The Research On The Function And Pathogenic Mechanism Of Apd Protein In Glaesserella Parasuis

Glaesserella parasuis(GPS)can cause Gl(?)sser’s disease,causing multiple cellulosic serositis in pigs,including arthritis and meningitis.The diseases caused by GPS have caused huge economic losses to the pig industry.Apd is considered to be an adhesive protein of Glaesserella parasuis and may be an important virulence factor of Glaesserella parasuis,but its function and its involvement in the pathogenesis of Glaesserella parasuis remain unclear.In this study,porcine alveolar macrophage 3D4/21 was selected as the cell research model.By constructing apd-deleted strain and expressing recombinant protein rApd,to study the interaction protein of adhesion protein Apd on the cell membrane,we used Co-IP,Western Blot and immunofluorescence.The effects of Apd on the pathogenicity of the Glaesserella parasuis were investigated by using q-PCR and infection test on weaned piglets.This study is to provide a theoretical basis for revealing the pathogenicity of the Glaesserella parasuis.The research mainly includes the following three aspects:1.Apd of GPS is involved in bacterial adhesion and invasion to the hostApd-deleted strain and its complement strain were constructed,we comfirmed that the adhesion and invasion ability of bacteria to cells was significantly weakened after apd was deleted through in vivo adhesion and invasion tests,and this phenomenon was also found in animal tests.When animals were infected with the mutant strain,the bacteria load in tissues and pathological sections were analyzed,and the invasion ability and pathogenicity of apd-deleted strain to the host were weakened.This suggests that Apd is involved in the process of bacterial adhesion to the host.2.There is interaction between the Apd of GPS and the host vimentinIn order to explore the interaction protein of Apd on cell membrane,recombinant protein rApd was expressed by prokaryotic expression system,and Co-IP was used to grab target protein.By protein mass spectrometry analysis,the interaction between Apd and vimentin.Co-IP,Western Blot and cellular immunofluorescence were used to verify the interaction between Apd and vimentin.On the other hand,vimentin was secreted by cells into the extracellular environment.In this study,we confirmed that the protein Apd of Glaesserella parasuis could interact with vimentin in serum.3.Apd of Glaesserella parasuis affects the pathogenicity of bacteriaBy measuring the growth curves of wild strain CF7066,deletion strain CF7066-Δapd and complement strain CF7066-CΔapd,it was found that the proliferation ability of the deletion strain was significantly reduced.In vitro biofilm tests also showed that apd was involved in the regulation of bacterial biofilm formation.After infection with apd deletion strain,the pathological reaction of animals was mild and the pathogenic ability was weakened.By detecting the changes of the m RNA expression levels of the virulence factors of the three strains,it was found that the expression levels of several important virulence factors of the bacteria were significantly decreased after apd deletion,which indicated that apd affected the virulence composition of the Glaesserella parasuis.In summary,this study discovered the Apd of Glaesserella parasuis interacts with the vimentin.Apd is involved in the adhesion ability of Glaesserella parasuis and affects the pathogenicity of bacteria.This study not only deepens our understanding of the pathogenic mechanism of Glaesserella parasuis,but also clarifies our understanding of the role of Apd.