The Role Of Tir In EHEC Adhesion And A/e Lesion

Enterohemorrhagic E. coli (EHEC) O157:H7 can cause a wide range of clinical illness, including diarrhea, haemorragic colitis and haemolytic uraemic syndrome. Infection by EHEC can cause A/E lesions and damage by Shiga toxins. Most studies now are focus on the two fields. The A/E lesion is required for a 34kb pathogenicity island called the locus of enterocyte effacement (LEE) located on the chromosome of EHEC which encodes a series of effector proteins and TTSS which helps the proteins to transport. The A/E lesion can form a pedestal structure beneath the bacteria adhesion and interact with the host protein in a complicated pathway. In our work, we try to understand more about the intimin translocated receptor (Tir) by expressing the recombinant Tir and constructing tir mutant. And we also want to know the role of Tir in bacteria adhesion. Then we detect the mRNA ofα-actin andα-actinin to study the relationship between them and Tir.Part I Prokaryotic Expression and Biological Activity of Tir and TccPThe EHEC protein intimin is essential for bacteria adherence and A/E lesion. And the Tir is one of the receptors of intimin encoded by EHEC. One of our works in this part is using genetic engineering to express full length Tir of EHEC in E. coli, and to identify the biological activity of Tir. We constructed the expression plasmid pET-22b (+)-tir. The expression vector was confirmed by sequencing and transformed into E. coli BL21 (DE3), and induced by IPTG to express the recombinant Tir. The recombinant Tir was mainly existed in the supernatant of lysis. We can obtain more than 10mg/L recombinant Tir during fermenting. After purified by Ni2+ affinity chromatography, we gain Tir with final purity of 90%. We connected FITC with the recombinant Tir. And then we incubate the Tir-FITC with HEp-2 cells. HEp-2 cells were identified by fluorescence microscope to confirm the biological activity of Tir for further research of the interaction of Int281 and Tir and the role of Tir in adherence.TccP (Tir-cytoskeleton coupling protein), secreting via TTSS plays an important role in Nck-independent way of A/E lesion, which takes place in EHEC. TccP was recruited to Tir and then activated N-WASP and further signal pathway in host cell. We expressed the recombinant TccP in the same way of Tir. The TccP was expressed as inclusion bodies. We denatured the recombinant TccP with 6 mol/L guanidine hydrochloride, and renatured by several grades of urea, and dialyzed into PBS solution. In the end we gain more than 1mg/ml, purity of 85% TccP solution. And TccP was detected by SDS-PAGE, confirmed by Western blotting.Part II The interaction of Tir, TccP, Int281 and the role of Tir in bacteria adhesion to host cellIntimin, which encoded by eae located on LEE (locus of enterocyte effacement) virulence island, anchored on the outer membrane of bacteria, with high conserved sequences in its N terminal, while on the other hand, the C terminal of intimin differs between pathogens. The C terminal of intimin is showed to be essential for interactions with host cell. The receptors of intimin include nucleolin andβ1-integrin which is encoded by the host cell, and Tir which is also encoded by LEE of pathogen. Tir which transfers via TTSS has a hairpin structure with its N terminal and C terminal inserting into the host cell membrane, and the extracellular part binding the bacterial outer membrane adhesion molecule, intimin. Several studies have reported that the interaction of intimin and Tir triggers the A/E lesion. Deleting of the binding domain of intimin, Tir can also insert into the host membrane but lose the ability to induce actin polymerization.In this part of our works, we try to show the role of Tir in EHEC adhesion on molecular and cell level. We assayed the interaction of recombinant Tir and Int281 which has been expressed by other fellows in our laboratory in vitro. Firstly, we product rabbit anti-Tir serum of which potency is more than 106 by immunizing white big ear female rabbit for three times. Secondly, we used ELISA to assay the binding activity of Int281 and Tir. We found that the binding activity of Int281 and Tir was positively related to the level of Tir concentration. Further more, we used SPR technology to measure the affinity of Int281 and Tir by coupling Tir to the surface of chip CM5, gradient dilution Int281 by HBS-EP injecting into the same chip to interacting with Tir, and calculating the affinity with BIAevalution software, which in final, we gain the data of 1.28E-8. The data showed that Tir is a specific with high affinity receptor of intimin. By using ELISA and SPR technology, we also detected the interaction of integrin and Int281, TccP and Tir. The result showed that the binding activity of integrin and Int281 is weak than than of Tir and Int281 which means that integrin is a receptor with low affinity of intimin, and TccP can hardly bind to Tir in a direct way which means that there may be other proteins from the host cell or the pathogen that involve in the interaction of Tir and TccP.To understand the role of Tir in bacteria adhesion, we constructed tir mutant by Red recombinant system. We found that the long homologous regions (500bp in this study) of tir can make a better way in recombination. After we gainΔtir:EHEC, we infect the HEp-2 cultured cells with different dose of wild type EHEC andΔtir:EHEC respectively, and then counting colonies growing on the LB plate following we put the adherent bacteria on the it. We found that the number ofΔtir:EHEC is less than the number of wild type EHEC (p<0.01) and both theΔtir:EHEC and the wild type EHEC enhance their abilities of adhering to HEp-2 cultured cells after we compensating Tir (final concentration less than 30ng/μl). The result showed that the high affinity receptor, Tir, is more important in bacteria adhesion than the receptor of integrin.Part III the role of Tir in transcript of the relevant mRNAs of A/E lesionsEPEC and EHEC can induce attaching and effacing lesions, known as A/E lesions. The host cell cytoskeleton rearrangement, which forms a pedestal structure, is seemed as the most significant feature of A/E lesions. Tir is the only known TTSS effector protein required for pedestal formation in EPEC. However, researchers found another important TTSS effector protein in EHEC which function as a Nck replacer, TccP/EspFu, to remedy the absence of tyrosine phosphorylated Y474. Tir interacts via its N-terminal domain with several focal adhesion proteins includingα-actinin, talin, and vinculin which usually connect actin cytoskeleton with the membrane, either directly through interacting with the membrane phospholipids or indirectly via interaction with membrane-associated proteins.In this part of our works, we focused on several genes of the host cell associate with A/E lesion, and tried to detect their mRNAs in order to find the connection between them and Tir. We settled the HeLa cultured cell model of A/E lesion by infecting HeLa cultured cells with wild type EHEC andΔtir:EHEC respectively, and then detecting through laser scanning confocal microscope to confirm the A/E lesion. On the base of this model, we found that the infection by 106 CFU and 4 hours can be the best condition of A/E lesion, which we can detect the significant feature of pedestal formation in the wild type EHEC infection group rather than theΔtir:EHEC infection group. We isolated the total RNA of HeLa cultured cells infected by wild type EHEC andΔtir:EHEC respectively every hour after infection. Following by reverse transcription, we gain cHNA of HeLa cells. Then we used the cDNA as template, detected the abundance of mRNAs ofα-actin andα-actinin. The result showed that the mRNAs ofα-actin andα-actinin are down regulated after infection with both wild type EHEC andΔtir:EHEC (p<0.01). The mRNA ofα-actin in HeLa cells infected byΔtir:EHEC down regulated faster than that infected by wild type EHEC (p<0.01). The mRNA ofα-actinin in wild type EHEC group down regulated much dramatically after one hour of infection. But then it restored a little and then kept decreasing with the relative value of mRNA more than that of theΔtir:EHEC group (p<0.01). The result suggested that Tir affect the transcription level ofα-actin andα-actinin and the conclusion need further research.Our study try to understand more about the interaction of Tir and intimin on molecular and cell level and the role of Tir in EHEC adhesion via expression of recombinant Tir and construction of tir mutant. We also preliminarily found that Tir, as a high affinity receptor of intimn, affect the transcription level ofα-actin andα-actinin.