Molecular and Cellular Dissection of Campylobacter Jejuni Effector Protein Function

标题：空肠弯曲菌效应蛋白对宿主细胞功能的调节 项目摘要 空肠弯曲菌感染是每年数百万例腹泻的原因， 格林-巴利综合征是后脊髓灰质炎时代导致弛缓性麻痹的主要原因。尽管研究人员 20多年来一直推测C.空肠介导的肠炎依赖于细胞的侵入 病原体和宿主会出现强烈的炎症反应 有助于C的反应。空肠介导的肠炎是不明确的。我的实验室发现这种病原体- 诱导事件（细胞侵入）需要从C.空肠至宿主细胞的胞质溶胶。我们 已经将这些分泌的蛋白质命名为弯曲杆菌侵袭抗原（Cia）。Cia蛋白协同 焦点复合物（细胞-基质粘附结构）的组分和MEK/ERK信号通路， 触发膜皱褶（片状伪足和丝状伪足延伸-由肌动蛋白支持的细胞突起 细丝）。我们的总体目标是确定C. jejuni利用来破坏 焦点复合物（FC）通过细胞骨架重组促进细胞侵袭。从上下文来看，这些研究 将深入了解微生物如何调节细胞信号通路，以促进病原体的生存， 传播。 我们已经证明CiaC和CiaD效应子是C.空肠细胞浸润。我们的工作 一种假设是，这两种效应物含有指导弯曲杆菌入侵形成的结构域 复杂.更具体地说，我们假设CiaC和CiaD增选FC和FC的组分- 相关蛋白质，使细胞通路的串扰（收敛）和触发细胞骨架 重组和趋化因子产生（IL-8）。CiaC和CiaD的精确机制 用于调节细胞骨架重排和细菌摄取是未知的。因此，我们的目标是阐明 CiaC和CiaD效应子如何驱动C。空泡细胞侵袭和趋化因子诱导对于 了解C.空肠介导的肠炎，是一个先决条件， 开发有针对性的方法来预防和治疗C.空肠疾病。 该提案的具体目标是： 1)建立CiaC和CiaD的宿主细胞靶标; 2)鉴定以CiaC-和CiaD-依赖性方式磷酸化的宿主细胞蛋白质，并确定CiaC-和CiaD-依赖性方式磷酸化的宿主细胞蛋白质。 这些磷酸化事件在调节宿主细胞信号传导途径中的作用;以及 3)测试C.空肠CiaC和CiaD效应子是仔猪疾病所必需的，并确定 任何一种蛋白质的缺乏都会导致独特的疾病表型。

Title: Modulation of Host Cell Function by Campylobacter jejuni Effector Proteins Project Summary Infection with Campylobacter jejuni is responsible for millions of cases of diarrhea per year and is associated with Guillain-Barré syndrome, the leading cause of flaccid paralysis in the post-polio era. Although researchers have surmised for more than two decades that C. jejuni-mediated enteritis is dependent on invasion of the cells lining the gastrointestinal tract and is accompanied by a robust inflammatory response, the pathogen and host responses that contribute to C. jejuni-mediated enteritis are ill-defined. My lab discovered that this pathogen- induced event (cell invasion) requires the delivery of proteins from C. jejuni to the cytosol of host cells. We have designated these secreted proteins the Campylobacter invasion antigens (Cia). The Cia proteins co-opt components of the focal complex (a cell-matrix adhesion structure) and the MEK/ERK signaling pathway to trigger membrane ruffling (lamellipodia and filopodia extensions – cellular protrusions supported by actin filaments). Our overall goal is to identify the mechanism(s) that C. jejuni utilizes to subvert components of the focal complex (FC) to promote cell invasion through cytoskeletal reorganization. Contextually, these studies will provide insight into how microbes modulate cellular signaling pathways to favor pathogen survival and dissemination. We have shown that the CiaC and CiaD effectors are necessary for C. jejuni cellular invasion. Our working hypothesis is that these two effectors contain domains that direct the formation of the Campylobacter invasion complex. More specifically, we hypothesize that CiaC and CiaD co-opt components of the FC and FC- associated proteins that enable the cross-talk (convergence) of cellular pathways and trigger cytoskeletal reorganization and chemokine production (IL-8) in host cells. The precise mechanism(s) that CiaC and CiaD use to modulate cytoskeletal rearrangement and bacterial uptake are not known. Thus, our goal of elucidating how the CiaC and CiaD effectors drive C. jejuni-cell invasion and chemokine induction is essential for understanding the molecular and cellular basis of C. jejuni-mediated enteritis and is a prerequisite for the development of targeted methods to prevent and treat C. jejuni disease. The Specific Aims of this proposal are to: 1) Establish the host cell targets of both CiaC and CiaD; 2) Identify the host cell proteins phosphorylated in a CiaC- and CiaD-dependent manner and determine the role of these phosphorylation events in modulating host cell signaling pathways; and 3) Test whether the C. jejuni CiaC and CiaD effectors are required for disease in piglets and determine if deficiency of either protein results in a unique disease phenotype.