Study Of Yersinia Pestis Proteins Influence On Host NF-κB Pathway And YpkA Kinase Activity

Objectives:1. Invasion of pathogenic bacteria may cause the activation of NF-κ B signalpathway in host, Yersinia pestis proteins SycE, YscL and YscS are component of T3SS,YscL is a regulatory protein to provide energy for the transport of T3SS effector proteinYscN ATTPase. YscS is a component of protein secretion apparatus. Through thedetection of Yersinia pestis proteins SycE, YPO2940, YPO3877, YscS and YscLinfluenting on the host NF-κ B signal pathway, it may provides the basis research for thepossible pathogenesis of Yersinia pestis.2. YpkA is an effector protein of Yersinia pestis type III secretion system, VASP is akind of cytoskeleton in human regulatory protein, through validation of YpkA on VASPphosphorylation in vitro, preliminary discussion is made on the mechanism of Yersiniapestis virulence potential.Methods:1. Yersinia pestis bacteria were grown in LB liquid medium and the DNA genomicwere extracted. After designing the primers with software, and yscL gene was amplificated with PCR. Inserted the PCR products into the plasmid pCMV-Myc, and to constructrecombinant plasmid pCMV-Myc-yscL, sequencing analysis right and the large quantityplasmid were extracted without endotoxin, used to transfect293T cells.2. The plasmid of pCMV-Myc/YscL, SycE, YPO2940, YPO3877or YscS of thelaboratory was respectively extracted. Then every plasmid without endotoxin wastransfected into293T cells.3. Using the technique of transient transfection, the extracted plasmids weretransfected into293T cells, western bloting was used to detect the expression of theabove5kinds of protein in293T cells with anti-Myc antibody. Expression is correct, then the5pasimids and the plasmid pBII-LUC expressing firefly luciferase and plasmidpRL-TK expressing renilla luciferase were cotransfected into293T cells respectively.Then the293T cells were stimulated with TNF-α, luciferase board activity was detectedon the chemiluminescence.4. YpkA was expressed in the E.coli BL21and purificated with GlutathioneSepharose4B beads; The expression of VASP in293cells, and Flag beads were used topurificating the VASP. The purified YpkA, VASP, ATP and kinase buffer were mixed,incubated at30℃for30min, phosphorylation of VASP was detected by western blotingmethod and isotope labeling method respectively by positive and negative control group.5. To analysis the experimental results wih statistical methods of variance analysis,SPSS13.0software was used.Results:1. The results of sequence showed that pCMV-Myc-yscL was constructed correctly;2. The WB results showed that SycE, YPO2940, YPO3877, YscS and YscLexpressed correctly in293T cells.3. Dual luciferase reporter assay results showed that the luciferase activity of SycE,YPO3877, YscS and YscL in the experimental groups were decreased, compared withthat of the negative control group, which means the difference was statistically significant(P <0.05), while YPO2940group were not (P>0.05).4. The phosphorylation of YpkA on VASP was not detected with westen blotingmethod, while the isotope labeling method was.Conclusion:1. SycE, YPO3877, YscS and YscL proteins of Yersinia pestis can inhibit NF-κBsignal pathway activation, while YPO2940do not.2. VASP can be phosphorylated with YpkA in vitro.