Construction And Evaluation Of Two-component Signal Transduction System AgrC/AgrA From Staphylococcus Aureus

Staphylococcus aureus as an important human pathogens that can cause a variety of infections. Accessory gene regulator system which regulate virulence factor system, plays an important role in the pathogenic process. The system consists of a two-component signal transduction systems(TCSTs, AgrC/AgrA), upon accumulation of AIP with increasing cell density, it induces phosphorylation of AgrC, followed by a second-step phosphorylation of the response regulator AgrA. AgrA can then bind to speci?c direct repeats in the intergenic region between the RNAII and RNAIII promoters. Activation of RNAIII, the agr effector molecule, results in repression of many surface-associated proteins while promoting exoprotein gene transcrip-tionand, to a lesser extent, translation. In vitro studies have shown: although obtain high level expression, easy to degrated by host protease or form inclusion body in the process of AgrA heterologous expression. It is against target protein function research in vitro. Further more, the membrane protein AgrC topological orientation is not clear in the proteoliposome, it make more difficult in vitro study of TCSTs. Research on the two-component signal transduction system of Staphylococcus aureus, it will provide a new method to screening new drugs in vitro.Firstly, we construct a expression vector using Restriction-free(RF) cloning. In order to monitor the protein expression level in real time, a C-terminal green fluorescent protein(GFP) was fused to AgrA to serve as a reporter. Host strain was screened by single-factor experiment. And then, combined with the Box-Behnken designed response surface method test, the cultured conditions which include culture time 、 RPM and IPTG concentration were optimized..Secondly, The AgrC proteoliposome reconstituted by detergent-mediated method. In order to built an artificial protein reconstitute system which not only maintain biological activity but has a high stability and high reconstitute efficiency. Based on a series of characterization methods(fluorescence microscope, dynamic light scattering and transmission electron microscopy), we investigate the stability and reconstitute efficiency of lipid influenced by hydration buffers and phospholipid complex which contains different porportion of negative phospholipid and cholesterol. Meanwhile, we explored AgrC topology orientation influenced by different dissolved state of liposome.Lastly, the activity of AgrA was assessed by using non-radioactive electrophoretic mobility shift assay(EMSA) based on Agr A protein Lyt TR region. we constructed artificial simulation of two-component signal transduction system model in vitro, verify the validity of the model through the electrophoretic mobility shift experiment(EMSAs). The results as follows:1. E. coli BL21-(DE3)-PlysS was screened as the best host cell. The optimum conditions selected are as follows: induction time is 22 h, speed is 222 rpm, IPTG is 0.5 mM. The expressed AgrA were purified using immobilized metal affinity chromatography(IMAC) and size exclusion chromatography(SEC) with the yield of AgrA is 5.56 mg/L. The activity of AgrA was cofirmed by using non-radioactive electrophoretic mobility shift assay(EMSA) based on AgrA protein Lyt TR region.2. HEPEs is the best hydration buffer. DOPC/DPPA/ chol(2/2/1, mol/mol/mol) is the best phospholipid complex. Protein /phospholipid(1/500, mol/mol) is the best proportion of reconstitution. Meanwhile, there has 60%~70% C-terminal cytoplasmic domain of AgrC protein reconstituted into liposomes are inside-orientated which not only remain a highly kinase activity but in keeping with AgrC protein orientation in biological membrane.3. The activity of Agr A was cofirmed by using non-radioactive electrophoretic mobility shift assay(EMSA) based on AgrA protein Lyt TR region. Two-component signal transduction system model can enhance AgrA delay effect of DNA proved AgrC/AgrA two-component signal transduction model is effective. By AgrC EMSA experimental analysis, because the proteoliposome can maintain a more activity of the membrane protein AgrC, so it has a significant influence on EMSA experiment.To sum up, through the construction of two-component signal transduction system model in vitro, not only successfully solved the problem of the transcriptional regulation factor AgrA soluble expression, but built an artificial protein reconstitute system which not only maintain biological activity and high stability but has a high reconstitute efficiency and oreintation controled. On the base of EMSA, we preliminaryly verified the validity of the TCSTs model.It laid a foundation to screen Staphylococcus aureus signal transfer inhibitors for the next step in vitro.