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

描述(由申请人提供)：嗜肺军团菌是一种兼性的细胞内病原体，是一种可能致命的退伍军人病的病原体。嗜肺乳杆菌在吞噬细胞中的细胞内复制完全依赖于Dot/ICM IV型分泌系统，该系统将数百个毒力因子(效应物)转移到宿主细胞中。这些效应器直接调节宿主细胞的功能，以创造一个允许的利基环境，支持细菌在敌意吞噬细胞内的生长。缺乏单个效应基因的突变体很少在细菌的细胞内生长中表现出明显的缺陷，这使得人们很难完全了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分析嗜肺乳杆菌细胞内复制重要的特定细菌活性的非常有用的线索。首先，一个sidJ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线虫中的细胞内生长。此外，含有SIDJ缺失突变体的液泡对内质网蛋白如Calnexin的获取被推迟。第二，在过量表达条件下，SidJ对酵母的毒性是由于钙调素的滴定所致。在一项旨在分析Dot/ICM底物对真核细胞影响的独立研究中，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膜运输的几种酵母蛋白可以抑制SdeA的毒性。更重要的是，在寻找可能调节SdeA活性的嗜肺乳杆菌蛋白时，我们发现SidJ是SdeA对酵母的毒性及其对哺乳动物分泌的抑制作用的强有力的抑制因子。因此，我们假设SdeA调节内质网(ER)和高尔基体之间的膜运输，而SidJ调节这种调节。我们将通过追求三个具体目标来验证这一假说：目标1：确定SdeA的细胞靶点和生化功能。工作假说是，SdeA以一种或多种参与内质网和顺式高尔基体之间膜运输的宿主蛋白为靶标。我们将确定SdeA针对的宿主蛋白，以及SdeA是否以及如何对宿主蛋白进行生化修饰。我们还将确定这种修饰如何改变宿主蛋白的功能。目的2：分析四逆汤对SdeA活性的调节作用。工作假说是，SidJ通过在时间和/或空间上调节SdeA的活动来保护SdeA的作用。我们将确定SidJ的生化活性以及这种活性在SdeA功能调节中的作用。目的3：评估SdeA(和Side家族成员)与参与内质网高尔基体转运的其他Dot/ICM底物之间的关系。工作假说是SdeA/SidJ与其他参与调节宿主膜转运的Dot/ICM底物协同作用，将膜物质的运输转移到细菌吞噬小体。我们将首先获得一组参与内质网高尔基体转运的Dot/ICM底物 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毒力因子对宿主囊泡运输的调控奠定基础，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.