The Study On Gene Function Of CobB, Gtr Ⅱ And YciD From Shigella Flexneri 2a 2457T

Glucosyltransferases (Gtrs) and O-acetyltransferase (Oac) belong to integral membrane proteins, and they are responsible for the differentiation of serotypes of Shigella spp. GtrⅡis a specific serotype, which can glucosylate the O-antigen repeat unit in rhamnose III, and was proved to be useful in discriminating serotype Y and. serotype 2a. In prokaryotic organisms, CobB deacetylates acetyl-CoA synthetase (Acs). As the homolog of eucaryota Sir2(Silent information regulator 2), the CobB was deduced to be related with chromatin silencing. YciD is a hypothetical protein belonging to the OmpW family. The virulent protein expression of Shigella flexneri is controlled by temperature i.e. expressing at 37℃or losing expression below 30℃. In our previous study, the comparative proteomic results of S.flexneri 2a 2457T cultured at 37℃and 30℃showed that the YciD expression was up-regulated at 37℃, which inspired us that the yciD might be a virulence-associated gene.To investigate the three genes'functions and make it clear whether they are associated with virulence, the cobB, gtrⅡhad been successfully knocked out by using an improvedλ-Red recombination system. The mutant of yciD is a strain constructed in our previous study. These mutants are termed asΔcobB::kan, AgtrⅡ::kanΔyciD::kan. The results of sereny tests showed thatΔcobB::kan,ΔgtrⅡ::kan caused a slighter inflammation than the wild strain, and theΔyciD::kan is the slightest one. Subsequently, the whole cell proteins of wild strain and these three mutants were prepared and compared by using two-dimension gel electrophoresis (2-DE), the comparative proteomics results showed thatΔcobB::kan,ΔgtrⅡ::kan possessed little differences with the wild strain; however, a down-regulated protein was observed inΔyciD::kan. A further research on AyciD::kan functions was carried out and the whole outer membrane proteins of mutant and wild strain were prepared and detected by 2-DE,3 down-regulated outer membrane proteins were observed. During virulent evaluation, the competition index (CI) of infected Hela is 0.8 and the murine lung-infected is 0.9, Thus, we can conclude that cobB and gtrⅡmight not affect the invasion S. flexneri, the absence of yciD decreased the S. flexneri virulence and had little effect on their invasive ability.13 phosphorylated proteins phosphorylated in 2457T were detected by Western blotting.This research developed a vision of the gene functions of Shigella and provided a methodology on researching the post-translational modification genes and OMP genes.