Identification And Characterization Of The Effector Of Type Ⅳ Secretion System In Streptococcus Suis Serotype2

Streptococcus suis is a gram-positive coccus which has been divided into35serotypesbased on the capsular antigens. Among all the serotypes, serotype2is regarded as the mostpathogenic and prevalent. S.suis2is an emerging zoonotic pathogen. It is not only a majorswine pathogen responsible for huge economic losses to the swine industry worldwide butalso can infect humans through the wound and respiratory tract dissemination. S.suis2infection will lead to a variety of diseases such as meningitis, pneumonia, arthritis andsepticaemia. So, it is also a serious threat to the close contacts especially abattoir workers.In the past, human cases of S. suis2infections have been often documented as sporadicones. However, two large-scale outbreaks caused by S. suis2have been reported in Chinain1998and2005. A high propotion of patients in these two outbreaks showed the typicalsymptoms of streptococcal toxic shock syndrome (STSS) characterized by acute high-fever,hypotension, shock, and multiple organ failure, that lead to a very short course of diseaseand high mortality. This is the first description of a larges-cale STSS outbreak caused by anon-group A streptococci (non-GAS) pathogen. And it makes the public health securityunder a great threat.The pathogenic mechanism of S. suis2is complicated and it is not illuminated so far.By using comparative genomics research, Chen et al. found that there is an uniquepathogenicity island(PAI) named89K PAI in the epidemic strains of98HAH12and05ZYH33which caused the two outbreaks in China. The related studies revealed that89KPAI is highly correlated with the high pathogenicity of the epidemic strains. Throughin-deep bioinformatics analysis, our group identified a gene cluster encoding a type Ⅳsecretion system(T4SS) in the5’ end of89K PAI. T4SS is widespread throughout theGram-negative and Gram-positive bacteria. It can translocate its pathogenic effectors acrossthe membrane of bacteria and finally into the host cells or external milieu. Our previousstudies imply that the T4SS of S. suis2may probably secret some effectors that contibute to its pathogenicity and induce STSS. Thus, finding the effectors of T4SS as well as studyingtheir functions, is significant to elucidate the mechanism of STSS caused by the highlypathogenic S. suis2.To this end, we firstly used a proteomics approach to screen the effectors of T4SS bycomparing the difference in secreted proteins between S. suis2wild-type and the keycomponent of T4SS deletion mutant strain. Then we verified the effector protein SspA bycomparing the transcription of sspA gene, the secretion of SspA protein and the interactionbetween SspA and a key component of T4SS. At last, the sspA gene knockout mutant strainwas constructed. Its pathogenicity and ability to trigger host immune response wasevaluated in an infection model of mice. The following experiments are included in thisdissertation:1. Screening of the effectors of T4SS: The extracellular proteins of S. suis2wild-type strain05ZYH33and T4SS mutant strain were precipitated with TrichloroaceticAcid (TCA) and acetone. Then we used LC-MS/MS and shot-gun strategy to analyze thesecretome of both strains. By comparison of the secretome of two strains,7potentialsecreted proteins of T4SS were discovered. In the7potential effectors, there were2subtilisin-like serine proteases (SspA),3ABC transporter family proteins,1prolyl-tRNAsynthetase and1hypothetical protein. According to the literature and actual situation, wechose05SSU1982protein (SspA) which got the highest scroe in MS for further research.2. Comparison of the transcription of sspA gene: mRNA of each strain(05ZYH33、virD4、 virB1、 virB4) was extracted from the late-exponential cultures. Then RT-PCRwas used to evaluate the transcription of sspA gene in each strain. The result showed nostatistical difference between wild-type strain and T4SS mutant strains. It suggested that thedeletion of the components of T4SS didn’t influence the transcription of sspA gene.3. Verification of the secretion of the effector: The gene encoding the major domainsof SspA was cloned into the plasmid pET-28a and the recombinant expression vector wastransformed into E.coli BL21. After induction of the expression, the recombinantprotein was purified by nickel affinity chromatography and ion exchange chromatography.Then the purified SspA was used as an antigen to immunize the BALB/c mice to producethe immune serum containing the polyclonal antibody of SspA. At last, this immune serum was used as primary antibody to detect SspA in the extracellular proteins of each strain.Result of Western blot demonstrated that SspA can be secreted via T4SS of S.suis2. It wasconsistent with the result of LC-MS/MS.4. Determination of the interaction between VirD4and SspA: The key componentof T4SS virD4gene was cloned into pGEX-6p-1. The recombinant protein GST-VirD4wasexpressed and purified. Purified proteins were used to observe direct binding betweenVirD4and SspA. In the pull-down assay, GST-VirD4coprecipitated with SspA, while GSTalone as a negative control did not. The result of pull-down assay confirmed that there wasa direct interaction between VirD4and SspA. It was also a strong evidence of SspA as aneffector of T4SS.5. Role of SspA in the pathogenicity of S.suis2: To investigate the role of SspA inthe pathogenicity of S.suis2, a gene knockout mutant of sspA gene was constructed byhomologous recombination. Then the virulence and ability to trigger host immune responseof sspA was evaluated in an infection model of mice. The inflammatory cytokines in theserum of infected mice were detected by ELISA. The results showed that deletion of sspAgene led an attenuation of the virulence of S.suis2and impaired its ability to trigger hostimmune response.In conclusion, in this study7potential effectors of T4SS in S.suis2were identified bya proteomics approach firstly. Then one of the potential effectors named SspA was verifiedby comparing the transcription of sspA gene, the secretion of SspA protein and theinteraction between SspA and a key component of T4SS. At last, deletion of sspA gene ledan attenuation of the virulence of S.suis2and impaired its ability to trigger host immuneresponse. It implied that SspA could induce the host to secret inflammatory cytokines invivo and therefore played an ipmportant role in STSS caused by S. suis2.