β1-integrin介导的幽门螺杆菌侵袭相关蛋白筛查及致病作用

More than 50% of the population worldwide have been infected by H. pylori, but only 15-20% of them developed into peptic ulcer disease or gastric cancer. Why had those people developed into gastroduodenal ulceration or gastric adenocarcinoma? In an attempt to explain this conundrum, the role of interaction between bacteria and host needs to be explored. .Although H. pylori was generally considered as an extracellular pathogen, current evidences indicate that it can invade epithelial cells in the gastric mucosa. Bacterial proteins encoded by virulence genes of H. pylori in host cells had been considered to be involved in pathogenesis of gastric diseases, and several lines of evidences suggested that, depending on specific host-pathogen interactions, a proportion of infected individuals will develop more severe consequences, including peptic ulcer disease, gastric cancer. Our previous study revealed that H. pylori could enter the AGS cells (human gastric adenocarcinoma epithelial cell line)，and the invasion intensity of H. pylori paralleled to the severity of digestive disease. To better understand of H. pylori pathogenicity, it is useful to detail the process of gastric invasion by bateria-host cells interaction...Integrins are presented on almost all types of mammalian cells. Many pathogens including bacteria and virus exploit their ligand-binding domain to interact with integrins on the host cell. It had been suggested that the invasion of H. pylori into gastric epithelial cells may though the similar pathway adapted by Yersinia which involved the β1-integrin receptor. .In the present study, T7 phage display system was used to screen for the bingding pattner for β1-integrin from a cDNA library encoding proteins or peptides of H. pylori. The potential candidate (designed as X) will be verified further by GST-pull down and Co-immunoprecipitation (Co-IP) technologies, and the bioinformatics methods will be used to predict the function of gene(s) X. Then AGS cell lines, the C57BL/6 mice will be infected with wild type strains of Hp NCTC11637, SS1 and their X gene deletion-mutants, the bacterial invasion rate will be evaluated by gentamicin protection assay. Finally, X gene of H. pylori and corresponded mRNA and protein from tissues of gastritis, peptic ulcer, and gastric cancer were detected by means of PCR ,real-time RT-PCR and western-blotting respectively, and relationship between X gene situation and progression of the H. pylori related gastric diseases was assessed.

我们前期研究及已有的报道均显示幽门螺杆菌（Hp）可侵入胃上皮细胞，且侵袭强度与消化性疾病的严重程度相关。已知介导Hp侵袭的胃上皮细胞受体为β1-integrin，但与之相关的Hp侵袭蛋白不明。本研究拟开展：1）应用噬菌体展示技术筛查、用GST-Pull down和Co-IP验证，获得与β1-integrin结合的Hp蛋白，生物信息学分析蛋白功能；2）通过庆大霉素保护性侵袭实验确定与Hp侵袭相关的蛋白，通过动物感染确定该侵袭相关蛋白的致病作用；3）对分离自临床不同消化性疾病的Hp（包括胃炎、胃溃疡以及胃腺癌），从DNA、RNA(转录)和蛋白（翻译）水平检测上述侵袭相关蛋白的基因及其表达，分析该基因存在及表达程度与Hp侵袭强度及临床消化性疾病类型的相关性。本研究旨在揭示Hp与侵袭相关的物质及其致病作用，为指导Hp根除性治疗和疫苗的研制提供新的思路，并有望就Hp作为胞内菌致病机制的研究奠定基础。

本项目首先以幽门螺杆菌（Hp）构建噬菌体cDNA展示文库，以β1-integrin 筛选与之结合的蛋白，获得13个蛋白，经测序和生物信息学分析筛选出的蛋白为：已知与β1-integrin 互作蛋白CagL和CagA，4个假定蛋白和7个已知功能蛋白。通过GST Pull-down和分子等离子共振技术，未发现新的与β1-integrin 结合的蛋白。选择CagA、CagL、CagI（已知与β1-integrin结合）作为研究对象，构建Hp NCTC11637 cagA、cagL、cagI敲除株及cagA/cagL 和cagI/cagL双敲除株，以及AGS细胞β1-integrin沉默株，研究CagA、CagL、CagI在Hp侵袭AGS细胞中的作用。结果表明CagA、CagL、CagI参与Hp侵袭AGS细胞作用,CagA和CagL为功能独立的侵袭基因，CagI可能通过改变CagL的表达量影响Hp侵袭作用；β5-integrin和β1-integrin共同介导幽门螺杆菌侵入胃上皮细胞。通过动物实验进一步证明CagA 和 CagL是幽门螺杆菌的侵袭蛋白。通过内化的Hp菌株对细胞功能影响的研究，证明侵入胞内的Hp可促进AGS细胞增殖，不影响细胞凋亡，内化的Hp菌株能刺激NF-κB的活化，继而诱导AGS细胞IL-8及AID 的表达。 此外，通过原核表达并纯化CagA，进一步阐析CagA—β1-integrin通路在CagA引导细胞功能改变忠的意义，证明 CagA与β1-integrin互助可诱导细胞IL-8的分泌，ERK信号通路可能参与该过程。分离来自不同严重程度消化道疾病的Hp菌株，分析目标蛋白的基因型与侵袭力的关系以及Hp侵袭强度及临床消化性疾病类型的相关性。结果表明：尽管分离自胃癌和胃溃疡的Hp侵袭力显著高于分离自胃炎的Hp，但它们均可测出cagA、cagL和cagI,其表达量无明显差异。