The Function Of HtrA In Shigella Flexneri

Shigella spp., commonly called Bacillus dysenteriae , are gram-negative enteric pathogens. The pathogenesis of S. flexneri is based on its ability to invade and replicate within the colonic epithelium, which results in severe the typical bacillary dysentery. Owing to the low infectious doses of Shigella (10-100 bacteria), the annual statutory report released by the Ministry of Health of infectious diseases in 2010 showed, the bacillary dysentery has become the fourth highest incidence, secondary to viral hepatitis, tuberculosis and syphilis, of which Shigella flexneri 2a was responsible for 50-70% .Currently the traditional way to treat dysentery is the use of antibiotics, but different serotypes have no cross-immunity and there are cross-resistant. The number of multiple drug-resistant strains which isolated from clinic is increasing, the conventional antibiotic therapy has met a huge challenge. While the most effective way is the use of vaccine treatment, few are known about its pathogenesis of Shigella and the host immune protection mechanism till now. No ideal and effective vaccine has been developed now. Therefore, it is great significance for the research about Shigella.We found that two protein spots with the same molecular weights and the different isoelectric points in the two-dimensional electrophoresis map represented HtrA protein. The abundance of these spots changed with the expression of the ArgT protein. This phenomenon might indicate that the post-translational modification of HtrA was closely related with its functions.HtrA, also known as DegP, is the periplasmic serine protease Do, a kind of heat shock protein. The chaperone function of HtrA dominates at temperatures below 35°C and that the proteolytic activity increases dramatically at temperatures above 35°C. We are trying hard to find its natural substrate proteins and its new functions. Given that Shigella invade epithelial cells at 37℃but not at 30℃, proteins with different functions between these two different temperatures could be related to Shigella virulence. In particular, HtrA protein has been reported as a virulence factor in other pathogens.Because the studies have shown that HtrA protein in E. coli has the function of both protease and molecular chaperones. We want to carry out an in-depth and detailed analysis of the function of HtrA protein in S. flexneri 2a 2457T and 301.Firstly, we knocked out htrA genes of S. flexneri 2a 2457T and 301 to construct htrA deletion mutants using an improved method based on theλ-Red recombination system. Then, we constructed recovery mutants with and without a special label using low copy plasmids. We use the Sereny tests to study the impact of the deletion of gene htrA on the virulence of S. flexneri 2a 2457T and 301. The results showed that the mutant strains could not cause corneal inflammation in guinea pigs, while wild type and the recovery strains were able to provoke strong kerotoconjunctivitis in guinea pig. After preparation of protein samples of the wild type and mutant strains grown at 30℃and 37℃, comparative proteomic analyses were performed using two-dimensional electrophoresis. The differential expressed proteins, which were most likely the natural substrates of HtrA protein, were identified by mass spectrometry. Particularly, those differences between two different temperatures might also be related to the virulence of Shigella. The results showed that there were lots of differential expressed proteins. Then, we conducted functional analyses of these proteins.Secondly, most proteins could not perform their functions independently, unless they formed a large complex with other proteins through protein-protein interactions. Here, the proteins interacting with HtrA were isolated by the method of GST pull down and coimmunoprecipitation. To rule out the influence of labels on the function of HtrA proteins, we use different labels marking the N-terminal and C-terminal of HtrA in this study. When the Flag tag was added to C terminal of the complete coding sequence of HtrA protein, HtrA protein was localized in the periplasmic space, like its natural conterpart. While, when the GST protein was fused to the N terminal of HtrA protein without signal peptide, the protein is localized in the cytoplasm. Moreover, in order to preventing HtrA from self-degradation, we change the 210-Ser into Ala. This kind of mutation could eliminate its protease activity without changing its structure. Then, we capture the interactive proteins in 30℃and 37℃, and separate the proteins though two-dimensional electrophoresis. These results might be more reliable then classic SDS-PAGE. Using this method, we found three proteins which may have a specific interaction with HtrA. These screening results need further verifications.In summary, we successfully constructed htrA gene deletion mutants of Shigella flexneri 2a 2457T and 301; we have constructed htrA recovery mutants and analyzed the pathogenesis by Sereny text; the natural substrates may possible be obtained by comparative proteomics; using GST pull down and immunoprecipitation, we captured the interactive proteins, and separate these proteins by two-dimensional electrophoresis. Since the virulence of Shigella are different at 30℃and 37℃, the proteins differential expressed at two temperatures might also have effects on virulence. All of results mentioned above can make further understandings about the biological functions of HtrA.