The Mechanism Of Berberine In Inhibition To Shigella Flexneri And The Function Of GadB

Shigella spp. is one of the most common causes of diarrhea of human pathogen. It is still a serious threat to human health. Since antibiotics have some advantages such as a quick effect and low cost, antibiotics treatment of Shigella spp. has its practical significance. But the antibiotic resistance plagued the development of antibiotic treatment. So to study the molecular mechanism of antibiotic resistance in Shigella spp. and pathogenesis posseses a certain theoretical significance and has potential applicable value. In the present thesis, Shigella flexneri 2a stain 301 was utilized as biomaterials, the effect of berberine on the growth and its mechanisms were investigated, and the data were showed as follows:First, effect of berberine on the growth of Shigella flexneri 2a stain 301 was dependent on the time of berberine treatment. The survival ratio of strains treated by berberine in the early logarithmic phase was much lower than that treated in early stationary phase, which was 62.45% (control, 152.6%) and 91.22% (control, 93.01%) respectively.The two-dimensional gel electrophoresis (2-DE) was applied to detect the effect of berberine on the expressing of proteins of Shigella flexneri 2a stain 301. The results revealed that effect of berberine on the expressing of proteins was dependent on the addition time. Nine proteins were identified as berberine influenced proteins when the strain was treated by 160μg/mL in early logarithmic phase, e.g. GadB, GadA, OsmY, SodC, Bfr, YgiW, YfiA, OspC2 and YqhE, which were down regulated. In these proteins, OsmY is involved in osmotic regulation; GadB and GadA are related to acid resistance, OspC2 is correlated to virology, and SodC is involved in antioxidant. Three proteins were identified as berberine influenced proteins when the strain was treated by 160μg/mL in early stationary phase, e.g. Dps and GapA were up-regulated and FabG was down regulated. It was interested that the expression of GadB, the glutamate decarboxylase was significantly down regulated by berberine. The enzyme might have important role in the pathogenesis of Shigella flexneri 2a stain 301.The null-mutant strain of gadB was successfully constructed. The mutant could not use glycerol as the only carbon source. Sereny test revealed that the virulence of gadB mutant were slightly weaker than wild-type strain. Deletion of gadB would influence the expressions of some proteins.A total of 10 proteins were identified as differentially expressed proteins according to the comparative proteomic results. The six proteins, Pgi, Pgk, Gntk, SdhB, LpdA and LldD were involved in energy and macromolecule metabolisms and two proteins, AphF and FkpA were related to shock responses. These data provided some trails for the function research of GadB.