大肠埃希菌VI型分泌系统的Hcp蛋白受体鉴定及信号通路研究

Escherichia coli is the leading cause of bacterial neonatal meningitis. Our previous study demonstrated that the neonatal meningitis E. coli model strain RS218 contained a new identified type VI secretion system (T6SS). The Hcp family proteins, including Hcp1 and Hcp2 proteins are the important effector and component protein of T6SS, respectively. Hcp1 protein as an effector can induce morphology changes, cytoskeleton rearrangement, cytokines release and caspase-8 involved apoptosis in human brain microvascular endothelial cells (HBMEC), and Hcp2 protein can increase the association with and invasion of HBMEC for E. coli strain RS218. Thus the hcp family proteins coordinately regulate the E. coli interaction with HBMEC and the pathogenesis of E. coli neonatal meningitis. However, the Hcp family proteins were not translocated into HBMEC by E. coli strain RS218 demonstrated by our recent translocation assay and the Hcp1 protein over-expressing strain inhibited the binding and invasion of HBMEC. So, a Hcp1 and/or Hcp2 specific receptor on HBMEC and some signaling moleculars were involved in the pathogenesis of meningitis-causing E. coli. In this project, we first intent to identify the specific receptor of Hcp family proteins on HBMEC through HBMEC cDNA library construction, yeast hybrid system and confocal microscopy methods. Then using real-time PCR, screening the signaling moleculars expressing changes in HBMEC for the cytotoxic of Hcp family proteins, including the signaling moleculars associated cytoskeleton rearrangement, caspase-8 involved cellular apoptosis and bacteria binding to and invasion of HBMEC. Last, using the signaling moleculars antibody, through western-blot and coimmunoprecipitation detection,or using the siganaling moleculars inhibitor to inhibit the cellular biological effect of Hcp family proteins, we will further identify the associated signal pathway, such as PI3K, AKT, Stat3 and so on. The research can demonstrate the receptor and molecular signal pathway in HBMEC for the Hcp family proteins associated with E. coli T6SS and give us a further understanding for the molecular pathogenesis mechanism of E. coli neonatal meningitis. Hope, the Hcp family proteins and/or its specific receptor and associated signaling moleculars in HBMEC may be the targets for the development of preventive or therapeutic drugs and/or vaccines against E. coli neonatal meningitis.

大肠埃希菌是新生儿脑膜炎首要病原菌。我们前期的研究表明脑膜炎模式菌株大肠埃希菌RS218中存在致病性VI型分泌系统，该分泌系统的Hcp家族蛋白，无需进入人脑微血管内皮细胞（HBMEC），即可诱导细胞骨架重排、凋亡和白细胞介素释放，影响细菌对HBMEC粘附和侵袭，且Hcp家族蛋白存在特异性受体并涉及胞内一系列信号传导，参与发病。为此，本研究拟首先构建HBMEC的cDNA文库，利用酵母双杂交系统、激光共聚焦等方法，找到并鉴定存在于HBMEC表面的Hcp蛋白特异性受体；利用实时定量PCR筛选出与细胞骨架重排和凋亡相关的（如PI3K、AKT、Stat3以及Caspase-8上游等）信号分子；结合抑制剂阻断和免疫共沉淀等实验验证信号通路。通过本课题的研究，明确大肠埃希菌VI型分泌系统Hcp家族蛋白的受体和HBMEC内部的信号传导，为细菌性脑膜炎的分子发病机制、疾病预防、靶向治疗提供可靠理论依据。