Research On The Function Of Effector IpaH9.8 In Shigella Flexneri 2a 301

Shigella (Shigella spp.). also known as dysentery bacillus, is a group of Gram-negative non-spore forming pathogenic bacteria which cause shigellosis. Shigella contains 4 species and 48 serotypes. including Shigella dysenteriae. Shigella flexneri, Shigella sonnei and Shigella boydii. Shigellosis spreads all over the world. The global number of annual incidence reaches 1.6 hundred million and over 95% of deaths occurred in developing countries. In China the incidence of shigellosis is over 4 hundred thousand, only next to hepatitis and tuberculosis. Shigella flexneri is the main strain of Shigella which causes shigellosis in developing countries, especially in China. Shigellosis also breaks out frequently in developed countries whose sanitation conditions are good. The incidence will be even higher in wartime or under natural disaster. It will weaken the power of armed forces and bring in unfavourable effects to disaster rescue. Although there are some medicine for shigellosis. the disease is still easily to change to be chronic or long-term bacteria carrier. Furthermore, the drug-resistance of the strain becomes increasingly seriously. At the present stage, the main therapeusis of shigellosis is antibiotic. But the appearance of the resistance and multidrug resistant strains raise the degree of difficulty in dysentery's prevention and cure, therefore, it is important for the development of new-medicine and vaccines to carry out the research on genetic and pathogenic mechanism of Shigella.Shigella causes shigellosis through infecting the human intestinal tract, on which pathogenicity relies the type III secretion system (T3SS). Shigella can activate the T3SS and inject the virulence proteins into the host cells when contect the epithelium. These virulence proteins can affect the functions of the cells, for example, which make the cell membrane shrunken, affect the skeleton of actin. activate the signal pathway of host cells, induce macrophage apoptosis and so on. The proteins secreted by T3SS can make the bacteria survived in host cells, but uninjured the host excessively. It can be seen that there is some concordance between the pathogenic mechanism of bacteria and the survival of host. Recently, it becomes prevalent that research on the T3SS of Shigella and the effectors functions of T3SS. The IpaH proteins of Shigella are encoded by chromatosome and plasmid. There are five copies of the ipaH genes exist on the big plasmid of S. flexneri 2a 301. which are ipaH1.4. ipaH2.5, ipaH4.5. ipaH7.8 and ipaH9.8. Previous researches show that IpaH9.8 can down regulate the expression of inflammatory factors and repress the inflammatory reaction obviously when Shigella invading the host cells. but which particular functions and the role in Shigella is still unknown now.Gene knock-out is an important skill platform on the study of functional genomics which is also one of the essential means on the research of pathogenic mechanism of bacteria. To explore the function of IpaH9.8 of S. flexneri 2a 301. the ipaH9.& gene was knocked out by the utilization of theλ-Red homologous recombination. Then we obtained some information about the functional correlation with the gene via analyzing the differences in the phenotype and functions between the deletion mutant and the wide-type strains. We successfully knocked out this gene using an improved method which based on theλ-Red recombination system. Then, a reverse mutant was also obtained by introducing a low-copy plasmid containing one copy of ipaH9.8 gene into the deletion mutant. Moreover, we verified the deletion mutant and the recovery mutant on molecular level by PCR and RT-PCR. It was proved that we had deleted ipaH9.8 gene, but remained the other genes of the ipaH family. Subsequently, we measured the growth curves and some basic biochemical events of wide-type strain, deletion mutant and reverse mutant respectively. To contrast the virulence phenotype of wide-type strain, deletion mutant and reverse mutant, we carried out the experiments as follows:the cell invasion test. Sereny test. E1ISA and the mouse pneumonia model. The results showed that there were no significant differences in the growth curves and the basic biochemical events among three strains, which illustrated IpaH9.8 has no influence on the growth and the basic biochemical metabolism of S. flexneri. All the strains could invade the Hela cells and the mouse macrophages J774.A.1 which made the nuclei disrupted, resulted in the cells death. There were no significant differences in colonv count after the strains invadine the Hela cells and J774.A.1, indicated that the absence of ipaH9.8 gene has little effect on the invasion of Shigella. The three strains also could cause inflammatory reaction of the guinea pigs' keratoconjunctivitis and the deletion mutant was much severely than the other two. We presumed that IpaH9.8 might inhibit the host inflammatory responses. Therefore, Then, we did a ELISA analysis of IL-1βand TNF-a in the supematants of J774.A.1 cells infected for 2 hours with wide-type strain, deletion mutant and reverse mutant. The result displayed that compared with the deletion mutant, the wide-type strain and the reverse mutant obviously repressed the secretion of the inflammatory factors. We confirmed that IpaH9.8 could inhibit the host inflammatory responses via the experiment of the mouse pneumonia model.After those analyses, comparative proteomic studies of the whole cell proteins of these three strains were performed by means of two-dimension gel electrophoresis (2-DE) combined with MALDI-TOF MS. The results showed that the expressions of 10 proteins were induced when ipaH9.8 gene were absent, while the expressions of 15 proteins were repressed, and 1 protein long-chain fatty acid outer membrane transporter exhibit clear post-translational modification. Among those proteins, key stress proteins DnaK and GroEL were down regulated and protein disaggregation chaperone was up-regulated. The most striking fact of our observation was many outer membrane proteins, shch as NmpC OmpF, OmpA. OmpA precursor, and OmpMxiD. were decreased or incresed in the ipaH9.8 gene deletion mutant, these resulta showed IpaH9.8 had an important effects on the invasion of Shigella and innate immunity of host. Furthermore. Pro-Q Diamond staining analysis revealed that IpaH9.8 trigger long-chain fatty acid outer membrane transporter phosphorylation at 131T/136T. These findings extend our knowledge of the function of IpaH9.8 protein and are useful for the research of pathogenesis of S. flexneri.In the above research, the growth curves and the basic biochemical events expand our knowledge about physiological nature of Shigella. The results of the cell invasion test suggest a very important clue on the invasion mechanism of Shigella. And the Sereny test also provides us many clues for the understanding of immune escape of S. flexneri Additionally, the comparative proteomic data also provides valuable information for screening new virulence-related proteins. Moreover, these results, the methods improved in this research and the experiences gained from this research would be helpful for the future research on pathogenicity of Shigella spp.