Function Of The Effector Protein EspO In Enterohemorrhagic Escherichia Coli

Background: Enterohaemorrhagic E.coli(EHEC)is one of the primary causes of childhood diarrhea.It can cause hemorrhagic colitis and hemolytic uremic disease,and seriously affect public health and life safety.EHEC belongs to the “A/E pathogenic bacteria” family,and its virulence depends on its Type Ⅲ Secretion System(T3SS).The system uses a needle-like structure to form a channel on the cell membrane of the host cell and secrete effector proteins into the cytoplasm of the host cell,thereby hijacking the physiological process of intestinal epithelial cells or regulating the immune response.T3SS plays a vital role in the pathogenesis of “A/E pathogens”.The study of effector proteins in the type Ⅲ secretion system is extremely critical for understanding the interaction between A/E pathogens and the host.Objective: EspO is an effector protein secreted into host cells by the EHEC type Ⅲ secretion system,and its homologous proteins are widely distributed.However,the current researches have limited cognition of its physiological functions.Therefore,the project will focus on studying the physiological substrate and biochemical function of EspO and its role in EHEC infection.Methods: The suicide plasmid pCVD442 was used to construct the espO gene knockout strain in a two-step method,and the espO gene replenishment mutant strain was constructed through the recombinant p Trc99a-EspO.Different EHEC strains were infected with HeLa cells to understand the biological function and lethal effect of EspO gene in the infection process.To explore the effect of EspO on the NF-κB inflammatory pathway and the subcellular localization of EspO at the eukaryotic co-transfection level.In addition,techniques such as co-immunoprecipitation and biological mass spectrometry were used to screen and identify the physiological substrates of EspO1.Results: In the process of searching for physiological substrates,combined with GST pull down,co-immunoprecipitation,and biological mass spectrometry,we identified a series of interacting proteins,including tubulin and HSP90.Immunofluorescence localization revealed that EspO aggregated into vacuoles or evenly dispersed in the cytoplasm when ectopic expression in HeLa cells.When EspO is ectopicly expressed in 293 T cells,it can activate the TNF-induced NF-κB signaling pathway,and this activation can be inhibited by the effector protein Nle B.We speculate from previous research reports of EspO and the function of HSP90 that EspO may be involved in the anti-apoptotic effect,but further experiments are needed to verify it.Therefore,espO-deficient strains were constructed and infected HeLa cells.EspO failed to inhibit cell death induced by TNF and TRAIL,indicating that EspO does not participate in death receptor-mediated apoptosis,or that EspO is not induced by these two pathways.Play a role in the process of apoptosis.Conclusion: This experiment initially studied the biological functions and substrate proteins of EspO,explored the influence of EspO on the biological characteristics of EHEC,confirmed that EspO can activate the NF-κB pathway induced by TNF,and explored the interaction EspO The role of TNF and TRAIL on the cell death pathway,and the exploration of the role of EspO in the process of EHEC infection will lay the foundation for the subsequent in-depth study of the function of EspO.