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

许多病原体通过建立膜结合的复制隔间在宿主细胞内生长 成长。这些隔室提供了重要的保护，不受宿主天然免疫细胞质的监视 并干扰将有机体贩运到可降解的隔间。隔间里的生长 是由微生物蛋白质驱动的，这些蛋白质促进了隔室的构建，保护了对先天免疫的保护 监视并对宿主细胞提供监管控制。病原体的这些不同的手臂是如何 人们对协调的理解很少。一种使用这种策略的细菌是嗜肺军团菌，它 肺炎期间在巨噬细胞内的空泡中生长。一组超过300种军团菌蛋白质 转移到宿主细胞中，控制细胞内生活方式的方方面面，包括形成 复制间隔室，阻止宿主细胞翻译，并阻止胞质识别 复制舱。生物体劫持宿主管状内质网(ER)是 复制间隔形成的最早步骤，这是由细菌Sde蛋白促进的事件。 拟议的研究将测试两个模型的SDE功能。首先，它将测试Sde蛋白的模型 通过促进内质网小管形成、复制间隔室的形成和免疫回避来控制 三步走。其次，本研究将检验东道主翻译启动的操作对翻译效果的影响 在协调复制、构建空泡和免疫回避中的作用。第一个模型提出 SDE脱泛素酶活性从复制间隔上的多聚泛素(PolyUb)中释放出游离泛素(Ub)。 这将作为ADPriBosyl转移酶和磷酸二酯酶结构域的池来促进Ub 小管ER蛋白Rtn4和宿主翻译起始因子(EIFS)的修饰。在最后一步中， 蛋白质家族用磷酸核糖掩蔽复制隔间，以防止宿主识别 自噬途径。该模型将通过操纵细胞中的PolyUb池进行测试，执行电子 显微镜观察在这一途径中错乱的突变体，并重建细胞中的管状ER重排- 自由系统。为了确定靶向eIF蛋白在这些过程中所起的作用，目标将是 使用提纯的成分进行验证，并确定受监管的启动步骤。此外，它将是 确定EIF蛋白是否被灭活以从粗略ER中移除核糖体并允许紧密对接 ER页到复制间隔，如果EIF操作使宿主翻译反应偏离 支持细菌复制。广泛的初步研究支持拟议的实验，提供了一个 为这项工作奠定了坚实的基础。在所有这些研究中，目标是找出 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.