Construction And Biological Characteristics Of The TccP Gene Deleted Mutants From Enterohemorrhagic Escherichia Coli O157:H7

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an enteric pathogen prevailing all over the world since it was first recognized as pathogens in 1982. It causes severe hemorrhagic colitis and the life-threatening extraintestinal complication of hemolytic uremic syndrome (HUS). EHEC O157:H7, produce Shiga toxin 1 or 2 (Stx1 or Stx2, respectively), or both. The major source of EHEC O157:H7 is contaminated food or drinking water. Treatment of this infection is difficult because antibiotics can not change the course of it's and may increase the incidence of HUS caused by the pathogen. This untoward effect has been proposed to be mediated by antibiotic-induced bacteriolysis such as vaccines and antibody.The interplay between bacterium and ruminant host probably has its main role in promoting infection and pathopoiesis.The adherence of EHEC on intestinal epithelial cells is believed to be the first step for developing these diseases.The adhesion is mainly mediated through the type III secretion system(TTSS).EHEC employ a TTSS to deliver effector virulence proteins translocated intimin receptor(Tir)and Tir-cytoskeleton coupling protein(TccP)into host cells in order to produce attaching and effacing (A/E)lesions.TccP is a novel EHEC effector that displays an Nck-like coupling activity following translocation into host cells.Once translocated,TccP directly binds and activates neural Wiskott-Aldrich syndrome protein(N-WASP)to stimulate actin polymerization,leading to pedestal formation.TccP is present in the sequenced EHEC genomes Z3072 is located at the 5′end of a cryptic prophage,CP-933U,have identical proline-rich repeats.TccP as only the second type III EHEC effector protein after Tir that is required for EHEC-induced actin polymerization and A/E lesion formation.In view of these, this study investigated the structure and function of EHEC O157:H7 from the following aspects:1. Constructing a targeting vector for the tccP gene of EHEC O157:H7: The gene of tccP DNA was amplified from EHEC O157:H7 by PCR, as a result two tccP homologous sequences of 1292bp and 1038bp were respectively inserted into plasmid pMD-18T in order to construct the displacement targeting vector. The chloramphenicol resistance gene (Cam) was used as an index of screening.1.1 pBlueScript SK(-)-RA: right homologous arm(RA) was cloned into the vector pBlueScript SK(-);1.2 pBlueScript SK(-)-Cam-RA: The chloramphenicol resistance gene (Cam) was cloned into the vector pBlueScript SK(-)-RA;1.3 pBlueScript SK(-)-LA-Cam-RA: the left homologous arm(LA) was cloned into the vector pBlueScript SK(-)-Cam-RA.2. Disruptting tccP gene by the genetic engineering technique.2.1 The sequence of LA-Cam-RA was transformed into EHEC O157:H7 by electroporation transformation, which was digested by BamHI,KpnI from the targeting vector . And the selection for the chloramphenicol resistance yielded the desired the knock-out mutant;2.2 Analysis of this mutant in vit ro was performed with PCR, RT-PCR analysis and Western blot hybridization.3. Function research of the mutatinΔtccP3.1 Adherence effect ofΔtccP with HeLa cell: HeLa cell adherence grown on the cover glass was incubated with the bacterium together for 4 hours with 5%CO2 at 37℃. Finally, adherence effect was observed by Giemsa's staining. There's no detection of aggregation adhesion;3.2 Detecting the adherence between O157 and HeLa cell by Fluorescent actin staining(FAS) to check the action polymerization. Result show thatΔtccP could effect the formation of"attaching and effacing"(A/E) lesion.3.3 Bacterial infection test ofΔtccP to host animal. Balb/c were infected with O157 live bacterium andΔtccP EHEC O157:H7 can not lead the mice to death. In a word,a tccP knock-out mutant vector of O157 has been constructed successfully, and the homologous sequences was found to be stably integrated into the O157 genome with the expected targeting fragment obtained, which is very important for the research of this pathogen. These results demonstrate the utility of the gene targeting approach in the study to investigate the gene function of the function of TccP in the"attaching and effacing"(A/E) lesion, and the mechanism by which this pathgen invades to the host cells.