The Effect And Mechanism Study Of Serine Protease HTRA/DEGQ Of Porcine Respiratory Bacterial Pathogens Breaking Through The Respiratory Mucosal Epithelial Barrier

Porcine Respiratory Disease Complex(PRDC)is a multifactorial syndrome that causes health problem in growing piglets and economic losses to farmers in the world.The etiological factors involved can be bacteria(like Actinobacillus pleuropneumoniae,Bordetella bronchiseptica,Pasteurella multocida,and Glaesserella parasuis),viruses(like swine Influenza A Virus,Porcine Reproductive and Respiratory Syndrome Virus,and Porcine Circo Virus type 2)or mycoplasmas(Mycoplasma hyorhinis and Mycoplasma hyopneumoniae).The role and impact of different microorganisms in the development of this disease can be complex,and these are not fully understood.The severity of lesions are a consequence of synergism and combination of different factors.Generally,the pathogenesis of viral respiratory disease is typically associated with destruction of the mucocilliary apparatus and with interference and decrease of the function of pulmonary alveolar macrophages.Bacterial pathogens often contribute to PRDC by activation of inflammation via enhanced cytokine responses.However,the molecular mechanism of PRDC pathogens destroy the respiratory barrier is still unclear.In our study,the newborn piglet tracheal(NPTr)epithelial cells were used as an in vitro model.The immunoblotting,immunofluorescence,ECIS,and Transwell assay were preformed to detect that PRDC bacterial pathogens destroy the tracheal epithelial barrier or promote their own paracellular transmigration.At the same time,the casein zymography assay and mass spectrometry were employed to identify the proteolytic protease in the G.parasuis strain of PRDC pathogens.Mass spectrometry data combined with amino acid homology analysis indicated that PRDC bacterial pathogen has serine protease HtrA/DegQ,and the biological function of serine protease HtrA/DegQ was confirmed by in vitro experiments.Finally,the serine protease HtrA/DegQ of PRDC bacterial pathogens plays an important role in disrupting the respiratory epithelial barrier and colonizing in tissues were revealed by piglet challenge experiments.The research details are as follows:1?PRDC bacterial pathogens infection disrupted the integrity of airway epithelial barrier in vivo and in vitro.As the Hematoxylin and Eosin(HE)staining shown,G.parasuis strain of PRDC bacterial pathogens infection caused the damage of tracheal epithelium,demonstrated by the loss of respiratory tract cilia and disruption of tracheal epithelial arrangement.The immunofluorescence showed that G.parasuis largely destroyed the distribution of the epithelial tight junction protein ZO-1 and the adherens junction protein E-cadherin in the pig tracheal tissues.In vitro,the immunofluorescence and immunoblotting showed that PRDC bacterial pathogens can significantly decrease of these tight junction proteins and adhesion junction proteins,and shedding ectodomian of E-cadherin into the supernatant of NPTr cells.Meanwhile,ECIS data showed that PRDC bacterial pathogens can significantly decrease the transepithelial electrical resistance(TEER)of the NPTr monolayer cells.2?E-cadherin maintains the barrier function of epithelial cells and affects the paracellular transmigration of PRDC bacterial pathogens.CRISPR/Cas9 gene editing technology was applied to specificly knockout E-cadherin in NPTr cells.The immunobloting,PCR,and real-time PCR showed that successful knockout E-cadherin in NPTr cells.The ECIS data showed that E-cadherin maintains the transepithelial electrical resistance(TEER)of the NPTr monolayer cells.Through the adhesion,invasion and paracellular transmigration experiments of PRDC bacterial pathogens on wild-type or E-cadherin knockout NPTr cells,we found that E-cadherin affects the paracellular transmigration of PRDC bacterial pathogens.Nuclear and cytoplasmic separation experiments and real-time PCR confirmed that the knockout of E-cadherin affects the distribution of the ?-catenin in the nucleus and the expression of chemokines.3?Degradation of E-cadherin was independent of the host cellular metalloproteases.To analyze the molecular mechanism of E-cadherin ectodomain shedding,the cellular metalloproteases activity was inhibited by metalloproteases family inhibitor during PRDC bacterial pathogens infection,and the immunobloting was performed to detect that the decrease of its full-length E-cadherin(E-Cad-FL)in whole cell lysates,as demonstrated by the increasing extracellular N-terminal fragment(NTF)in cell.The results presented that the metalloproteases family inhibitor did not alleviate the infection-caused E-cadherin degradation.Meanwhile,the immunobloting was used to detect that the protein levels of matrix metalloproteases MMP7 and ADAM10 after PRDC bacterial pathogens infection.The results showed that the MMPs member MMP7 was significantly and time-dependently downregulated in response to the infection,while the ADAM10 was almost not affected.To investigate the effect of ADAM10 on E-cadherin ectodomain shedding,siRNA or specific inhibitors was used to inihibits ADAM10 fuction and then infected with PRDC bacterial pathogens.The immunobloting showed that ADAM10 inhibition and knockdown could not significantly restore the E-cadherin degradation by these respiratory pathogens.Meanwhile,the gelatin zymography experiment showed that the cellular metalloproteases activity did not increase during PRDC bacterial pathogens infection.4?Screening of potential proteases with proteolytic activity in PRDC bacterial pathogens.To explore the factors involved in the release of the ectodomain shedding of E-cadherin by PRDC bacterial pathogens,casein zymography experiment and mass spectrometry were employed to identify proteolytic proteases in G.parasuis strain,and the serine protease HtrA was identified as a possible contributor.Multiple sequence alignment and structure prediction indicated that all PRDC bacterial pathogens have DegQ serine protease homologous to HtrA.To characterize HtrA/DegQ activities in more detail,we clone and purified different wild-type(WT)HtrA/DegQ from PRDC bacterial pathogens and generated corresponding inactive mutants by exchanging serine to alanine(SA)in the active center.Activities of purified recombinant proteases were determined by casein zymography.All tested recombinant proteases HtrA/DegQ displayed caseinolyotic activities,whereas the corresponding inactive HtrA/DegQ mutants were completely inactive.Moreover,the immunobloting showed that HtrA/DegQ serine protease can degrade the extracellular domain of recombinant porcine E-cadherin.Through the preparation of mouse HtrA polyclonal antibody,the immunobloting was used to confirm that the serine protease HtrA of G.parasuis can be secreted to the extrancellular and cleave E-cadherin of NPTr cells.Finally,ECIS showed that the HtrA/DegQ serine protease of PRDC bacterial pathogens affects the TEER of NPTr monolayer cells.5?Deletion of htrA gene of G.parasuis strain affects its colonization in piglets.Firstly,PRDC bacterial pathogens G.parasuis htrA deletion mutant was generated by using natural transformation system.PCR,immunobloting and real-time PCR assays were performed to confirm the gene deletion in ?htrA strain.In vitro,the immunofluorescence,immunoblotting,adhesion,and Transwell experiments confirmed that the htrA gene deletion affects the ectodomain shedding of E-cadherin and paracellular transmigration of G.parasuis.In vivo,HE staining,bacteria isolation,immunohistochemistry and immunofluorescence showed that the htrA gene deletion affects the pathological damage of G.parasuis to respiratory tissues,the destruction of tracheal epithelium E-cadherin,the ability to transmigrate the respiratory epithelial cells barrier and colonize in the systemic tissues.6?Type ? AAA+ ATPase active protease ClpX regulates the protein level of HtrA.The analysis of the casein zymography and mass spectrometry results indicated that the protease ClpX directly or indirectly participates in the hydrolysis of casein.Multiple sequence alignment and ATP hydrolysis experiments,it is found that ClpX is a protease with ATPase activity.To characterize ClpX activities in more detail,we clone and purified different wild-type(WT)ClpX from G.parasuis and generated corresponding inactive mutants by exchanging glutamic acid to alanine(EA)in the Walker B domain.ATP and casein hydrolysis experiments showed that ClpX protein does not have caseinolyotic activity.By constructing the clp X gene deletion strain and overexpressing the ClpX protein in the htrA gene deletion strain,we found that it is not ClpX protein that directly degrades casein,but the clp X gene deletion affects the total and secreted HtrA protein.Similarly,we infected NPTr cells with clp X gene deletion strains,and the immunoblotting showed that the deletion of clp X affected the cleavage of E-cadherin.