Study On The Mechanism Of Escherichia Coli O157:H7 EspF Protein Inducing Apoptosis In Host Cells Via Endoplasmic Reticulum Stress

Background and ObejectiveEnterohemorrhagic Escherichia coli O157:H7 is a foodborne zoonotic intestinal infectious disease pathogen that can be transmitted through the fecal-oral route and can cause diarrhea,hemolytic uremic syndrome,hemorrhagic colitis,and acute kidney disease in humans.The bacteria mainly cause intestinal mucosa colonization to cause adhesion-erasure of epithelial cells and stx expression to produce Shiga toxin.Since the first discovery in 1982,the continued spread of outbreaks of enterohemorrhagic E.coli poses a huge threat to human health and has also challenged global public health.Among them,EspF protein,as a key effector protein of EHEC O157:H7,plays an important role in the pathogenic process of pathogenic bacteria,but its specific pathogenic molecular mechanism is not clear.Recent studies have found that the virulence protein has a certain relationship with the endoplasmic reticulum of the host cell.This topic mainly explores whether EspF targets the endoplasmic reticulum and then triggers the endoplasmic reticulum stress response to eventually cause host cell apoptosis.There is no vaccine or effective interventions to prevent or treat diseases caused by EHEC,so it is necessary to further understand the mechanism of EspF-induced cell death in order to develop effective vaccines or therapeutic strategies.Methods（1）Targeting the endoplasmic reticulum of host cells by EspF protein.The λRed homologous recombination system was used to construct the EHEC O157:H7 espF gene deletion strain（ΔespF）and the complement strain（ΔespF/pespF）.In combination with the wild type EDL933w（Wild type）,HT-29 cells were selected for infection,the relative and absolute quantitative technology iTRAQ combined with high-resolution mass spectrometry system to detect intracellular protein expression levels and regulatory changes.The recombinant plasmid EGFP-EspF was used to perform subcellular localization experiments to determine the function of EspF protein to target the endoplasmic reticulum.（2）Study on the pathway of ER stress response triggered by EspF protein.In terms of morphology,transmission electron microscopy confirmed that the pathological state of endoplasmic reticulum stress expansion caused by EspF protein in host cells.Second,after the cells of different experimental groups were infected with the treated cells,the stress pathway was detected using real-time quantitative PCR and western blot.Expression of related molecules such as glucose-regulated protein-78（BiP）,C/EBP homologous protein（CHOP）,and protein apoptotic enzyme 12（Caspase 12）at the mRNA and protein levels;Fluo-4 AM staining was used to help compare calcium ion aggregation production;Qualitative and quantitative identification of specific signal pathways of endoplasmic reticulum stress response triggered by EspF protein.（3）Study of host cell apoptosis induced by EspF protein.Annexin V-FITC/PI double staining combined with flow cytometry was used to analyze the apoptosis of HT-29 cells infected by each group of strains;the relative survival rate of the cells was compared using MTT colorimetry;differential expression of apoptotic enzyme 9/3 detected by western blot.The above determined the effect of the presence of EspF protein on the ability of EHEC O157:H7 to induce apoptosis in host cells.Results（1）iTRAQ protein quantification combined with bioinformatics analysis,and subcellular localization combined with transmission electron microscopy observations jointly determined that EspF targets the endoplasmic reticulum of the host cell.（2）EspF protein can trigger the host’s endoplasmic reticulum stress response via PERK,IRE 1α,and ATF-6 pathways.Deletion of the espF gene can cause down-regulation of ER stress-related proteins,and decrease NF-κB activation and phosphorylation.（3）EspF protein can specifically activate Caspase apoptosis response via Caspase 12.Deletion of espF gene caused a decrease in the apoptosis rate and relative survival rate of HT-29 cells,and the expression of apoptotic proteases 12,9 and 3 decreased.ConclusionEHEC O157:H7 EspF protein binds to the host’s endoplasmic reticulum through targeted localization,triggering an endoplasmic reticulum stress response,and eventually induces apoptosis.Through the research in this article,it provides new clues for the study of drug treatment of EHEC infection and the prevention of outbreaks.