Molecular Mechanisms of Host Function Exploitation by Type IV effectors of Legion

描述（由申请方提供）：嗜肺军团菌是潜在致死性军团病的病原体，是一种兼性细胞内病原体。细胞内复制L.噬菌体中的嗜肺菌完全依赖于Dot/Icm IV型分泌系统，其将数百种毒力因子（效应子）易位到宿主细胞中。这些效应子直接调节宿主细胞功能，以产生支持细菌在敌对吞噬细胞内生长的容许小生境。缺乏单个效应基因的突变体很少在细菌的细胞内生长中显示出显著缺陷，使得很难完全理解它们在感染中的作用。与Dot/Icm底物SidJ相关的几个特征使其成为解剖对L.嗜肺菌细胞内复制。首先，一个cDNAJ缺失突变体是有缺陷的， 在小鼠骨髓来源的巨噬细胞和阿米巴宿主盘基网柄藻的细胞内生长中。此外，收购内质网蛋白，如钙连接蛋白的空泡窝藏的ESTJ缺失突变体被延迟。第二，SidJ在过表达条件下对酵母的毒性是由于钙调素的滴定。在一项旨在分析Dot/Icm底物对真核细胞的影响的独立研究中，我们发现SdeA强烈抑制酵母生长和哺乳动物细胞中的分泌途径。此外，我们发现，参与内质网和顺式高尔基体之间的膜运输的几种酵母蛋白可以抑制SdeA的毒性。更重要的是，在筛选中寻找L。pneumophila的蛋白，可以调节SdeA的活性，我们确定SidJ作为一个强大的抑制剂的毒性SdeA酵母和哺乳动物分泌的抑制作用。因此，我们假设SdeA调节内质网（ER）和高尔基体之间的膜运输和SidJ调节这种调制。我们将通过以下三个具体目标来验证这一假设：目标1：确定SdeA的细胞靶点和生化功能。工作假设是SdeA靶向一个或多个参与内质网和顺式高尔基体区室之间的膜转运的宿主蛋白。我们将确定SdeA靶向的宿主蛋白以及SdeA是否以及如何对宿主蛋白进行生物化学修饰。我们还将确定这种修饰如何改变宿主蛋白质的功能。目的2：分析SidJ对SdeA活性的调控。工作的假设是，SidJ的功能，以保障SdeA的影响，通过时间和/或空间调节其活动。我们将确定SidJ的生化活性和这种活性在调节SdeA功能中的作用。目的3：评估SdeA（和SidE家族成员）和其他参与ER-高尔基体转运的Dot/Icm底物之间的关系。工作假设是SdeA/SidJ与参与调节宿主膜运输的其他Dot/Icm底物一起发挥作用，以将膜材料的运输转移到细菌吞噬体。我们将首先获得一组参与ER-高尔基体转运的Dot/Icm底物，通过它们的能力， 抑制哺乳动物细胞的分泌，然后构建缺乏这些基因的突变体，以评估它们在细胞内细菌复制中的作用。这些研究将为研究细菌β-烯因子对宿主囊泡运输的调节奠定基础，并将在军团菌感染的治疗和预防方面具有翻译意义。

DESCRIPTION (provided by applicant): Legionella pneumophila, the etiological agent for the potentially fatal Legionnaires' disease, is a facultative intracellular pathogen. Intracellular replication of L. pneumophila in phagocyte is entirely dependent upon the Dot/Icm type IV secretion system, which translocates hundreds of virulence factors (effectors) into host cells. These effectors directly modulate host cellular functions to create a permissive niche that supports bacterial growth within hostile phagocytic cells. Mutants lacking individual effector genes rarely display significant defect in intracellular growth of the bacterium, making it difficut to obtain a complete understanding of their role in infection. Several features associated with the Dot/Icm substrate SidJ make it a very useful lead to dissect specific bacterial activity important for L. pneumophila intracellular replication. First, a sidJ deletion mutant was defective in intracellular growth in both mouse bone marrow-derived macrophages and the amoebae host Dictyostelium discoideum. Further, acquisition of endoplasmic reticulum proteins such as calnexin by vacuoles harboring the sidJ deletion mutant was delayed. Second, the toxicity of SidJ to yeast under overexpression condition was due to the titration of calmodulin. In an independent line of study aiming at analyzing the impact of Dot/Icm substrates on eukaryotic cells, we found that SdeA strongly inhibits yeast growth and the secretory pathway in mammalian cell. Further, we found that several yeast proteins involved in membrane transport between the endoplasmic reticulum and the cis-Golgi apparatus can suppress the toxicity of SdeA. More importantly, in a screening to search for L. pneumophila protein that may regulate SdeA activity, we identified SidJ as a strong suppressor of the toxicity of SdeA to yeast and of its inhibitory effect on mammalian secretion. Thus, we hypothesized that SdeA modulates membrane transport between the endoplasmic reticulum (ER) and the Golgi apparatus and that SidJ regulates such modulation. We will test this hypothesis by pursuing three specific aims: Aim 1: Determine the cellular target and the biochemical function of SdeA. The working hypothesis is that SdeA targets one or more host proteins involved in membrane transport between the endoplasmic reticulum and the cis-Golgi compartment. We will determine the host protein targeted by SdeA and whether and how SdeA biochemically modifies the host protein. We will also determine how such modification alters the function of the host protein. Aim 2: Analyze the regulation of SdeA activity by SidJ. The working hypothesis is that SidJ functions to safeguard the effects of SdeA by temporally and/or spatially regulating its activity. We will determine the biochemical activity of SidJ and the role of such activity in the regulation of SdeA function. Aim 3: Assess the relationship between SdeA (and members of the SidE family) and other Dot/Icm substrates involved in ER-Golgi transport. The working hypothesis is that SdeA/SidJ function in concert with other Dot/Icm substrates involved in modulating host membrane trafficking to divert the transport of membrane materials to the bacterial phagosome. We will first obtain a set of Dot/Icm substrates involved in ER-Golgi transport by their ability to inhibit secretion by mammalian cells, followed by the construction of mutants lacking these genes for evaluation of their roles in intracellular bacterial replication. These studies will lay he foundation for the investigation in the modulation of host vesicle trafficking by bacterial virulene factors and will have translational implications in the context of treatment and prevention of Legionella infections.