Study On The Effects And Pathogenesis Of EspF In Haemorrhagic Colitis Of Intestinal Epithelial Cell Induced By Escherichia Coli O157:H7

Enterohemorrhagic Escherichia coli(EHEC)O157:H7 is a new foodborne zoonotic pathogen that predominantly colonizes human and animal colorectum.It is transmitted through the fecal-oral route and causes severe diarrhea,hemorrhagic colitis(HC),hemolytic uremic syndrome(HUS),thrombotic thromobocytopenic porpura(TTP),and other gastrointestinal symptoms.EspF exists in EHEC,enteropathogenic Escherichia coli(EPEC),and Citrobacter rodentium(CR),which causes severe disease in humans.The N-terminal of EHEC O157:H7 EspF(length:1-73 amino acids)is relatively conserved and the C-terminal(length:74-248 amino acids)is composed of four highly homologous proline-rich sequences.However,the molecular pathogenetic role of the N-and C-terminal domains during Escherichia coli O157:H7 infection remains unclear.(1)Construction of EHEC O157:H7 mutant and complementation strains.We knocked out the espF sequence(747bp,?espF),N-terminal sequence(219bp,?espEN,and C-terminal sequence(528bp,?espFc)separately using the pKD46-mediated ? Red homologous recombination system.Then,we built the corresponding complementation strains,namely,?espF/pespF,?espFN/pespFN,?espFc/pespFc by overlap PCR.(2)The N-Terminal domain of EspF plays an important role in apoptosis.The level of reactive oxygen species(ROS),cell apoptosis,mitochondrial trans-membrane potential were evaluated by DCFH-DA,double staining of Annexin V-FITC/PI,JC-1 staining.The results showed that deletion of the E.coli O157:H7 espF gene and its N-terminal led to a decrease in ROS levels in the host cells.The espF-N mutant strain exhibits a slower rate of reduction in ?m.EspF plays an important role in promoting apoptosis in the host cells,and its N-terminal domain is indispensable.(3)The N-Terminal of espF is involved in tight junction disruption and TNF-?secretion.The level of inflammatory factors(TNF-?,IL-6,IL-1 ?)and transepithelial electrical resistance(TER)were evaluated by ELISA kit and epithelial voltohmmeter.The results showed that a progressive decrease in TER was observed in HT-29 cells infected with WT,?espFc andpespF but not ?espF and ?espFN.The deletion of the espF-N gene resulted in a decrease in TNF-? expression within the host cells.These findings indicate that the N-terminal domain of EspF plays an important role in the infection process.(4)The espF-mutant strains attenuate pathogenicity to BalB/c mice.The animal mortality and mean colon weight were evaluated by mice assay.The results indicated that the WT and ?espFc groups developed more severe pathological reactions than the other groups.These findings reveal that the deletion of the espF gene attenuates the pathogenicity of EHEC O157:H7 to BalB/c mice,particularly the N-terminal domain.(5)The espF gene plays an important role in the regulation of the host cell protein.We used the isobaric tags for relative and absolute quantitation(iTRAQ)to explore the protein regulation function of EspF.The iTRAQ combined with 2D LC-MS/MS is emerging as a powerful methodology in the search for disease specific targets and biomarkers using cell lines,tissues,body fluids and so on.In this study,we found 145,230,229 proteins differentially expressed in sample pair ?espF:Control,?espF:WT,WT:Control.Furthermore,we adopted function annotation analysis of all identified proteins and function enrichment analysis of all differentially expressed proteins to explore more meaningful proteins and pathways.In summary,EspF has multiple functions;its N-terminal promotes ROS generation,disrupts membrane potential of mitochondria and tight junction,increases the expression of inflammatory factor TNF-?,resulting in the cell apoptosis and death of mice.The findings of the present study provide information on the pathogenesis and molecular mechanisms of EHEC O157:H7.