Salmonella-host interaction is mediated by a novel Cdc42 effector and lipidations

项目摘要 由沙门氏菌引起的胃肠道疾病。鼠伤寒） 每年在全球范围内导致许多疾病、住院和死亡。S.鼠伤寒成功 通过注射调节宿主肌动蛋白细胞骨架网络的毒力效应子侵入宿主细胞， 宿主细胞机器如Rho GTP酶。Rho GTdR家族成员Cdc 42已被鉴定为一种 S的目标。鼠伤寒沙门氏菌;然而，其招募的具体机制及其在体内的功能， 沙门氏菌感染仍不清楚。初步研究表明Cdc 42效应蛋白1 （Cdc 42 EP 1）是从Cdc 42相互作用体中分离的一种新的结合蛋白，定位于沙门氏菌的进入位点， 上皮细胞此外，其与缺乏脂质修饰的Cdc 42变体的相互作用降低。 虽然S.已发现鼠伤寒沙门氏菌利用宿主脂化酶来修饰其自身的蛋白质， 目前尚不清楚Cdc 42的异戊二烯化和棕榈酰化修饰如何影响沙门氏菌的侵袭能力。 我们假设S.鼠伤寒通过Cdc 42 EP 1招募Cdc 42，并改变Cdc 42的脂质 修饰影响体内细菌侵入和疾病易感性。提出了两个目标， 确定沙门氏菌体外感染过程中Cdc 42 EP 1-Cdc 42机制的细胞功能和影响 和体内。该项目的具体目标是了解沙门氏菌是否利用Cdc 42 EP 1-Cdc 42 机器获得进入宿主细胞和生存，以及这种细胞机器如何影响病原体的 体内侵袭能力。 目的1将阐明S.鼠伤寒操纵和招募 Cdc 42和Cdc 42 EP 1的表达。Cdc 42 EP 1在沙门氏菌入侵过程中的作用及其空间和生物学特性 感染后与Cdc 42时间关系及其与脂质缺陷型Cdc 42突变体的相互作用 所有的感染都将被研究。 目标2将利用我们独特的基因工程Cdc 42小鼠模型，以确定是否 改变Cdc 42机制影响体内对沙门氏菌感染的易感性。cdc 42基因敲除小鼠将 用于确定Cdc 42的缺失如何影响沙门氏菌入侵。我们还将利用我们的新 开发了表达棕榈酰化Cdc 42的转基因小鼠，以了解这种修饰如何影响 细菌感染和疾病易感性。除了分析这些小鼠中的细菌入侵，我们 将使用酰基生物素交换试验确定沙门氏菌是否修饰宿主蛋白。 总之，这些目标将揭示沙门氏菌-Cdc 42相互作用的细胞生物学机制， 生理水平。

PROJECT SUMMARY Gastrointestinal diseases caused by Salmonella enterica serovar Tymphimurium (S. Typhimurium) lead to numerous illnesses, hospitalizations, and deaths globally every year. S. Typhimurium successfully invades host cells by injecting virulence effectors that modulate the host actin cytoskeletal network, through host cell machinery such as Rho GTPases. The Rho GTPase family member Cdc42 has been identified as a target of S. Typhimurium; however, specific mechanisms for its recruitment and its function in vivo during a Salmonella infection remain unclear. Preliminary studies demonstrate that Cdc42 Effector Protein 1 (Cdc42EP1), a novel binding protein isolated from Cdc42 interactome, localizes to Salmonella entry sites in epithelial cells. Additionally, its interaction with Cdc42 variants deficient of their lipid modifications is reduced. Although S. Typhimurium has been found to utilize host lipidation enzymes to modify its own proteins, it is unknown how the prenylation and palmitoylation modifications of Cdc42 impact Salmonella’s invasive ability. We hypothesize that S. Typhimurium recruits Cdc42 via Cdc42EP1, and that altering Cdc42’s lipid modifications impacts bacterial invasion and disease susceptibility in vivo. Two aims are proposed to determine the cellular function and impact of Cdc42EP1-Cdc42 machinery during Salmonella infection in vitro and in vivo. The specific goal of this project is to understand whether Salmonella exploits Cdc42EP1-Cdc42 machinery to gain entry and survival in host cells, and how this cellular machinery impacts the pathogen’s invasive ability in vivo. Aim 1 will address the molecular mechanisms by which S. Typhimurium manipulate and recruit both Cdc42 and Cdc42EP1 during invasion. The role of Cdc42EP1 during Salmonella invasion, its spatial and temporal relationship with Cdc42 upon infection, and its interaction with lipidation-deficient Cdc42 mutants during infection will all be studied. Aim 2 will utilize our unique genetically engineered Cdc42 mouse models to determine whether altering Cdc42 machinery affects the susceptibility to a Salmonella infection in vivo. Cdc42 knockout mice will be used to determine how deletion of Cdc42 impacts Salmonella invasion. We will also utilize our newly developed transgenic mice that express palmitoylable Cdc42 to understand how this modification may impact a bacterial infection and disease susceptibility. In addition to analysis of bacterial invasion in these mice, we will determine if Salmonella modify the host protein using an acyl biotin exchange assay. Together, these aims will reveal the mechanism of Salmonella-Cdc42 interaction at cell biology and physiology levels.