Study On The Pathogenesis Of Streptococcus Suis SsPepO, SsPspC And The Immunogenicity Of ΔSsPepO/ΔSsPspC Mutant Strain

Streptococcus suis(S. suis) is a major pathogen causing streptococcosis in swine which leads to enormous losses in breeding industry worldwide. Streptococcus suis serotype 2 (S. suis 2) is also a threat to human health. At present, S.suis 2 is considered to be the first, second and third main pathogen of bacterial meningitis in Vietnam, Taiwan and Thailand respectively. Although S.suis 2 has been gained high attention worldwide, several virulence factors have been characterized. However, further research of its pathogenesis is still needed to investigate the immune evasion mechanism of S. suis 2, which implies some new uncharacterized virulence factors may play important roles. In hence, in order to control S. suis 2 effectively, it is urgent to explore, identify new virulence factors and clarify the pathogenesis and immune mechanism, development of a more safe and effective vaccine against swine S.suis 2 is extremely urgent.S.suis 2 could develop a series of strategies to escape the host innate immune response, and stimulated the host inflammatory reaction, finally resulted in typical symptom such as sepsis, meningitis, and toxic shock syndrome, etc. How to find and explain the genes or proteins which induce inflammatory reaction is the molecular basis of explanation for S.suis 2 inducing toxic shock syndrome. Previously, our group constructed the two-competent system deletion mutant ResS/ResR, and scanned the gene chip of the mutant. We found that the expression level of genes SSU05 SsPepO and SSU05_SsPspC were down-regulated 44 folds and 10.5 folds, respectively. By blast analysis, we found that the SsPepO and SsPspC showed 62% and 43% identity to PepO and PspC of Streptococcus pneumonia respectively, which play important roles in the innate immune evasion. In previous study, the single gene mutant SsPepO showed less virulent than the WT strain in piglet model, but the mechanism was unclear. In order to reveal the mechanism, in this study we constructed single gene deletion strain ΔSsPspC and double gene deletion strain ΔSsPepO/ΔSsPspC, using highly pathogenic strain SC19 as a parent strain. Then the biological characteristics of mutant strains were analyzed. We further evaluated the pathogenicity of the ASsPepO, ASsPspC and ΔSsPepO/ΔSsPspC, and detected the expression level inflammatory factors in mice modle. The immune effect of double mutant ΔSsPepO/ΔSsPspC was evaluated through mouse and piglet model. The main contents of the study include:1. Construction of temperature sensitive recombinant suicide plasmidThe upstream and downstream fragments flanking regions of SsPspC were amplified from S.suis 2 WT strain SC19 genomic DNA by PCR. The PCR products were digested with the corresponding restriction enzymes and cloned into pSET4s vector to create recombinant plasmid pSET4s-ΔSsPspC.2. Construction of the gene deletion strain ΔSsPspC and ΔSsPepO/ΔSsPspCThe recombinant plasmid was transformed into competent cells of SC19 and ΔSsPepO. The selected mutants are confirmed by PCR, RT-PCR and DNA sequencing, et al. Two mutants ΔSsPspC and ΔSsPepO/ΔSsPspC was successfully constructed.3. Biological characteristics and pathogenicity of the gene deletion strain ΔSsPepO, ΔSsPspC and ΔSsPepO/ΔSsPspCIn order to compare the biological characteristics of ΔSsPepO, ΔSsPspC, ΔSsPepO/ΔSsPspC and the WT, the strains were cultured at the same condition. The growth curves of the ΔSsPepO, ΔSsPspC, ΔSsPepO/ΔSsPspC and the WT were similar after 12 h incubation, the growth ability, hemolysis ability and colony morphilogy and hemolysis ability of ΔSsPepO, ΔSsPspC and ΔSsPepO/ΔSsPspC were consistent with that of the WT in vitro.Using BALB/c mice as a model, the virulence of mutants were assessed, and 50% lethal does (LD50) results indicated that the deletion of both SsPepO and SsPspC resulted in a more significant reduction of virulence than the single gene SsPspC deletion mutant. According to the results, mice were infected with same doze (5×108CFU) of different strains respectively. The mice infected with mutant showed reduced morbidity, and mice infected with ΔSsPepO/ΔSsPspC showed only 50% mortality. The blood samples of mice infected with 1×108 CFU of ASsPepO, ASsPspC and ΔSsPepO/ΔSsPspC strains were collected at 3h,6h,9h, and 12h after infection. The expression levels of inflammatory factors such as MCP-1, IL-8, IL-6, TNF-α, IL-1β, were analyzed. The results showed that at 9h-post infection, the level of MCP-1 in ΔSsPspC and ΔSsPepO/ΔSsPspC infection group was significantly higher than that in parent strain infection group (P<0.05). Besides, at 3 h-post infection, the level of IL-8 of mutant strain infection group was also significantly higher (P<0.01). The above results lay a foundation to the further exploration of the role of.SsPepO and SsPspC in inflammatory storm.4. Evaluation of the protective efficacy of the double gene deletion strain ΔSsPepO/ΔSsPspC in mice6-week-old Balb/c mice were immunized with 1×108CFU of ΔSsPepO/ΔSsPspC. The blood samples were taken 14th d after the booster immunization, the levels of antibody were measured, antibody titer of was 1:40960, and that IgG2a level was higher. As Lymphocyte proliferation shows, after 72h, splenocytes stimulated with ConA of ΔSsPepO/ΔSsPspC were significantly proliferated than those of the negative and blank control groups:both the IFN-γ and IL-2 levels were obviously higher than the non-stimulated group, however, the IL-4 levels was low. All those results suggested that immune response stimulated by ΔSsPepO/ΔSsPspC was a Thl-type immune response. Mouse whole-blood killing bacteria assay indicated the sterillization rate of immunized group was 52%. All groups were challenged with a lethal dose 1.29×109CFU of SC19, the survival rate of immunized group was 50%, as the passive immunization showed the rate was 62.5%. After the challenge, there was no obvious pathological changes of immunized group.5. Evaluation of the protective efficacy of the double gene deletion strain ΔSsPepO/ΔSsPspC in pigThe Satety test of piglets were infected with a dose of 2×108 CFU and 4×108 CFU of ΔSsPepO/ΔSsPspC. A week after challenge, body temperature of piglets quickly returned to normal level after a slight rise of 1℃, spirit and feeding of piglets were normal. The piglets were inoculated (i.m.) with about 4×108CFU of ΔSsPepO/ΔSsPspC, the levels of antibody could be observed a week after immunization, and the levels, especially IgG2a level, were significantly higher after booster immunization. Whole-blood bacterial killing assays indicated that antiserum against ΔSsPepO/ΔSsPspC played an essential role of germicidal action and Phagocytosis. Fourteen days after the second boost, piglets were challenged with virulent S. suis serotype 2 and serotype 7. Survival of piglets immunized with ΔSsPepO/ΔSsPspC against S. suis serotype 2 and serotype 7 were 100% and 60%, respectively. Compared to the negative and blank control group, pathological changes and lung injury index of immune group were obviously lighter. Moreover, no obvious pathological changes in immune group were observed in piglets after chanllenge. As a whole, ΔSsPepO/ΔSsPspC can induced immunoprotection against S.suis 2 and provide a novel strategy against infection of S. suis 2.