Preliminary Study Of Effector Nlef From Escherichia Coli O157:H7

Enterohaemorrhagic E.coli is an important gram-negative enteropathogen,the serum O157:H7 has caused widespread outbreak,and now it has become a global public health problem.EHEC is an extracellular parasite bacteria,which has Type III secretion system(T3SS). EHEC translocates T3SS effectors into intracelluar by T3SS to perform pathogenicity and virulence transmission.There are 39 effector proteins which have been identified by experiments,Among them,NleF is one of the effectors which function and cellular target is not clear.The molecular weight of NleF is 21.4kD,which gene is z6020,and it lies on O-island 71 of EHEC O157:H7 genome.By sequence analysis,we found that the sequence of z6020 is exactly the same with the nleF of the EPEC,and with 89% similarity with the nleF of the Citrobactor rodentium.Previous studies have shown that,compared with the wild type,the colonization ability of the Citrobactor rodentium decreased significantly when infected mice.This indicates that NleF may be an important virulence factor of EHEC,studying its function plays an important role in exploring the pathogenesis of EHEC.In this study,according to the classical lambda Red homologous recombination and overlap extension PCR,we amplified the recombinant DNA fragment,which integrates the upstream and downstream homologous arm of z6020 and kanamycin resistance gene;And then we transfered the recombinant DNA fragment into the EDL933 competent cell,which contains pKD46;thus we constructed nleF knockout strains by kan gene replacement;And then pCP20 were transferred into the knockout strain competent,which containing kan;and by resistance screening and seqencing,and we construct knockout strain An/eF.We transfered pET-24a(+)-NleF into knockout strain AnleF competent to construct recovery strain Δn/eF/NleF.In order to explore whether NleF influences EHEC growth or not,we drawed the growth curve of the wild type,the knockout strain AnleF and the recovery strain AnleF /NleF,we found that there is no significant difference among the three bacterium whether in LB or DMEM(10%FBS) culture medium.This result showed that NleF has no effect on EHEC growth.In order to explore the effect of NleF on EHEC adhesion, we infected HeLa with three kinds of bacterium,namely wild type, ΔnleF and ΔnleF/NleF,by immuno-fluorescence assay to analyse their adhension ability to HeLa cells.The results showed that there is no significant difference in the number of three kinds of bacterium adhesion to HeLa.This result reflected that NleF has no effect on EHEC adhesion.In order to further explore the effect of NleF on cell death caused by EHEC infection,we infected HeLa cells with three kinds of bacterium;and then collected the supernatant after infection 1.5h and 5h,and determined LDH release,and calculated the cytotoxicity,and compared the difference. The experimental results showed that after 5h infection,knockout strain compared with wild strain increased cytotoxicity clearly,but the cytotoxicity of recovery strain had no significant change; This reflected that NleF may have the ability to suppress cell death to facilitate bacteria escap from the host immune system.The Caspase-4 was reported important in the process of cell death caused by bacterial infection,the prokaryotic expression vector pGEX-2TK-NleF was constructed and induced with IPTG. By GST pull down assay,NleF was identified has interaction with Caspase-4. Except all,the eukaryotic expression vector of the subunits of Caspase-4 p19 and p10 were constructed,namely pcDNA3-Flag-p19 and pcDNA3-Flag-p10.In this study, nleF gene knockout strain AnleF and its recovery strain ΔnleF/NleF were constructed,and the effects of NleF on bacterial growth,adhesion and cell death caused by EHEC infection were analysed.The interaction between NleF and Caspase-4 were found,and laid the foundation for further study the effect of NleF on the pathogenicity of EHEC.