| Enterohemorrhagic Escherichia coli (EHEC) O157:H7is one of many pathogenic bacteria that will cause severely damages on intestinal system. O157:H7,which under the classification of extracellular bacteria, is infect host mainly by means of injecting effector proteins through Type Ⅲsecretion system(T3SS)into the host and influencing physiological functions of host. Those influences can be listed as changing metabolize of cells, adjusting immune responses, affecting signaling pathways inside cells and so on. At present days, it is became widely accepted by most that there are62effectors inside EHEC O157:H7by analyzing it through bioinformatical ways,39were confirmed by experiments. Analyses upon pathogenic mechanism of NleE, NleC and NleH was careful and thorough, which indicates that those effectors’ pathogenetic characteristics were by means of affecting signaling pathways of NF-κB,MAPK, ubiquitin and apoptosis at different levels. On the contrary, research on pathogenic mechanism of effector NleB of EHEC O157:H7is still at its preliminary stage until today, which bears far-reaching significance to carry on exploring pathogenetic characteristic of O157:H7.The effector NleB of Enterohemorrhagic Escherichia coli (EHEC) O157:H7is consist of two members:NleBl and NleB2(NleB1has been confirmed by experiments while NleB2is yet to be proved),and both of them were proved to be transcribable and expressible. With close relationship of EHEC, both of Enteropathogenic Escherichia coli (EPEC) and Citrobacter rodentium contains one NleB effector. The NleB of C.rodentium functions as a translocated N-acetyl-D-glucosamine (O-G1cNAc),with its transferring abilities which modifies GAPDH, therefore it sabotages the interaction of TRAF2-GAPDH and TRAF2,yet suppresses the activation of NF-κB and polyubiquitination of TRAF2.Through sequence-checking,it is come occur that effector NleB1of EHEC O157:H7bears a huge resemblance to effector NleB1of Citrobacter rodentium---around89%. This discovery sets the fundamental of further researching process of analyzing connections between effector NleB1、NleB2of O157:H7and signaling pathway of NF-κB, with details of how it is functioning.Research on transcriptional rate of nleBl and nleB2by using Real-Time PCR shows that both of them are transcriptional but differs in efficiency. Differences are obvious:nleB2is much less efficient in transcriptional process than nleBl,with quantity of expression is just about0.0076of its counterpart.In order to take the analyses on functionality and pathogenic mechanism of NleB in EHEC O157:H7to a further more in-depth level, we create the genetical knockout strain of nleBl and nleB2though the method of Lambda Red recombination. The outcome of virulence assessment indicate that compared with wild strain, growing speed of knockout strain shows no significant changes through its period in LB medium, however, things will be different if it has been nourished in DMEM medium.No significant changes occur to the numbers of adherent cell HeLa before and after the knockout of gene.To examine conditions of IL-8through ELISA, it is safe to conclude that the efficiency of expression of IL-8is uninhibitable if it has been infected by knockout strain along with some stimulation from TNF-a.Then, we construct the vector of pAcGFP-nleB1, pAcGFP-nleB2to assay the effect of the signaling pathways of NF-κB,we found that nleBl and nleB2can inhibit the signaling pathways of NF-κB with the stimulation of TNF-a,without effection of stimulation of IL-1β.Of this research, Gene knockout strains of nleBl and nleB2has been established with an early-stage virulence analysis carried out on those basis, we prove that NleBl,NleB2can inhibite the signaling pathways of NF-κB with the stimulation of TNF-a,without effection of stimulation of IL-1β. Those preparations sets the fundamentals of analyzing functionality on protein of NleB1and NleB2along with demonstration of the pathogenetic characteristic of Enterohemorrhagic Escherichia coli (EHEC) O157:H7. |
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