分泌蛋白到宿主细胞调节宿主免疫反应，是许多病原菌感染机体的重要策略。寻找肠出血性大肠杆菌（EHEC）感染过程中，调控宿主免疫反应的效应分子，阐释其分子机制具有重要意义。前期构建EHEC分泌蛋白系列缺失突变株，通过感染细胞实验，发现NleP（暂命名）能够上调IL-8的表达。本课题在前期工作基础上，利用细胞和小鼠感染模型，进一步探究NleP在EHEC感染过程中对宿主肠道上皮与免疫细胞的影响，及其在病原菌感染定植和组织损伤中的作用。在前期发现NleP能激活NF-κB通路的基础上，观察NleP是否还能调控其它信号通路；通过检测抑制特定信号通路时IL-8的表达变化，分析NleP上调IL-8表达与信号通路激活的关系；通过RNAi沉默通路关键基因，筛选其作用靶点；通过体外磷酸化和质谱分析，鉴定NleP与靶分子的相互作用及磷酸化位点。从而阐明NleP上调IL-8等细胞因子表达的分子机制。

Enterohemorrhagic Escherichiacoli (EHEC) belongs to a group of pathogens which employ their type III protein secretion system (TTSS) to inject (translocate) toxic proteins (effectors) into the host cell. The injected effectors subvert normal host cell innate immunity to benefit the bacteria. Upon infection, EHEC cause intestinal hyper-activated inflammatory responses characterized by infiltration of immune cells and damage to the lumen of the epithelium. However, no EHEC effectors are demonstrated to directly activate the host innate immune pathway. In our previous study, using the deletion mutant of EHEC, an effector named NleP was identified that significantly activated IL-8 secretion by epithelial cells. Then, with the TEM β-lactamase translocation assay and luciferase reporter assay, we found that it could translocate into host cells by the EHEC T3SS and harbored a potent NF-κB signal pathway stimulating activity. Thus, we infer that NleP may play important roles in the increased immune reaction of host intestinal. To validate the hypothesis, in this research, the role and molecular mechanism of NleP from Enterohemorrhagic E. coli in the activation of the innate immunity of intestinal cell was systematically analyzed. First, animal or cell infection models with NleP deletion mutant of EHEC were used to observe the expression of immune molecules, cells or tissue pathological injury changes over time, which were used to investigate the role of NleP in host intestinal cell innate immunity and virulence of this pathogen. Then, immunoblot was used to test which signal pathway could be induced by NleP. Finally, the interaction targets of NleP in host intestinal cell were identified through RNAi technology. And phosphorylation sites in target were identified by in vitro phosphorylation experiments and mass spectrogram analysis.