| Streptococcus suis(SS)is an important Gram-positive pathogen in swine industry,and also an emerging zoonotic pathogen for humans.Herein,33 reference serotypes(1-19?21?23-25?27-31 and 1/2)and 27 novel cps loci(NCL1-26 and Chz)SS strains have been identified,according to the capsular polysaccharides.Many clinical manifestations caused by SS infection have been reported,such as meningitis,arthritis,pneumonia,endocarditis,and septicemia.SS is considered to be an important pathogen that causes economic and public health problems globally.With the frequent outbreak of SS infection and the increasing severity of SS resistance,researching on the pathogenic mechanisms and effective vaccine of SS is increasingly necessary.In this study,we clarified a new SS pathogenesis mechanism mediated by the secretion protein SssPl.The candidate immunogens of SS have been screened by the immunoproteomics assays.This study will promote a better understanding of the pathogenesis of SS.1 Comparative genomic analysis shows that Streptococcus suis serotype Chz meningitis isolate CZ130302 contains the unique 50K and 58K Pathogenicity IslandsIn our previous work,we found a virulent SS strain CZ130302,belonging to a novel serotype Chz,was associated with acute meningitis in piglets.CZ130302 caused a high fatality rate during that outbreak in pig farm and showed a higher virulence level than SS2 virulent strains P1/7,ZY05719 and HA9801 in the BALB/c mice infection model.CZ130302 is also a multi-drug resistant strain,which is resistant to many antibiotics,such as ?-lactams,aminoglycosides,macrolides and tetracyclines.In this study,we sequenced and analyzed the complete genomes of three Chz serotype,including strain CZ130302 and two avirulent strains HN136 and AH681.The well-known virulence factors in two avirulent strains HN136 and AH681 is similar to that of CZ130302 in terms of the proportion and distribution.Among them,the ATP binding protein SstF is unique to the virulent strain CZ130302.The sstF deletion mutant was created via natural DNA transformation.The results showed that SstF was involved in the resistance to bacitracin.Further investigations revealed a large-scale genomic rearrangement in CZ130302,which was proposed to be mediated by prophages and/or transposase genes.By genome comparison,we found two putative genomic islands(GI)uniquely encoded in strain CZ130302,and designated them as 50K and 58K GIs.In mouse infection model,the deletion of 50K and 58K GIs caused 270-fold and 3-fold attenuation of virulence,respectively.In conclusion,our results suggested that the 50K and 58K pathogenicity islands are the key virulence components of the virulent strain CZ130302.2 SssPl,a Streptococcus suis fimbriae-like protein transported by SecY2/A2 system,Contributes to bacterial virulenceBy genome comparison,we found the 50K?58K pathogenicity islands uniquely encoded in strain CZ130302.Notably,we identified a complete SecY2/A2 system,coupled with its secretory protein SssP1 encoded in 50K GI,which contributed to the pathogenicity of strain CZ130302.Immunogold electron microscopy and immunofluorescence analyses indicated that SssPl could form fimbriae-like structures that extend outward from the bacterial cell surface.The sssP1 mutation also attenuated bacterial adherence in HEp-2 and HBMEC cells when compared with the wild-type.Furthermore,we showed that two analogous Ig-like subdomains of SssP1 have sialic acids binding capacities,which is also important in HBMEC cell adhesion.The results of our research will help to understand the pathogenesis of SS and provide theoretical basis and technical means for prevention and control of SS.3 Binding of adhesin SssP1 of Streptococcus suis to vimentin promotes attachment to host cellsStreptococcus suis is thought to be one of the important pathogens that cause bacterial meningitis in pigs and humans.Penetrating the blood-brain barrier(BBB)and evading host immune defenses are the preconditions for SS to cause meningitis.In vitro,compared with CZ130302,the ability of the sssP1 mutation to adhere to both HBMEC and bEnd.3 cells was significantly attenuated.Infection of BALB/c mice with CZ130302 strain could cause bacteremia and the bacteria were also detected in the cerebrospinal fluid(CSF),while the colonization ability of the SssP1 mutant strain in the blood was significantly attenuated;and no bacteria were found in the cerebrospinal fluid.Transwell chambers containing HBMEC and/or HA1800 were used to model the blood-brain barrier(BBB).We observed that the SS wild-type CZ130302 strain crossed the BBB model more readily than the SssP1 mutant strain.SssP1 is considered to be an important adhesin in the pathogenesis of meningitis,the molecular basis for this interaction is not well defined.We used pull-down and Far-western blot to demonstrate that SssPl contributes to SS attachment to HBMEC via the direct interaction of its binding region(NR)with vimentin.Furthermore,it was confirmed that the 216-781 amino acid and the 1711-2214 amino acid of the SssP1-NR domain have specific binding ability to vimentin.Our data suggest that Streptococcus suis adhesin SssPl plays an important role in crossing the BBB and the development of streptococcal meningitis.4 Screening the candidate immunogens of Streptococcus suis and evaluation of immune response and protective effectDue to the existence of many serotypes and a wide range of immune evasion capabilities,vaccines that provide effective cross-protection against different strains are the focus of current research.Bacterial wall proteins play an important role in invasion and infection,and can be good vaccine candidates due to their high expression and accessibility to antibodies.To screen for the immunogenic cell wall proteins of Streptococcus suis as candidate immunogens,the two-dimensional electrophoresis(2-DE)and immunoblotting were applied.In this study,16 immunogenic protein spots were screened,and 8 distinct proteins(GH25,Pk,PdhA,Ldh,ExoA,Pgk,MalX and Dnak)were successfully identified by MALDI-TOF-MS.Subsequently,the 8 recombinant proteins were successfully expressed and showed strong immunogenicity.The results of the challenge test revealed that the 8 recombinant proteins could induce higher IgG antibody titer and effectively inhibit the adhesion of CZ130302 to HEp-2 and HBMEC cells,and also support immunoprotective effects against SS infection in BALB/c mouse model.In addition,based on the conservation and immunoprotection of the eight protective antigenic proteins,the PdhA,Ldh and MalX proteins were screened to form a triple subunit vaccine as a candidate vaccine for cross protection.Challenge studies showed that the three protein-immunized mice were able to provide protection against challenges by SS2 virulent strain ZY05719(62.5%protection)and the SSChz virulent strain CZ130302(75%protection).In conclusion,a triple immunogen consisting of PdhA,Ldh and MalX proteins can be used as a candidate for Streptococcus suis subunit vaccine. |
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