Study On The Biological Function Of MSLP53,a Secretory Protein Of Mycoplasma Synoviae

Mycoplasma synoviae(MS)infection is a respiratory tract infectious disease caused by Mycoplasma synoviae in chickens.Some secreted proteins belong to amphitypy protein,which exhibit the characteristics of membrane proteins in addition to their functions as secreted proteins.Lipid-associated membrane proteins(LAMPs)and secreted proteins play a key role in the pathogenesis of mycoplasma against the host.Previous laboratory studies have found that LP53 is a secreted protein of MS,and it is speculated to be related to the pathogenicity of MS.This study aims to explore the role of the secreted protein LP53 during the pathogenesis of MS by researching its biological function,whcih lays a molecular foundation for the study of the pathogenisis of secreted proteins in mycoplasma.Firstly,the full-length of the mutated MSlp53 gene was obtained by Overlap PCR amplification,and was used to construct two different prokaryotic expression systems,including pColdI-MS/p53-BL21 and pGEX-4T-MS/p53-BL21;Purified MSLP53 proteins with different tags,namely His-MSLP53 and GST-MSLP53,were obtained.The anti-His-MSLP53 rabbit/rat positive sera were prepared by immunizing rabbits and rats with purified His-MSLP53 protein.The results of indirect immunofluorescence assay showed that His-MSLP53 protein had strong cell adhesion ability,and the anti-His-MSLP53 serum not only significantly inhibited the adhesion of this protein to DF1 cells,but also inhibited the adhesion of MS bacteria to DF1 cells with the adhesion inhibition rate of 61.8%,which showed that MSLP53 protein played an important role in the adhesion of MS to host cells.In addition,the Western blot and ELISA results demonstrated that His-MSLP53 protein could bind to the extracellular matrix protein cPlg,and it was speculated that this binding effect was one of the mechanisms of its adhesion to host cells.Then,the cytotoxic effect of GST-MSLP53 protein on host cells was measured by MTT assay,and GST was used as a control.The results showed that GST-MSLP53 protein caused a significant decrease in HD-11 cell activity compared with GST,and the cytotoxic effect of GST-MSLP53 protein on HD-11 increased significantly with the increase of protein concentration(40,60,80,100,120 μg/mL).Next,the pro-inflammatory response of MSLP53 on HD-11 cells was examined.The effect of GST-MSLP53 protein on the expression of IL-1β in HD-11 cells at the transcriptional level as well as at the translational level was examined by qPCR and ELISA.The results showed that the pro-inflammatory cytokine IL-1β in HD-11 cells was significantly up-regulated at the transcriptional level as well as at the translational level after GST-MSLP53 treatment,using GST-treated HD-11 cells as a control.In addition,NF-κB is a common signaling pathway in cellular inflammatory and apoptotic responses,and its downstream signaling molecule p65 nucleus-entrance assay confirmed that GST-MSLP53 protein stimulation could induce significant p65 nucleus-entrance in HD-11 cells,thus tentatively demonstrating that MS upregulates the expression of pro-inflammatory cytokines through secreting MSLP53 via NF-κB signaling pathway.Finally,the effect of GST-MSLP53 on HD-11 cell was observed by microscopy,and it was found that HD-11 cells were crinkled,rounded,or even crumpled and died after 24 h of GST-MSLP53 protein treatment compared with the GST-treated group,and the damage of the cells increased with the increase of treatment concentration(50,100,150 μg/mL).Then,the apoptogenic effect of MSLP53 protein on HD-11 cells was examined.Cleaved-Caspase 3 was detected after rMSLP53 stimulation and the expression of cleaved-Caspase 3 increased with increasing treatment concentrations(50,100,150 μg/mL),indicating that GST-MSLP53 protein could induce apoptosis in HD-11 cells.Annexin V-FITC/PI staining assay detected the apoptosis of p3×Flag-CMV-14-MSlp53 transfected HD-11 cells,and the results showed that the cells produced apoptosis after 24 h of transfection,which further demonstrated the apoptogenic effect of MSLP53 on cells.In summary,rMSLP53 is a secreted protein as well as a membrane protein of MS WVU1853,which has good immunogenicity and its antiserum can mediate in vitro complement bactericidal.The protein has strong cell adhesion ability and its antiserum can significantly inhibit its adhesion to DF1 cells.Meanwhile,MSLP53 can bind to cPlg,which may be one of the mechanisms of its adhesion.In addition,MSLP53 has cytotoxic effects on HD-11 cells,causing significant morphological changes in HD-11 cells.It not only induced up-regulated expression of IL-1β at transcriptional and translational level in HD-11 cells via NF-κB signaling pathway,but also had an apoptogenic response to HD-11 cells,indicating its participation in the adhesion and pathogenic process of MS to host cells.It is worthwhile to research it at a deeper level and provide a molecular basis for the study of the biological functions of MS secretory proteins.