Role of SipC in manipulating host signaling during Salmonella infection

肠道沙门氏菌是引起一系列人类疾病的革兰氏阴性肠道细菌属， 从肠胃炎到伤寒和败血症。沙门氏菌在全球范围内， 健康负担，以及治疗和预防疾病的新疗法的开发将需要详细的 了解沙门氏菌致病的分子机制。 沙门氏菌是通过被污染的食物和水传播的，而细菌侵入非吞噬细胞 肠上皮细胞是建立感染的关键步骤。沙门氏菌诱导它们自己的 通过注射操纵肌动蛋白动力学和宿主细胞的“效应”蛋白， 通过III型分泌系统（T3 SS）直接向宿主细胞中发出信号。多个效应器的协调 它们的靶点对于有效入侵至关重要，但其潜在机制还不清楚。 SipC是T3 SS的核心组成部分，也作为效应器发挥作用。本提案中概述的研究 测试SipC支架促进细菌入侵的信号复合物组装的假设 和细胞内存活。 该实验室先前的工作通过酵母双链体鉴定了十几种SipC相互作用蛋白， 杂交筛选，其中几种直接结合或调节肌动蛋白。从这个组中，我们已经证实细丝蛋白， FHOD 1和MAP 4K 4与HeLa细胞提取物中的SipC相关。在目的1中，它们在肌动蛋白重组中的功能 感染期间细菌的摄取将使用RNAi结合免疫荧光来表征 和细菌内化测定。此外，我们将探讨SipC在调节其定位中的作用 和活性的测定。 沙门氏菌感染诱导Akt激酶活化以促进细胞内细菌生长。我们有 发现SipC直接与Akt结合，并且SipC相互作用蛋白Exo 70是Akt的组成部分。 外囊复合物是沙门氏菌诱导的Akt活化所必需的。目标2描述了实验， 表征SipC和外囊复合物在调节Akt信号传导中的作用。SipC突变体缺乏 与Akt的结合将用于缺失/互补实验以确定SipC如何调节Akt的表达。 感染期间Akt的定位、激活和下游信号传导。共沉淀和 将使用免疫荧光测定来确定Exo 70的消耗是否影响Akt的募集或 其上游激酶与细菌侵入位点和/或Akt与上游激酶之间的复合物形成的结合。 总的来说，这些研究将确定SipC作为宿主细胞组装支架的新作用。 在沙门氏菌感染期间参与肌动蛋白重组和细菌信号传导的蛋白质。重要的是这些 结果可能对革兰氏阴性菌的发病机制有广泛的影响，并将作为一个 为今后细菌致病机理的研究奠定了基础。

Salmonellae enterica are a genus of Gram-negative enteric bacteria that cause a range of human diseases, from gastroenteritis to typhoid fever and septicemia. Salmonella spp. present a significant global health burden, and development of new therapies to treat and prevent disease will require a detailed understanding of the molecular mechanisms underlying Salmonella pathogenesis. Salmonella is spread through contaminated food and water, and bacterial invasion of non-phagocytic intestinal epithelial cells is a key step in the establishment of infection. Salmonella bacteria induce their own uptake and survive within host cells by injecting 'effector' proteins that manipulate actin dynamics and host signaling directly into the host cell through a type III secretion system (T3SS). Coordination of multiple effectors and their targets is essential for efficient invasion, but the underlying mechanisms are not well understood. SipC is a core component of the T3SS that also functions as an effector. The studies outlined in this proposal test the hypothesis that SipC scaffolds the assembly of signaling complexes that promote bacterial invasion and intracellular survival. Previous work in the lab identified more than dozen SipC-interacting proteins through a yeast-two- hybrid screen, several of which directly bind or modulate actin. From this group, we have confirmed that filamin, FHOD1, and MAP4K4 associate with SipC in HeLa cell extracts. In Aim 1, their function in actin reorganization and bacterial uptake during infection will be characterized using RNAi in combination with immunofluorescence and bacterial internalization assays. In addition, we will explore the role of SipC in regulating their localization and activity through depletion/rescue assays using proteins deficient in binding to SipC. Salmonella infection induces activation of Akt kinase to promote intracellular bacterial growth. We have discovered that SipC binds directly to Akt and that the SipC interacting protein Exo70, a component of the exocyst complex, is essential for Salmonella-induced activation of Akt. Aim 2 describes experiments to characterize the roles of SipC and the exocyst complex in regulating Akt signaling. SipC mutants deficient in binding to Akt will be used in deletion/complementation experiments to determine how SipC regulates the localization, activation, and downstream signaling of Akt during infection. Co-precipitation and immunofluorescence assays will be used to determine whether depletion of Exo70 affects recruitment of Akt or its upstream kinases to sites of bacterial invasion and/or complex formation between Akt and upstream kinases. Collectively, these studies will define a novel role for SipC as a scaffold for the assembly of host proteins involved in actin reorganization and bacterial signaling during Salmonella infection. Importantly, these results are likely to have broad implications for pathogenesis of Gram-negative bacteria and will serve as a foundation for future research on bacterial pathogenesis.