肠出血型大肠埃希菌EspF蛋白通过内质网应激导致肠上皮细胞凋亡及自噬机制研究

EspF protein of Enterohemorrhagic Escherichia coli can cause hemorrhagic colitis, but the exact mechanism is unknown. My previous experiments confirmed that the N-terminus of EspF protein could target mitochondria to induce apoptosis and destroy epithelial barrier. And it was firstly discovered by iTRAQ proteomics that EspF can up-regulate multiple endoplasmic reticulum stress and autophagy-related proteins in host cells, especially the expression of the marker protein Bip. In view of this, I hypothesize that EspF can promote endoplasmic reticulum stress through Bip protein to accelerate colorectal cell apoptosis and autophagy. To verify the hypothesis, my project is intended to: (1) Detecting whether EspF protein activates endoplasmic reticulum stress pathways such as IRE1, ATF6, and PERK; (2) Observing the localization of EspF protein in endoplasmic reticulum and lysosomes by immunofluorescence; (3) Detecting the expression of Caspase12, pJNK, BAX and CHOP in host cells to investigate whether EspF induces intestinal cell apoptosis through endoplasmic reticulum stress; (4) Studying on the dynamic regulation of autophagy-associated protein LC3I/LC3II by EspF. I hope my project will reveal the intrinsic relationship and signaling pathway of EspF and endoplasmic reticulum stress and provide a new idea for the prevention and treatment of hemorrhagic colitis caused by EHEC.

肠出血型大肠埃希菌EspF蛋白可导致出血性结肠炎，确切机制未明。申请人前期实验证实EspF蛋白N端可靶向定位线粒体诱导细胞凋亡、破坏上皮屏障，并通过iTRAQ蛋白质组学首次发现EspF可上调宿主细胞多种内质网应激及自噬相关蛋白的表达，尤其是标志蛋白Bip。鉴此，申请人推测EspF可由Bip蛋白促进内质网应激从而加速结直肠细胞凋亡和自噬。为验证该假说本项目拟:(1)检测EspF蛋白可否激活IRE1、ATF6、PERK等内质网应激通路;(2)免疫荧光观察EspF蛋白在细胞内质网、溶酶体的定位情况;(3)检测宿主细胞内Caspase12、pJNK、BAX、CHOP表达来研究EspF可否通过内质网应激引起肠细胞凋亡;(4)研究EspF对细胞自噬相关蛋白LC3I/LC3II等动态调控作用。以期揭示EspF诱导内质网应激的信号通路和内在关系，为源头防治EHEC引起的出血性结肠炎提供新思路。

在世界各地，经常发生与肠出血性大肠杆菌(EHEC)相关的大规模食源性疫情。在其毒力蛋白中，由LEE毒力岛编码的EspF是其功能强大的效应蛋白之一。在本项目中，我们用EHEC野生型菌株和espF基因缺陷型菌株分别感染宿主细胞。通过同位素标记的相对和绝对定量的新兴蛋白质组学技术(iTRAQ)，我们探索了宿主细胞内EspF蛋白的致病特征。我们的研究结果表明，EspF蛋白主要涉及的细胞差异反应包含：免疫调节、蛋白质合成、信号转导、细胞骨架重排、内质网应激和细胞凋亡等。值得注意的是，与espF基因缺陷株相比，野生株(WT)感染时宿主细胞内质网和核糖体中的蛋白质加工通路显著上调。因此，我们进一步利用分子生物学实验证明：肠出血性大肠杆菌的EspF蛋白可激活IRE1、ATF6、PERK等内质网应激通路，诱导宿主细胞出现内质网应激反应；且EspF蛋白可增强内质网应激下游pJNK、BAX、CHOP、Caspase3/9/12等细胞凋亡通路的表达，激活宿主细胞内质网应激依赖性细胞凋亡途径。本研究对EspF蛋白的致病机制进行了深入地探索，这将大大加深我们对A/E大肠杆菌与宿主蛋白相互作用的机制了解。