Study On Effects Of Dnak And EF-Tu In Mycoplasma Hyorhinis’s Hijacking Of Host Fibrinolytic System To Break Through The Extracellular Matrix Barrier

Mycoplasma hyorhinis(Mhr)is a common pathogen of the upper respiratory tract of pigs with a high clinical infection rate.Under certain conditions,it can cause polyserositis(pericarditis,pleuritis and peritonitis),arthritis,pneumonia,otitis media and etc,which seriously affects economic returns.Meanwhile,Mhr has been found to be associated with gastric cancer and other human tumors,which can enhance the migration and invasion abilities,as well as the drug resistance of tumor cells.It poses a potential public health threat.The previous data of our laboratory and literature reports suggest that the systemic infection plays a key role in the pathogenesis of Mhr.But how Mhr breaks through the barrier to form systemic dissemination remains unclear.In this paper,we investigated the ability of Mhr to hijack host fibrinolytic system to help itself degrade the extracellular matrix(ECM)barrier.Subsequently,we studied the moonlighting functions of two Mhr proteins,heat unstable elongation factor(EF-Tu)and heat shock protein 70(DnaK)on Mhr surfaces and their roles as plasminogen receptors(PlgR)in Mhr’s hijacking of fibrinolytic system breaking the ECM barrier.The main research contents and results are as follows:1.Mhr hijacks the host fibrinolytic system to degrade and crosse the ECM barrier: Firstly,it was found that Mhr can bind multiple ECM components,including fibronectin,laminin,type IV collagen and vitronectin;Mhr could bind plasminogen(Plg)to the bacterial surface,which was indicated by indirect immunofluorescence and ELISA.The Plg bound on the surface of Mhr could be activated to fibrinolytic enzyme(Plm)by host Plg activator(tPA),but Mhr could not directly activate Plg by itself.Transwell model was further used to prove that Mhr treated with Plg and tPA could effectively hydrolyze ECM and penetrate ECM barrier.2.As one of the PlgR of Mhr,DnaK helps Mhr to hijack the fibrinolytic system,and has the function to adhere host cells and bind ECM: Firstly,a recombinant E.coli expressing rDnaK was constructed.and the recombinant protein rDnaK was expressed and purified.The specific anti-DnaK rabbit serum was prepared by immunizing rabbits.The exposure of DnaK on Mhr surface was proved by colony immunohybridization and flow cytometry.The ELISA result indicated that rDnaK protein bound Plg,which was competitively inhibited by ε-aminocaproic acid.The result of the substrate hydrolysis test proved that the rDnaK-bound Plg could be activated by tPA to form proteolytic Plm.Further studies showed that rDnaK promoted the process of Plg activation.These data suggested DnaK as one of the PlgR on Mhr surface.Furthermore,indirect immunofluorescence and microplate adhesion tests showed that rDnaK bound porcine PK-15 cells and human NCI-H292 cells,indicating the role of DnaK as one of the adhesins of Mhr.It could also bind various ECM components.3.As one of the PlgR of Mhr,EF-Tu helps Mhr to hijack the fibrinolytic system,and has the function to adhere host cells and bind ECM: Firstly,a recombinant E.coli expressing rEF-Tu was constructed.and the recombinant protein rEF-Tu was expressed and purified.The specific anti-EF-Tu rabbit serum was prepared by immunizing rabbits.The exposure of EF-Tu on Mhr surface was proved by colony immunohybridization and flow cytometry.The ELISA result indicated that rEF-Tu protein bound Plg,which was competitively inhibited by ε-aminocaproic acid.The result of the substrate hydrolysis test proved that the rEF-Tu-bound Plg could be activated by tPA to form proteolytic Plm.Further studies showed that rEF-Tu promoted the process of Plg activation.These data suggested EF-Tu as one of the PlgR on Mhr surface.Furthermore,indirect immunofluorescence and microplate adhesion tests showed that rEF-Tu bound porcine PK-15 cells and human NCI-H292 cells,indicating the role of EF-Tu as one of the adhesins of Mhr.It could also bind various ECM components.In summary,the results of this study indicate that Mhr can bind Plg to hijack host fibrinolytic system to help it breaking through the ECM barrier.Further study found that DnaK and EF-Tu are two kinds of Mhr surface PlgR,which participate in helping Mhr to hijack the fibrinolytic system.Meanwhile,they also have the multiple moonlighting functions of cytoadhesion and binding ECM.These results lay a foundation for further systematic identification of Mhr PlgRs and studies on their interactions with host fibrinolytic system.This study provides a new idea for the analysis of the systemic Mhr infection,and also provides an important reference for the interpretation of the pathogenesis of Mhr and related vaccine development.