Kocking Out Suilyin Gene And It Contribute To Invasive Infection Of Streptococcus Suis Serotype2

ABSTRACT:Streptococcus suis is an important zoonotic pathogen, endemic in nearly all countries with an extensive pig industry, while serotype2(SS2) with the strongest virulence. Since the relativity between time and space, the virulence factor of S.suis is difficult to identified fully. So far, the mechanisms of S.suis for SS2invasive disease have not been well understood. The capsular polysaccharide (CPS) is the only recognized virulence factor, and extracellular factor (EF), muramidase released protein (MRP) and suilyin (SLY) are accepted for virulence factors for most researchers. Suilysin is a pore-forming cholesterol-dependent cytotoxin (CDC) expressed by many virulent S. suis strains and a secreted protein.In this study, The sly replacement vector was constructed and transformed into05ZYH33by electroporation. One sly deleted strain was obtained. In order to further understanding and confirming virulence of SLY, the role of SLY played in virulence was analyzed by comparing the biological characteristics of wild strain05ZYH33and mutant Δsly. Moreover, the recombinant mutants of SLY were contructed by site-directed mutation. Successfully constructed the non-hemolytic suilysin mutant SLY(P353V) with immunogenicity, which might be further studied as a promise candidate for S.suis vaccine. The results are as follows: 1. Knock out of sly from SS2epidemic strain05ZYH33and its biological activities.DNA fragments corresponding to the upstream and downstream regions of SLY gene were amplified from SS2epidemic strain05ZYH33, The Cm cassette was amplified from plasmid pSET1.Then the PCR amplicons were cloned into the temperature-sensitive S. suis-E.coli shuttle vector pSET4s one by one, giving rise to the knockout vector pSET4s::sly. One sly deleted strain(Δsly) was obtained by double cross-over and Cm resistance selection. The growth patterns of WT and mutant have no significant differences in liquid THB. SLY wasn’t detected in the supernatant of mutant by western blotting. Unlike the WT, Δsly is lack of hemolytic activity.2. The role of SLY in virulence2.1SLY is associated with enhanced severity of S.suis infectionTo assess the roles of SLY in virulence in detail, we performed experimental infections in SPF ICR mice. The mices infected with05ZYH33died within the experimental time, however, the group challenged with ASLY mutants were not noted obvious signs of disease. The data indicate that ASLY decreased substantially in virulence in this infection model. Moreover different infection pathways resulted in different Survival rate. Survival rate of S.suis through intraperitoneal injection was higher than intravenous injection after4h and ASLY mutants were much less than05ZYH33in blood of mice through intraperitoneal injection. The pathological sections of Peritoneal showed that the abdominal epithelial cells of mice infected by SS205ZYH33lost their normal morphological features and integrity of the tight junctions or died, while the epithelial cells infected by S.suis Asly had few changes. These data demonstrate that SLY is associated with enhanced severity of S.suis infection. 2.2Suilysin increased the release of inflammatory cytokine and contributed to the traversal across the epithelial barrier in TLR4dependent mannerSS205ZYH33could induce the release of the inflammatory cytokines in the blood of ICR mices more than its mutant Asly. These data indicated SLY played important roles on inducing the release of inflammatory cytokines which might be one reason that SLY enhanced severity of S.suis infection. An in vitro model of epithelial barrier showed that cholesterol (the receptor of SLY) and CLI-095(specific inhibitor of TLR4) could significantly inhibit the traversal of05ZYH33across the epithelial barrier. These data demonstrated that SLY contributed to the traversal across the epithelial barrier in TLR4dependent manner.3. Construction and activities analysis of Suilysin mutantThe proline in353site of Suilysin was site-directed mutated to alanine leucine and valine, respectively. The recombinant mutants were refolded and purified by immobilized metal ion affinity chromatography, and purified proteins were evaluated the hemolysis activity and immunogenicity. Results showed that SLY mutant SLY(P353V) lost hemolytic ability but immunogenicity. Compared to wild-type SLY, SLY(P353V) has similar function of protection to Streptococcus suis infection and might be further studied as a promise candidate for Streptococcus suis vaccine.