EHEC Virulence Factor-Nlep Acting On Host Inflammatory Pathways Such As NF-?B

Enterohemorrhagic Escherichia coli(EHEC)as an important food-borne pathogens,its infection is characterized by:the world epidemic,the high incidence and mortality[1-3].EHEC cause haemorrhagic colitis and the potentially fatal haemolytic uraemic syndrome(HUS),and can result in a serious clinical harm[4].such as in 2011 in Germany and other European countries,most lethal outbreak of E.coli caused great panic in society,known as"poison cucumber incident".Pathogen interact with host cells,which is the key step to its infection[5].EHEC invaded eukaryotic cells,the host cell initiated the inherent and acquired immune responses,both against each other,the final result is EHEC successful colonization in host or cleared by the host.When EHEC interacted with the host immune system,a variety of cellular responses will start signaling pathways,such as nflammatory pathways(the first signal NF-?B pathway:response very quickly to inflammatory).Many scientists have focused on inflammatory pathways,triggering a new hotspot.There are two research teams were concerned about two virulence factors NleC and NleH,which interacted with the host when EHEC infection occurred,their findings:two virulence factors inhibit NF-kB inflammatory pathways,conducive to EHEC colonization in early period of infection and subsequent infection diffusion,and their genes located Bacteriophage BP-933W[6,7].As a movable genetic elements,EHEC prophage is an important source for bacterial to obtain new virulence factors.Lethal Shiga toxin(Stx)gene is located on the prophage[8].We are concerned about EHEC 0157:H7,the whole genome sequencing has been completed.Scanning the whole genome after sequence analysis,we focused on genes upstream the gene of Shika toxin coding serine/threonine kinase.we tentatively named Nlep.Now the existing literature about Nlep showed it encoded phosphorylation kinase providing a selective advantage to prophage[9,10].And phosphorylation signals paly an important regulatory role for opening and closing the passage,we hypothesized that Nlep located prophage is an important virulence factor,interacts with the host signal path after infection.In order to verify the speculation,the paper elaborated from two parts:The first part:As a EHEC virulence factor,Nlep involved in bacterial infection,the research will be divided the following three aspects:1.Nlep was translocated to the cell by the type ? secretion system when the bacterial infecting host.Hela cells were infected with the EHEC bacteria,Marking specific fluorescence antibodies we found Nleplocated in the cytosol of target eukaryotic cells;The reporting system of ?-lactamase detected that Nlep can not enter the cells,when knockouting out EspN the EHEC T3SS key protein,These results suggest that Nlep as T3SS effector protein early EHEC gets into cytoplasm.2.Nlep promotes host inflammatory cytokine secretion when EHEC infection happen.First screening cytokines in EHEC infection cell lysates was found expression levels of inflammatory cytokines IL-8 and IL-1? significantly increased.Subsequently EHEC infected two typical cells HT-29 and U937,ELISA results showed Nleppromoted inflammatory cytokines IL-8 expression,Nlep mutant significantly reduced the expression level of IL-8(P<0.01).The results show Nlep is a key regulatory factor to the expression of inflammatory factors after infection.3.Nlep exacerbated inflammatory response in mice infected with EHEC.When mice infected with EHEC,Nlep promote the secretion of inflammatory cytokines,as infection days increasing.When mice death occurs,residual rate between wild group and ?Nlep group has a significant difference,ANlep group has a immune protective effect.The results showed that Nlep is closely associated with the inflammatory response after the bacterial infected mice.These results suggest that Nlep as an important virulence factor play an important role after EHEC infected host cells,it is closely related to the host inflammatory response.The second part starting from biological phenomena,shows the molecular mechanisms of Nlep acting on NF-?B pathway the first inflammation signal:1.Nlep acted on NF-?B signaling pathway.Both in bacterial infectionand cells transfected,Nlep efficiently promoted the phosphorylation of NF-?B pathway inhibiting factor IkBa,the medium induced the p65 nuclear translocation,activated NF-?B inflammatory pathway.2.Nlep phosphorylated I?B?.In vitro kinase assay and MS confirmed Nlep promoted IkBa phosphorylation in sites not only the Ser32/36 but also the new Tyr28/55.3.Nlep phosphorylated I?B?,which is not dependent on the upstream IKK complex.Knockout Assay found Nlep promoted IkBa phosphorylation,which are unique,does not depend on IKK complex.Nlep kinase activated NF-?B signaling pathwayindependently.4.The action of Nlep in the NF-?B non-canonical pathway.Immunoblotting and interference experiments found Nlep activated NF-?B non-canonical pathway independently.In summary conclusion:We found virulence factor Nlep induces a potent activation of the NF-?B pathways,which can phosphorylate IkBa target at Ser32/36 and the new Tyr28/55.After EHEC infected host,the virulence factors Nlep activated the NF-?B inflammatory pathways,causing high secretion of inflammatory cytokines,further feedback regulation of signaling pathways,thereby exacerbating the infection.The findings of this paper suggests there are other pathways coordinating role of cytokines triggered "cascades" exacerbated infected host cells,leading to serious death of the host.