Regulation Of Virulent Plasmid On Chrosome In Shigella

Shigella spp. are gram-negative, facultative anaerobes, non-spore forming pathogens. The Shigella spp. mainly spread through fecal-oral route. After entering the digestive system through food and water they release virulence factors to break the barriers of gastrointestinal and invade into and replicate within the colonic epithelium, which results in an infection characterized by fever, dehydration, hematochezia, abdominal cramps and diarrhea. The bacteria cause a disease called shigellosis in humans and it will even lead to bacteremia. The children are Susceptible population owing to their low immunity. The infection dose is low and normally 10-100 bacteria can cause classic infection symptom. That's one of the causes of why shigellosis is the main infectious disease in the developing countries. According to rough statistics, 163 million of 165 million cases are in developing countries each year and the infection rate is also high in China. Furthermore, the current treatment for bacterial infections relies on antibiotics which were effective at first. However, the resistance strains emerge because of mutations, leading to over 90 percent of strains with the resistance to many kinds of antibiotics. Although vaccines can be effective methods for treating shigellosis, the detailed pathogenesis of Shigella spp. and the host's immune response are still not completely revealed until now, which has been the difficulty of the study of vaccines and the clinical applications. Owing to the harm of Shigella spp. and the limitation of therapies, it is important and urgent for us to carry out the basic research of Shigella spp..Escherichia coli and Shigella spp. have high homology and resemblance on the level of genome although there are differences between them in phenotype and pathogenicity. There has been reported that Shigella has evolved from Escherichia. The pathogenicity of Shigella is closely related to the virulent plasmid which is about 220 kb possessing most of the virulent genes.It has been found that virulent plasmid can not induce K-12 to have the pathogenicity similar to that of Shigella, which indicates that the evolution from Escherichia to Shigella involves the mutual enhancement between virulent plasmid and chromosome. The pathoadaptive mutation is a kind of coevolution which enables the mutant strain owning the plasmid to better adapt the environment.The report about the regulation of chromosome by plasmid is rare at present, which is exactly the original intention of our research. With the development of genomics in recent years, the sequence analysises of many typical Shigella strains, such as S. flexneri 2a 2457T and 301, have been completed, which paves the way for the further advancement of proteome. The comparative proteomic-based approach applied in research mainly focuses on the mutual regulation between chromosome and plasmid and will provide us many important clues for the understanding of pathogenesis.Firstly, in this study, using the principle of plasmid incompatibility, two derivative strains without large virulent plasmids, 301△pCP and M90T△pWR501, were constructed. And then, three mutants (MG1655/pXL275-virG:pSF, MG1655/pXL275-virG:pCP and MG1655/pXL275-virG:pWR501)were obtained by the means of conjugation which transferred recombinant virulent plasmids of 2a 2457T, 2a 301 and 5a M90T to Escherichia K12 MG1655. To eliminate the effects of pXL275 upon the research, a negative control mutant, MG1655/pXL275, was consequently constructed.On this basis, API-strep biochemical test were applied and the results showed that the virulent plasmid and pXL275 have no significant effects on the metabolism of MG1655.What's more, the comparative proteomics research was carried out to analyze the proteomic changes of strains. In this study, 2D-gel electrophoresis was adopted to identify the differential protein expressions between mutants and wild-type strains cultured at 37℃. The protein spots between mutants and wild- type strains were excised from gels, in-gel digested and indentified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF/TOF) in combination with Mascot search program. The following conclusions were drawed from the comparisons of protein expression profiles between mutants and wild-type strains: 1) The pXL275 has no obvious effects on the expression of MG1655. 2) The conjugation of plasmids into MG1655 can induce the expression of many virulence related proteins while inhibit the production of glutamate decarboxylase isozyme which is closely related with the survival of bacteria in intestinal flora. 3) The curing of virulent plasmid leads to the absence of IpaA, IpgC, VirA, Mxi-Spa and many other virulence proteins.What the 2D-gel electrophoresis shows is the message of denatured proteins, but most of the proteins play the biological roles in the form of complex within the cell. In this study, Blue Native PAGE combined with SDS-PAGE technology was applied to get a better understanding of protein complex. The results show that pXL275 does not influence the expression of protein complex in MG1655, while the conjugation of virulent plasmid into MG1655 leads to notable change of the variety of complex. In summary, this study has not only successfully constructed virulent plasmid deletion mutants, conjugation mutants and negative control mutant, but also gets the message related to differential protein expression of monomeric proteins and complex. All these work provides us many important clues for the understanding of mutual regulation between chromosome and virulence plasmid. What's more, the conjugation technology applied in this study can also provide new perspective for the construction of mutants.