The Research On The Function Of Shigella Flexneri 2a GlpR,deoR Gene

Shigella spp. is a group of Gram-negative, non-spore forming, facultative pathogens. The bacteria cause a disease called shigellosis in humans, an infection of the large bowel characterized by abdominal cramps, diarrhea, and fever. There are four different species of Shigella based on the differences in O antigen and some biochemical reactions. Among them, the Shigella flexneri and Shigella sonnei are distributed more wider than the Shigella boydii and Shigella dysenteriae. According to statistics of WHO, there're approximately 164.7 million cases every year, of which 1.1 million are dead , most of which are children under 5 years old.Serum type 2a of Shigella spp. is responsible for the majority of cases of endemic dysentery prevalent in developing countries where sanitation is poor.In recent years , about 20 million people are infected every year and its one-year cumulative incidence rate is at the third place merely inferior to the pulmonary tuberculosis and hepatitis B. Owing to the low infectious dose (10 to 100 bacteria) , person-to-person transmission is probably the most common route , and the emergence of multiple resistance strains, the traditional treatment by antimicrobial agents encounters big problems. Because the mechanism of pathogenicity and immunoprotection are unknown, no ideal and effective vaccine has been developed till now.It was reported that the availability of invasion into HeLa cells when cultivated at 37 oC but not at 30 oC. The compared protein proteome study of S. flexneri 2a 2457T in laboratory cultured in different temperatures found that: When the temperature of culture rose to 37℃, GlpQ, GlpB proteins expressed increasely. When the virulent plasmid of S. flexneri 2a 2457T was removed, GlpQ, GlpB proteins also expressed increasesly significantly. Obviously, GlpQ, GlpB proteins have some influence on the virulence of S. flexneri 2a 2457T. And GlpQ, GlpB proteins are regulated by the GlpR, DeoR, H-NS proteins,owing to some research about the H-NS proteins have been reported which is discussed behind, and GlpR, DeoR protein belonged to DeoR protein family, which is a bacterial regulatory protein family (PF00455). DeoR is a transcriptional regulator, which regulate the metabolism of deoxyribose. GlpR is also a transcriptional regulator to regulate the glycerol-3-phosphate.To explore the function of glpr, deoR genes of S. flexneri 2a 2457T, we firstly successfully knocked out the genes using an improved method based on theλ-Red recombination system established by Datsenko and Wanner. Then, the recovery mutants were also obtained by introducing a low-copy plasmid containing one copy of glpR, deoR genes into the deletion mutant. Subsequently, some experiments were carried out as following: 1) the growth curves of wild-type strain, deletion mutant and recovery mutant were measured respectively. 2) some basic biochemical events were also investigated. All of the results of above studies showed no significant differences between three strains. We use the Sereny tests, HeLa cell model and the mouse model to study the impact of the deletion of gene glpR, deoR on the virulence of shigella. We found that wild strain, the mutant strains as well as the recovery strains were able to provoke keratoconjunctivitis in guinea pig and the deletion of glpR gene will markedly enhance the virulence of Shigella. The whole-cell protein proteome were studied by means of two-dimension gel electrophoresis (2-DE) combined with MALDI-TOF MS. The analysis of comparative proteomics showed that the expressions of 14 proteins were induced while the expressions of 3 proteins were repressed in the deletion mutant and recovery mutant of the glpR and deoR genes. The results showed that the deletion of glpR, deoR could induce the expression of a series of proteins which were inhibited by them. In the glpR gene deletion mutant, the expression of glycerol kinase (GlpK), 3 - phosphoglycerate dehydrogenase B subunit (GlpB) and phosphodiesterase (GlpQ) was increased. And in the deoR gene deletion mutant, the expression of thymidine phosphorylase (DeoA), purine nucleoside phosphorylase (DeoD) and 2 - deoxyribose - 5 - phosphate aldolase (DeoC) were increased.YciD (NP-836950) is a hypothetical protein (22kDa) containing 212 amino acids. It was one of the members of OmpW family according to its conserved sequences. However, its function is still unknown. The results of comparative proteomics between the S. flexneri 2a 2457T wild strain and the gene hns (also known as virR) deletion mutant showed that the expression of YciD was decreased gene hns deletion mutant under 37℃. We think that the protein is controlled by H-NS. The yciD gene deletion mutant was constructed by improvedλ-Red recombination system in order to study the further function of gene yciD.