The Crystal Structure Of EHEC O157:H7 IntiminC188 And It's Function Analysis

Enterohemorrhagic Escherichia coli(EHEC) O157, an emerging pathogen, causes severe hemorrhagic colitis and the life-threatening extraintestinal complication of hemolytic uremic syndrome (HUS) in 5 to 10% of patients. The source of E. coli O157 is mainly of contaminated food or drinking water . Treatment of infection with E. coli O157 has been difficult because antibiotics do not change the course of the enteritis of E. coli O157 and may increase the incidence of HUS caused by the pathogen. This untoward effect has been proposed to be mediated by antibiotic-induced bacteriolysis and release of intracellular Shiga toxins.E. coli O157:H7 has been shown to attach to the cytoplasmic membranes of intestinal epithelial cells, to efface their microvilli (attaching and effacing/AE lesion), and to cause actin to accumulate beneath sites of bacterial attachment. These features are shared with several other enterohemorrhagic E. coli (EHEC) serotypes and members of the enteropathogenic E. coli (EPEC) group. The eae gene, which has been shown to be necessary for attaching and effacing activity, encodes a 96-kDa outer membrane protein (OMP) which is termed Intimin. And Intimin is necessary for intimate attachment of the bacteria to epithelial cells . Analysis of the nucleotide sequences of the Intimin genes from different EHEC and EPEC strains has shown a high degree of homology in the 5'two-thirds of the genes and a significant degree of heterogeneity in the 3'one-third of the genes.After initial attachment of E. coli O157:H7 with intestinal epithelial cells, Tir is translocated to the host cell and localizes in the host cell membrane. Tir acts as a receptor for the bacterial outer membrane protein intimin. The interaction of Tir and intimin triggers the final step of AE lesion formation, leading to actin condensation, pedestal formation and intimate bacterial attachment.In view of these, this study investigated the structure and function of EHEC O157:H7 IntiminC188 from the following aspects:1. Gene cloning, expression, refolding and purification of EHEC O157:H7 IntiminC188 and Tir-M. The primers were designed and synthesized to amplify theIntiminC188 and Tir-M coding gene eae-c188 and tir-m by PCR. The target gene werecloned into pET-21a(+) plasmid through NdeI and XhoI restriction site. Then therecombinant prokaryotic expression plasmid was transformed into E.coli BL21(DE3) andthe target protein induced by IPTG was detected by PAGE. After refolding and purification,IntiminC188, a mutant IntiminN916Y of accident and Tir-M have been gotten.2. Crystals of intiminC188 and mutant intiminN916Y were successfully grown usingthe method of hanging-drop vapor-diffusion. Native data were collected on RigakuMicroMax007 to 2.8 A and 2.66 A.The crystal structure was solved by molecularreplacement and difference Fourier analysis in X-ray crystallography, and then, was refinedsmoothly alternating steps of automatic and manual adjustment. The final model hasreasonable crystallographic R and Rfree values with good stereochemistry and the mainchains fit well with the electron density map.Despite the amino acid sequence of intiminC188 and mutant intiminN916Y showscomparatively low identity to that of EPEC intimin, the three-dimensional structure issimilar. Although the folds of the main chains are approximately the same in intimin of bothEHEC and EPEC, there are still a large number of residues are different, especially theresidues that are adjacent to the active site(C-terminal). Combining with detailedbioinformatics study, we pointed out several residues that may be important to receptorbinding. This will be helpful to further functional research upon the protein.3. BIAcore^? 3000 apparatus (surface plasmon resonance technology) was used toanalyse the affinity between Tir-IBD(intimin binding dormain) and its ligand intiminC188or mutant intiminN916Y. The kon, koff, and KD were obtained by fitting the data with theBIAcore evaluation program (Version 4.1). The affinity change of intiminC188 andmutant intiminN916Y correspond with their structure difference.In a word,structural and functional analysis of EHEC O157:H7 intimin and Tirinteraction are very important for the research of this pathogen. This work has resolved thecrystal structure of EHEC intiminC188 and shed light on the structure and function researchof EHEC intimin and Tir interaction.