Study On Adhesive Characterization Of GrpE In Mycoplasma Gallisepticum And The Mechanism Of Induction Of The Expression Of IL-1?

Mycoplasma gallisepticum(MG),a primary pathogen of causing chronic respiratory disease in chickens and infectious sinusitis in turkeys,adheres to and infects the host cells by LAMPs(lipid-associated membrane proteins,LAMPs).To find vital adhesion protein of the MG LAMPs,our previous study separated and identified components of LAMPs including GrpE through FPLC(fast protein liquid chromatography,FPLC)and MS(mass spectrometry,MS)analysis.Herein,Grp E was regarded as the candidate target by relative tests.Recombinant protein GrpE(rGrpE)was expressed by BL21 cells and the rGrp E was used to prepare for the monoclonal antibody(MAb).Flow cytometry test and Laser confocal microscope confirmed that rGrpE could obviously adhere to the surface of the DF-1 cells,which could be significantly inhibited by MG positive serum.ELISA tests indicated that rGrpE could adhere to DF-1 cells in a dose-dependent way,which could be significantly suppressed by MG positive serum.In conclusion,our results suggest that GrpE is an adhesion related protein of MG.The PAMPs(pathogen associated molecular patterns,PAMPs)of pathogens can interact with PRRs(pattern recognition receptors,PRRs)located in the surface of host cells,resulting in induction of the expression of pro-inflammatory cytokines,such as,IL-1?,TNF-a,IL-6,IL-12.These inflammatory cytokines have a positive impact on defending the invasion of pathogens.Previous studies indicated that not only MG LAMPs played a significant role in promoting the process of MG adhering to host cells,but PRRs located in surface of host cells could recognize MG LAMPs,leading to the activation of NF-?B signal pathway and the expression of pro-inflammatory cytokine IL-1?.However,which component of LAMPs plays such an important role in this process remains obscure.Therefore,to address this question,we took advantage of the DF-1 cells to investigate the function of rGrpE.SYBR Green I Real-time PCR and ELISA tests displayed that rGrpE could induce the expression of IL-1? in a dose-dependent way in DF-1 cells.Western blot test confirmed that rGrp E could induce the phosphorylation of p65 in DF-1 cells.Nuclear translocation of p65 induced by rGrpE was detected by confocal laser microscope.The expression of IL-1? was decreased significantly after the function of NF-?B was inhibited by SYBR Green I Real-time PCR and ELISA tests.In conclusion,these results show that rGrpE can induce the expression of IL-1? in DF-1 cells through activation of the NF-?B signal pathway.Taken together we conclude that GrpE is an adhesion related protein of MG and can induce the expression of IL-1? in DF-1 cells through activating NF-?B signal pathway.