Research On Expression Regulation Of ArgT And Its Function In Shigella Flexneri 2a 2457T

Shigella flexneri is a gram-negative facultative pathogen which causes the most communicable bacterial dysenteries by invading the epithelial cells. The traditional treatment to dysentery is use of antibiotics, but more than 95% strains produce the resistance to various antibiotics. The best treatment is use of vaccine, but few are known about Shigella's pathogenesis and the host's immune response till now. However, the virulence of Shigella spp. is the interaction result of many proteins encoded by both its chromosome and the virulent plasmid, and the proteins involved in must be more than the known virulence factors. Therefore finding the novel virulence genes and confirming their expression regulatory is the most important works.In the former study of comparative proteomic analysis of S. flexneri 2a 2457T and its derivative strain without large invasive plasmid at 30℃and 37℃in our lab, a strange protein spot was found. Then a high-resolution comparative proteomic analysis was performed to definite the expression changes of these region proteins. The results showed that the strange spot is corresponding to two proteins. One is PhoN2, a known virulence factor, which is upregulated at 37°C; the other is ArgT, a lysine-arginine-ornithine binding protein, which is upregulated at 30°C. At present, there is no report about the expression regulation of ArgT related to temperature. Given that the virulence factors of S. flexneri are produced at 37°C, but not at 30°C, we suggest that ArgT should be related to the virulence of S. flexneri.First, a series of experiments were disigned to uncover the regulatory mechanism of ArgT differential expression. We proved here that: 1) the differential expression of gene argT was not regulated at RNA level, but at protein level; 2) ArgT was degraded in periplasm at 37°C. 3) ArgT was degraded by a periplasmic protease HtrA directly, which was the only protease to response to the proteolysis, and whose activity, but not synthesis, was affected by temperature; 4) the cleavage site in ArgT was between position 160 (Val) and position 161 (Ala); and 5) the tyrosine residue at position 225 of ArgT might be a key site recognized by protease HtrA.Furthermore, we estimated the effect of ArgT to the virulence of S. flexneri. As is well known, the evolution of pathogen from its non-pathogenetic ancestor involves in acquire of virulence genes and lose of anti-virulence genes. An anti-virulence gene is not necessary in pathogen, and its compulsive expression will attenuate the virulence. In S.flexneri 2a 2457T, gene argT was normally expressed at 30?C, but degraded by HtrA at 37?C; and it is normally expressed in E.coli, but mutated and degraded in S.flexneri. All of the evidences seemed to suggesting the relationship between the function of ArgT and the virulence of S. flexneri. So, the competitive invasion assays to HeLa cells were performed and the results indicated that the deletion of argT would not effect the virulence but the compulsive expression of ArgT would attenuate the virulence of 2457T. At the same time, another competitive invasion assay to lung cells of Balb/c mice were also carried out, and the results indicated the compulsively expression of ArgT would attenuate the virulence. Thus the results suggested that argT was a potential novel anti-virulence locus in S. flexneri,and represented a novel mode by proteolysis.Finally, we further explored other functions of ArgT. Proteins could not perform their functions independently, unless they formed large compound with other proteins by interactions to complete the functions. Here, the proteins interacting with ArgT of the whole proteins from S.flexneri 2a 2457T were isolated by the method of GST pull-down and 2DE, among which OmpR and Cyp were identified by mass spectrum. OmpR is a part of osmotic response regulator OmpR-EnvZ and also a virulence factor, which regulates the expression of vir and OmpF/C. In addition, the deletion and over-expression of genes must affect other proteins expression. Then the comparative proteomic analysis of S. flexneri 2a 2457T, argT deletion mutant and ArgT over-expressed mutant were performed. 10 differential expression spots, representing 9 proteins were discovered. In the argT deletion mutant, 2 proteins were up-regulated, 3 were down-regulated; while in ArgT over-expressed mutant, 3 proteins were up-regulated, and 2 were down-regulated.In conclusion, the regulatory mechanism of argT differential expression at 30?C and 37?C in S.flexneri 2a 2457T is that argT was normal expressed when cultured at 30?C (strains without invading capacity), but degraded by the periplasmic protease HtrA when cultured at 37?C (trains with invading capacity) because HtrA has protease activity at 37?C, but not at 30?C. Moreover, argT is normally expressed in E.coli (non-pathogenetic strains), but mutated at three sites and degraded in S.flexneri (pathogenetic strains). Furthermore, the competitive invasion assays in HeLa cells and in Balb/c mice indicated that the compulsively expression of ArgT would attenuate the virulence of 2457T, suggesting that argT was a potential novel anti-virulence locus in S. flexneri. In addition, protein interaction and comparative proteomic analysis provided the reference to the research on ArgT function.