Identification And Immunoprotection Analysis Of Host Binding Proteins Of Streptococcus Suis Serotype 2

Streptococcus suis(S.suis,SS)is an emerging zoonotic pathogen which causes severe infections in both pigs and humans.Among its 33 serotypes,Streptococcus suis serotype 2(SS2)with the strongest pathogenicity was most widely studied and reported as the most important pathogenic serotype in the world.Prevention is the key to control S.suis infections.So,the development of different types of vaccines is essential.The attenuated vaccines have a good immunization effect but the danger to return to be virulent and strict transport conditions limited their application.Inactivated vaccines are safer than attenuated vaccines and easy to save.However,because of its poor immune effect,multiple immunizations are necessary.Subunit vaccines,made of immunogenic protein constructs of bacteria or virus,can avoid many antibodies induced by unrelated antigens so as to reduce the side effects and the associated diseases caused by the vaccine.Research on subunit vaccines has brought hope to the discovery of newly effective S.suis vaccines.Bacterial surface proteins play an important role in the adhesion of bacteria to the host by binding to host extracellular matrices(ECM)proteins such as fibronectin(FN)and laminin(Laminin,LN),which is the first step of bacterial colonization and pathogenesis.In consequence,blocking the ECM binding proteins on the surface of bacteria with their corresponding antibody may degrade the adhesion and reduce the pathogenicity of the bacteria.In this study,recombinant FN/LN binding proteins of Streptococcus suis were expressed and purified.Then,their immunoprotective properties as subunit vaccines were evaluated,which includs Part 1-3.Before bacterial invasion of the host,bacteria bind to the fixed ECM molecules of host and the free molecules such as Factor H(FH)in complement system.ECM molecules such as FN and LN play an important role in the pathogenesis of bacteria,while FH is also associated with anti-SS2 infection.To survive and proliferate in the blood,SS2 must escape the innate immune defense system of host,such as complement system,which is necessary in causing meningitis,sepsis and other diseases.Factor H-binding proteins(FHBPs)recruit and gather FH,a complement negative regulator,to inhibit the activation of C3b in the complement alternative pathway and degrade the activated C3b to inactivated iC3b,which contributes the escape from the phagocytosis mediated by complement and the survival of Streptococcus suis in the blood.However,the mechanism of SS2-FH binding is unclear,which may be mediated by FHBPs on SS2 surfaces.In this study,Excellular proteins that bind to FH were screened and their biological functions were preliminarily studied,which includs Part 4.1.Recombinant expression,purification and antibody preparation of Streptococcus suis FN/LN binding proteinsPreviously,we identified 15 potential LN binding proteins and five potential FN binding proteins by the method combining proteomics and Far-Western Blotting.Among them,six proteins including elongation factor Tu(EF-Tu),lactate dehydrogenase(LDH),fructose-bisphosphate aldolase(FBA),Enolase,oligopeptide-binding protein OppA precursor(OppA)and 3-ketoacyl-ACP reductase(KAR)were first reported to bind to LN.The FN binding activity of EF-Tu and LDH was also reported for the first time.In this study,six proteins including EF-Tu,FBA,LDH,Enolase,OppA and KAR were selected for prokaryotic expression and purification.Six fusion proteins obtained in our study were used to immunize experimental rabbits to gain the polyclonal antisera.2.Analysis of in vitro properties of recombinant FN/LN binding proteins.The bactericidal assay was used to evaluate the role of SS2 surface proteins in pathogenic process of bacteria.The results showed that the survival rate of SS declined obviously after its corresponding surface proteins were blocked by antisera against EF-Tu,FBA and LDH,which demonstrated a protective effect on the body.So,EF-Tu,FBA and LDH were selected for the following research.Furthermore,the immunogenicity of EF-Tu,FBA and LDH were analyzed.On the one hand,the western blotting results of recombinant proteins and convalescent serum showed that EF-Tu,FBA and LDH could react with serum.On the other hand,the western blotting results of rabbit polyclonal sera and native proteins of SS showed that the polyclonal sera were able to recognize native proteins EF-Tu,FBA and LDH in SS2 lysates.In conclusion,EF-Tu,FBA and LDH with immunogenicity can be expressed in vivo,which laid a theoretical foundation for the following experiments.3.Protective evaluation of recombinant FN/LN binding proteins on two mice modelsTwo mice models(BALB/c and ICR)were used to further evaluate the immunoprotective effects of EF-Tu,FBA and LDH.After three immunizations,the blood was harvested from mice and the antibody titer was determined by ELISA.After a high level of antibodies were produced in mice,they were challenged with SS2.And then,the survival rate and mortality rate were observed and recorded.The results showed that average antibody titers of EF-Tu,FBA and LDH in BALB/c mice were 1:21333,1:42667,1:42667,repectively and 1:170667,1:42667?1:149333,respectively in ICR mice model.However,the high level of antibodies in mice did not produce an effective immune protection from SS2 infection.4.Screening of FHBPs from excellular proteins and preliminarily functional studies of themIn the previous study,14 potential novel FHBPs were identified in the cell wall proteins of Streptococcus suis by the proteomics and Far-Western blotting assays.And the biological functions of eight proteins among them were preliminarily analyzed to explore the role of FH in the pathogenic process of SS2.The results showed that the binding of FH to the cell surface contributed to the adhesion and invasion of SS2 to HEp-2 cells and enhanced the SS2 virulence,which contributed to SS2 resistance to killing.In this study,the same method of 2D-Far-Western bloting was used to screen FHBPs from excellular proteins of SS2.Biological functions of these FHBPs were preliminarily studied,which aims to provide evidence for the exploration of pathogenicity of SS.