Study On The Mechanism Of Escherichia Coli O157:H7 EspF Protein Interacts With ANXA6 Protein To Regulate Autophagy Via ER Stress

Background and ObjectiveEnterohemorrhagic Escherichia coli(EHEC)O157:H7 is a foodborne pathogen causing a variety of extra-intestinal diseases,such as hemolytic uremic syndrome,thrombotic thrombocytopenic purpura as well as life-threatening cases.EspF,one of the major virulence effector proteins of EHEC O157:H7,is primarily responsible for the development of inflammatory colitis.Our previous study revealed that EspF targets the endoplasmic reticulum(ER)and regulate ER stress.Also,ER stress is a molecular switch that regulates autophagy.However,the interaction among EspF protein,ER stress and autophagy remains unclear.In this study,the relationship among EspF-ANXA6-ER stress-autophagy was investigated,aiming to elucidate the mutual effects of EHEC O157:H7 and the host,clarify its role in cell-cell communication,host defense,and virulence.Our research provides a theoretical basis for development of ER stress-autophagy therapeutics in the treatment of EHEC infections.Methods(1)The involvement of EspF protein in regulation of endoplasmic reticulum stress.Caco-2 cells were infected with EHEC O157:H7 and ΔespF strains to construct bacterial infection models in vitro.The endoplasmic reticulum stress protein expression levels were detected by Western blot.Plasmid pEGFP、pEGFP-EspF、pEGFP-EspF-T2A-ANXA6 were transfected in host cells to explore the subcellular localization of EspF and BiP protein by immunofluorescence.(2)The interaction of EspF and ANXA6 proteins in the regulation of ER stress signaling pathways.ER stress level in host cells was detected by western blot and qPCR after transfection with indicated plasmids.Calcium ion distribution and cell activity were detected by Rhod-2/AM calcium ion staining and AnnexinV-633 apoptosis detection kit,separately.(3)The interaction between EspF protein and ANXA6 protein in regulating autophagy.Caco-2 cells transfected with indicated plasmids were used to construct the ER stress research models.The protein expression of LC3,Akt,p-Akt,mTOR and p-mTOR were detected by western blot.Results1.espF-deletion O157:H7 strain infection enhances ER stress in Caco-2 cells.Wild EHEC strains infection can stimulate the expression of BiP protein in host cells.espF-deletion O157:H7 strain infection can further activate BiP protein,improve eIF2α phosphorylation,and up-regulate the expression of ATF4 and CHOP,suggesting that EspF protein may down-regulate of ER stress by inhibiting PERK pathway.In addition,the EspF protein co-localizes with BiP protein,but the interaction between EspF and BiP weakens the co-localization effect.2.EspF-ANXA6 interaction regulates endoplasmic reticulum stress by inhibiting PERK pathway.EspF significantly inhibits eIF2α phosphorylation,down-regulates ATF4 and CHOP expression,stimulates Ca2+ release,and induces apoptosis,depending on the interaction between EspF and ANXA6.3.EspF-ANXA6 interaction regulates autophagy by inhibiting Akt pathway.EspF and ANXA6 protein interaction inhibits the transformation from LC3-I to LC3-II and the phosphorylation of Akt,suggesting that the formation of autophagy is inhibited which may depend on the inactivation of Akt pathway.ConclusionEHEC O157:H7 EspF protein is the bridge to inhibit ER stress and regulate autophagy,which depends on the interaction with host ANXA6 protein.Through EspF-ANXA6-ER stress-autophagy pathway,it disrupts ER stress,breaks calcium homeostasis,inhibits autophagy,cut off communication between host cells so as to destroy the antibacterial reaction.This search provides theoretical basis for the prevention and control of EHEC O157:H7 infectious diseases.