分泌蛋白NleP在EHEC感染过程中调控炎症信号通路的作用及其分子机制

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.

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。

分泌蛋白质到宿主细胞调节宿主免疫反应，是肠出血性大肠杆菌（Enterohemorrhagic Escherichia coli, EHEC）等许多病原菌感染机体的重要策略。寻找病原菌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。在本项目研究中，我们发现了一个新的肠出血性大肠杆菌效应分子NleP (又名Stk)。研究发现NleP具有真核样丝氨酸/苏氨酸激酶活性，能够在肠出血性大肠杆菌感染时通过III型分泌系统进入宿主细胞，诱导宿主产生过激的炎症反应。同时，我们发现了NleP在宿主细胞中作用的信号通路和靶点：NleP通过磷酸化IκBα及其家族分子来激活NF-κB经典或非经典通路，提高IL-8等细胞因子的表达水平。在感染EHEC的小鼠中，NleP能增加血清细胞因子KC（IL-8在小鼠中的同源分子）的水平，并能激活结肠炎症反应，加剧结肠的病理损伤。进一步，明确了NleP的毒力是依赖于其真核样磷酸激酶活性。同时发现NleP的同源蛋白质广泛分布于多个病原体中，具有类似的作用机制。综上所述，通过本课题研究我们发现了一类对病原菌感染具有重要影响的新的真核样效应分子，明确了其调控宿主免疫反应的分子机制和致病效应，对于解释细菌致病机制和制定病原菌的防控策略具有重要意义。在本项目执行其中共发表论文12篇，其中SCI或SCIE论文6篇，获得授权专利2项。特别是以本项目研究内容为主体的论文于2017年在The Journal of Infectious Diseases（IF=6.3）杂志以封面文章形式发表。同时在本项目执行期间，本人以本课题研究为基础申请并成功入选2018年度北京市科技新星计划。