| Streptococcus suis is an important zoonotic diseases mainly caused by Streptococcus suis type 2(S.suis 2). Bacterial two-component regulatory system (TCS) is an important sensing and control systems by which bacteria can sense the complex outside environment, and maintain their own survival, growth and reproduction.1358hk/rr is one of the 15 pairs of S.suis 2 TCS, also common in other bacteria. Sequence alignment showed that S. suis 2 1358hk/rr is highly homologous with Streptococcus pneumoniae VicK/R that has 81% same amino acid sequence. In VicK histidine kinase gene deletion Streptococcus pneumoniae, the current result found that the growth, biofilm formation, bacterial growth slows down and the virulence is reduced. Therefore, the VicK gene is necessary for the growth of Streptococcus pneumoniae. We do a lot of work to make the gene deletion, but failed. So we assume that 1358hk/rr may affect the growth or survival of the S.suis 2.At present, there are some homology models about Streptococcus pneumoniae and Staphylococcus epidermidis for the histidine kinase YycG, and some effective inhibitors have been screened from Compound library, while there is still nothing relevant report about S.suis 2. Therefore, this study is focused on S.suis 2 histidine kinase (1358hk). After the homology modeling, we do the virtual screening for the pilot inhibitors which against the target that can inhibit the growth of S.suis 2. The main contents are as follows:1.1358hk Homology Modeling and AnalysisUsing homology modeling approach constructed three-dimensional structure of protein 1358hk, and verified the rationality of this model by the software Procheck. The results showed that 1358hk and Thermotoga maritima X numbered 3DGE in the PDB have 33% consensus sequence,57% similarity about the diffraction structure (resolution 2.8 A). The modeling structure of 1358hk can be well superimposed with the template. The results showed that:The model of S.suis 2 to 1358hk is qualified, so it can be used to do molecular docking by Sybyl7.3 to screen small molecule compounds.2. Virtual screening for inhibitor of S. suis 2 1358hkIn this study, we used docking algorithms FelxX to screen SPECS database for compounds with high scores. Through scoring with Xscore block of Syby17.3, we got some compounds. Then the 108 compounds selected by medicine analysis were screened by FlexX refering to artificial experience. After four rounds of screening in different strategies, we finnaly got 40 compounds from SPECS database.3. Enzyme activity identification of S. suis 2 1358'As 1358HK is a transmembrane protein, the intracellular fragment 1358'of kinase domain was expressed. By PCR amplification of 1358', the pET28a-1358'recombination plasmid was constructed. Then it was transformed in E. coli BL21 cells and induced by IPTG. The recombinant protein was expressed, purified and identified by SDS-PAGE and Western Blot. We applied fluorescence kinase assay to test the purified protein activity. The result showed the recombinant protein 1358'was approximately 35kDa and the purified protein could react with ATP.4. Experimental verification of lead compound in vitroBy screening the 40 compounds selected from SPECS database with purified 1358', we hoped for small molecular compound could inhibit hydrolysis of ATP. The result came out to be compound 1 inhibited 66% reaction when the concentration was 500μM. When the concentration declined to 100μM, the inhibition rate was 50%. However, we detected no inhibition of S. suis 2. In this study, we found the compound which could inhibit 1358' activity at low concentration, which providing experimental evidence for further selection of new antibacterial drugs. |
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