells, controlling all aspects of the intracellular lifestyle, including formation of the replication compartment, blocking host cell translation, and preventing cytosolic recognition of the replication compartment. The organism hijacks host tubular endoplasmic reticulum (ER) as one of the earliest steps in replication compartment formation, an event promote by the bacterial Sde proteins. The proposed studies will test two models for Sde function. First, it will test the model that the Sde proteins controls ER tubule formation, replication compartment formation, and immune avoidance by promoting a three-step pathway. Secondly, it will test the model that manipulation of host translation initiation plays a role in coordinating replication vacuole construction and immune avoidance. The first model proposes that the Sde deubiquitinase activity liberates free ubiquitin (Ub) from the polyUb on the replication compartment. This would serve as a pool for ADPribosyltransferase and phosphodiesterase domains to promote Ub modification of the tubular ER protein Rtn4 and host translation initiation factors (eIFs). In the final step, the protein family masks the replication compartment with a phosphoribose to prevent recognition by the host autophagy pathway. The model will be tested by manipulating polyUb pools in the cell, performing electron microscopy on mutants deranged in this pathway, and reconstructing tubular ER rearrangements in a cell- free system. To determine the role that targeting of eIF proteins plays in these processes, the targets will be verified using purified components and the regulated initiation step will be identified. In addition, it will be determined if eIF proteins are inactivated to remove ribosomes from rough ER and allow close docking of ER sheets to the replication compartment, and if eIF manipulation skews the host translational response to favor bacterial replication. Extensive preliminary studies support the proposed experiments, providing a solid underpinning for this work. Throughout these studies, the objective is to identify a weak link in the microbial strategy of manipulating ER tubules to promote intracellular growth, with an eye toward developing antimicrobials that target this process.