Screening And Functional Analysis Of The Host Binding Proteins Of Streptococcus Suis Serotype 2

Streptococcus suis(SS)is a major invasive pathogen in swine and a zoonotic agent for humans that can cause meningitis,arthritis and septicemia.It is also a potential public health threat through zoonotic transmission and is responsible for significant economic losses to the porcine industry worldwide.SS strains have been characterized on the basis of the capsular polysaccharide(CPS).Among the 35 serotypes(serotypes 1-34 and 1/2),serotype 2(SS2)is regarded as the most commonly isolated and the most closely associated with the diseases,which attracted considerable international attention to the potential public health threat.So far,there are no less than 100 putative virulence factors have been reported to be involved in the virulence of SS2,of which at least 35 of these have been described as critical virulence factors.Unfortunately,the pathogenic mechanism of infection with SS2 is still not well understood.The interactions between host extracellular matrix(ECM)proteins and bacterial surface adhesins is a necessary step in the colonization of epithelial surfaces.SS can also interact with the host immune system to evade complement-mediated phagocytosis.In this study,a method combining proteomic analysis and Far-Western blotting assays was developed to screen directly for host-binding proteins,including ECM(laminin and fibronectin)-binding proteins and Factor H(FH)-binding proteins.Then,we selected the newly identified proteins and further confirmed their binding activity to host proteins.The basic biological functions of these proteins provide a theoretical basis for further study on the pathogenesis of SS infection.Additionally,We selected some interesting virulence-associated proteins with host-binding capacity to assess the phenotypic changes between the parental strain and the mutant strains.This study provided some newly valuable insights into the pathogenesis of SS through biological function analysis of the virulence-associated host-binding proteins.1 Identification of novel laminin-and fibronectin-binding proteins by Far-Western blot:capturing the adhesins of Streptococcus suis type 2Bacterial cell wall(CW)and extracellular(EC)proteins are often involved in interactions with extracellular matrix(ECM)proteins such as laminin(LN)and fibronectin(FN),which play important roles in adhesion and invasion.In this study,an efficient method combining proteomic analysis and Far-Western blotting assays was developed to screen directly for bacterial surface proteins with LN-and FN-binding capacity.With this approach,fifteen potential LN-binding proteins and five potential FN-binding proteins were identified from SS2 CW and EC proteins.Nine newly identified proteins,including oligopeptide-binding protein OppA precursor(OppA),elongation factor Tu(EF-Tu),enolase,lactate dehydrogenase(LDH),fructose-bisphosphate aldolase(FBA),3-ketoacyl-ACP reductase(KAR),Glyceraldehyde-3-phosphate dehydrogenase(GAPDH),Inosine 5'-monophosphate dehydrogenase(IMPDH)and amino acid ABC transporter permease(ABC)were cloned,expressed,purified and further confirmed by Far-Western blotting and ELISA.Five proteins(OppA,EF-Tu,enolase,LDH and FBA)exhibited specifically binding activity to both human LN and human FN.Furthermore,seven important recombinant proteins were selected and identified to have the ability to bind HEp-2 cells by the indirect immunofluorescent assay.In addition,four recombinant proteins and their corresponding polyclonal antibodies were observed to decrease SS2 adhesion to HEp-2 cells,which indicates that these proteins contribute to the adherence of SS2 to host cell surface.Collectively,these results showed that the approach described here represents a useful tool for investigating the host-pathogen interactions.2 Factor H specifically capture novel Factor H-binding proteins of Streptococcus suis and contribute to the virulence of the bacteriaFactor H(FH),a regulatory protein of the complement system,can specifically bind to factor H-binding proteins(FHBPs)of SS2,which contribute to evasion of host innate immune defenses.In the present study,we aimed to identify novel FHBPs and characterize the biological functions of FH in SS2 pathogenesis.Here,a method that combined proteomics and Far-western blotting was developed to identify the surface FHBPs of SS2.With this method,fourteen potential novel FHBPs were identified among SS2 surface proteins.We selected eight newly identified proteins and further confirmed their binding activity to FH.The binding of SS2 to immobilized FH decreased dramatically after pre-incubation with anti-FHBPs polyclonal antibodies.We showed for the first time that SS2 also specifically interact with mouse FH.Furthermore,we found that FH play an important role in adherence and invasion of SS2 to HEp-2 cells.Additionally,using a mouse model of intraperitoneal challenge,we confirmed that SS2 pre-incubated with FH enhanced bacteremia and brain invasion,compared with SS2 not pretreated with FH.Taken together,this study provides a useful method to characterize the host-bacteria interactions.These results firstly indicated that binding of FH to the cell surface improved the adherence and invasion of SS2 to HEp-2 cells,promoting SS2 to resist killing and leading to enhance virulence.3 The non-conserved region of MRP is involved in the virulence of Streptococcus suis serotype 2Muramidase-released protein(MRP)of SS2 is an important epidemic virulence marker with an unclear role in bacterial infection.To investigate the biological functions of MRP,three mutants named ?mrp,?mrp domain 1(?mrp-dl)and ?mrp domain 2(?mrp-d2)were constructed to assess the phenotypic changes between the parental strain and the mutant strains.The results indicated that MRP domain 1(MRP-D1,the non-conserved region of MRP from a virulent strain,a.a.242-596)played a critical role in adherence of SS2 to host cells,compared with MRP domain 1*(MRP-D1*,the non-conserved region of MRP from a low virulent strain,a.a.239-598)or MRP domain 2(MRP-D2,the conserved region of MRP,a.a.848-1222).We found that MRP-D1 but not MRP-D2 could specifically bind to fibronectin(FN),factor H(FH),fibrinogen(FG)and immunoglobulin G(IgG).Additionally,we confirmed that mrp-dl mutation significantly inhibited bacteremia and brain invasion in a mouse infection model.The mrp-dl mutation also attenuated the intracellular survival of SS2 in RAW246.7 macrophages,shortened the growth ability in pig blood and decreased the virulence of SS2 in BALB/c mice.Furthermore,antiserum against MRP-D1 was found to dramatically impede SS2 survival in pig blood.Finally,immunization with recombinant MRP-D1 efficiently enhanced murine viability after SS2 challenge,indicating its potential use in vaccination strategies.Collectively,these results indicated that MRP-D1 is involved in SS2 virulence and eloquently demonstrate the function of MRP in pathogenesis of infection.4 Fibronectin-/fibrinogen-binding protein(FBPS)is not a critical virulence factor for the Streptococcus suis serotype 2 strain ZY05719Fibronectin-/fibrinogen-binding protein(FBPS)of SS2 is an atypical anchorless microbial surface components recognizing adhesive matrix molecules(MSCRAMM)for which the role in bacterial infection are not clearly established.To investigate the biological functions of FBPS,an fbps knockout mutant was constructed in SS2 strain ZY05719 to explore the phenotypic changes between the wild-type and mutant strains.Cell morphology analyses combined with the basic growth curves showed that deletion of fbps does not significantly influence neither the thickness of the capsule of SS2 nor the cell growth characteristic.In addition to three previously identified host components fibronectin(FN),factor H(FH)and fibrinogen(FG),we also found that both laminin(LN)and immunoglobulin G(IgG)could specifically bind to FBPS.Furthermore,we confirmed that FBPS plays an important role in adherence of SS2 to host cells.The in vitro assays demonstrated that an inactivation of fbps does not inhibit the intracellular survival of SS2 in RAW246.7 macrophages,attenuate the ability of invasion of host cells or the growth ability in pig blood.Additionally,the fbps mutation failed to decrease the virulence of SS2 in both BALB/c mice and zebrafish.Finally,immunization with recombinant FBPS showed no significant difference from the control groups in terms of murine viability after SS2 challenge.Taken together,we concluded that FBPS is not a critical virulence factor for the SS2 strain ZY05719,suggesting that many of the virulence-associated proteins are not really critical virulence factors.5 Characterization and functional analysis of PnuC that is involved in the oxidative stress tolerance and virulence of Streptococcus suis serotype 2Streptococcus suis,an important swine pathogen and a major zoonotic agent,is responsible for severe financial losses in the global swine industry.Although a multitude of virulence factors have been reported,the pathogenesis of SS infections remains poorly understood.In our previous work,we identified a potential virulence-associated protein,named PnuC,unique to virulent strains of SS2.To investigate the functions of PnuC,the pnuC gene deletion mutant(?pnuC)was constructed in SS2 strain ZY05719 to assess the phenotypic changes between ?pnuC and the parental strain.The mutant strain ?pnuC exhibited highly sensitive to H2O2 stress and reduced the growth ability under vigorous shaking,suggesting that PnuC contributes to the oxidative stress tolerance.Additionally,zebrafish infection model showed that the virulence of pnuC+strains were significantly higher than pnuC-strains.Mouse infection experiments demonstrated that the abilities of ?pnuC to colonize the tissues were significantly attenuated compared with the parental strain.Histopathology of brain tissues showed that the deletion of pnuC significantly impairs the development of SS2 meningitis.Furthermore,the pnuC mutation decreased the virulence of SS2 in both BALB/c mice and zebrafish infection models.Taken together,these results indicated for the first time that PnuC is involved in the oxidative stress tolerance and virulence of SS2 during infection.