Important Pathogenic Factors Of Enterohemorrhagic Escherichia Coli O157:H7: Crystal Structures, Functions And Applications

Enterohemorrhagic Escherichia coli O157: H7 (EHEC O157), an important zoonotic pathogen, colonizes on epithelia of large intestine, and causes a wide spectrum of diseases containing diarrhea, life-threatening hemolytic uremic syndrome (HUS), etc. in human and animals. Outbreaks of EHEC infection have been reported worldwide since it was first identified as pathogen in 1982. Different from other bacteria infections, conventional use of antibiotics can enhance pathogenesis, which makes prevention and treatment of EHEC infection remains problematic. Additionally, as EHEC O157 is easy to grow, and can spreads in multiple pathways, it can be use as bio-weapons and bio-terrior angentsPoor knowledge on the pathogenic mechanism of EHEC O157 is the main reason for our inability on EHEC O157 control. Thus, it is in urged need to fully understand its pathogenesis to guide the research and development of new drugs, vaccines and treatments for EHEC prevention.EHEC causes diseases in human and animals mainly by intimate attachment of bacteria to host cell, triggering of local inflammation and inducing attaching and effacing (A/E) lesions. EHEC O157 is able to stickly attached to intestinial endothelial cells, and trigger the local inflammation and disturb the balance of electrolyte and water. Meanwhile, EHEC cause local effacement of brush border microvilli and induces actin accumulations beneath the attached bacteria, termed A/E lesions. These effects lead to death of infected cells and tissue injury and infectious diseases in final.In this study, with the help of crystallographic and biochemical tools, we investigated key proteins involved in the process of EHEC O157 caused inflammation and A/E lesions, in order to fully understand EHEC pathogenesis. In addition, based on the evidences from our crystal structures and precious immunology studies on EHEC protective antigens, we develop a vaccine procedure for EHEC control. The details were described as the following.1) The crystal structure determination and biochemical analysis of the C-terminal domain of outer membrane protein A (OmpA). OmpA plays multiple roles in EHEC O157 physiology. Furthermore, OmpA helps EHEC O157 to cause disease by induction of bacteria adhesions, stimulation of the host immune response, etc. OmpA is composed of two domains: N-terminal domain and C-terminal domain. The N-terminal domain is aβ-barrel structure and embeds in the outer membrane of bacteria. The C-terminal domain is likely to be the key domain for its multiple pathogenic roles, but no structural information about the C-terminal domain ompA is available currently.In this study, we purified and crystallized EHEC OmpAC, collected a diffraction data set to 2.7 (A|。) resolution and determined its crystal structure by molecular replacement (MR) method. In the structure we found a special domain swapped dimer of OmpAC, and a characterized peptidoglycan binding sites. Meanwhile, we analyzed the oligomer status of OmpAC in solution by size-exclusion chromatogram and cross linking. Results show that OmpAC performs as a dimer which in consensus with the crystal structure data. These finding help us to better understand the base of OmpA related mutiple functions.2) Studie on the contribution of Tir IBD dimerization in the intimin-Tir interaction. EHEC and Enteropathogenic Escherichia coli (EPEC) are able to induce A/E lesions in similar ways. The formation of A/E lesions is an outcome of signal transduction and intimin-Tir interaction palys a central role in this process. Studies on EPEC showed that the dimerization ofβ-barrel domain from intimin was able to induce the cross linking of intimin-Tir complex and activates the downstream signal transduction. Our precious crystallographic studies showed that the intimin binding domain of Tir (Tir IBD) is also a dimer. As Tir IBD of EPEC also behaves as a dimer, we deduced that the Tir IBD dimerization could perform as theβ-barrel domain of intimin and induce the cross linking of intimin-Tir complex.In this study, we prepared the recombinant proteins EHEC intimin, EHEC Tir IBD, EPEC Intimn, EPEC Tir IBD. And by size-exclusion chromatogram, we purified the intimin-Tir IBD complex of EHEC and EPEC. By electronic microscopy and single particle reconstruction method we found that intimin-Tir IBD complex from both EHEC and EPEC form a tyre like structure with a diameter of about 10 nm, which is similar to that induced by intiminβ-barrel domain. Following that we mutated the core amino acids in Tir IBD dimerization, and found that the Tir mutant of EHEC and EPEC were unable to induce the cross linking of intimin-Tir IBD complex of EHEC and EPEC, respectively. These findings indicates that Tir IBD dimerization is able to induce the cross linking of intimin-Tir complex, which is similar to the contribution of intiminβ-barrel domain.3) Preliminary crystallographic studies on TirN, TirN-SH3 complex, N-WASP, and TCCP-N-WASP complex. As described in the previous section, the formation of A/E lesions is an outcome of effector interplays both from bacteria and host cells. Beside the participation of intimin-Tir interation, the cross talks betweent N terminus of Tir (TirN) and SH3 domain of cortactin, TCCP (Tir coupled cytoskeletal protein)and N-WASP (Neural Wiskott-Aldrich syndrome protein) also play an important role in A/E lesions formation. No available structural data to explain the interaction patterns of these proteins in the present.In this study, we carried out the preliminary crystallographic research on TirN, TirN-SH3 complex, N-WASP, and TCCP-N-WASP complex and got the following results. (1) We successfully purified the recombiant TirN and Se-methionine substituted TirN, and got the high-quality protein crystals. After X-ray diffraction, we successfully collected data set to a resolution of 2.4 (A|。) and 2.5(A|。). The structure determination of TirN is continued. (2) By size-exclusion chromatogram, cross linking and dynamic light scattering, we found TirN performs as a dimer in solution, which is similar to the intimin binding domain of Tir. This information provides further evidence of Tir dimerization mediated Intimin-Tir complex cross linking. (3) We successfully purified the TCCP binding domain of N-WASP160, and grow the protein crystal. (4) We successfully purified the TCCP-N-WASP complex by size-exclusion chromatogram. The crystal screening of this complex is continued.4) Vaccine development based on crystal structure and previous immunology studies.Evidences from our crystal structures and immunology studies suggested that the pathogenic factors EspA, the C-terminal 300 amino acids of intimin (IntiminC300) and the B subunit of Stx2 (Stx2B) were immunogenic and confered partial protection. In our previous study, we combine EspA (E), C-terminal 300 amino acids (IntiminC300, I) and Stx2B (S) together to generate trivalent and bivalent recombinant proteins EIS, EI, ES and SI, and tested them as vaccine candidates in mice. Result showed that immunization with EIS strongly protected mice from EHEC challenges. In addition, EIS-producing attenuated Salmonella vaccine was able to elicit significant systematic immunity, which protects mice when challanged with live EHEC O 157.In this study, we prepared a anti-EIS muti-clone antibodies and found that it is capable of inhibiting EHEC induced actin polymerization, which gave an explanation of the protection of EIS vaccine. Addtionally, we found that a new procedure containing a combination of recombinat EIS protein and EIS-producing Salmonella provide enhanced imunity and protection. These data lay a solid foundation for EHEC O157 control in the near future.