Identification Of Novel Virulence Factors Of Streptococcus Suis Serotype 2 In China

Streptococcus suis ?S. suis? is an important zoonotic pathogen. Streptococcus suis Serotype 2 ?SS2? is the most virulent and the most frequently isolated serotype among the 35 serotypes. In 1998 and 2005, two S. suis outbreaks in humans occurred in local region of Jiangsu and Sichuan provinces, respectively. It is worth noticing that most of severe cases present typical symptoms of streptococcal toxic shock syndrome ?STSS?. So far, the pathogenesis of S. suis type 2 are not well known, several putative virulence factors have been described, including the capsular polysaccharide ?CPS?, muramidase-released cell-wall protein ?MRP?, extra-cellular protein factor ?EF? and suilysin ?SLY?. To find new virulence factor is a hot issue in this field, which due to the various virulence factors and the relativity of the space-time of pathopoiesis.After SS2 disease prevelent in our country in 2005, Chinese scientific institute sequenced the epidemic strains ?98HAH12 and 05ZYH33? that isolated in 1998 and 2005 and the comparative genomics show that a Pathogenicity Island ?PAI? with 89kb was detected, designated 89kb. This PAI only exists in the Chinese popular strains. Immunogenic proteins usually play important roles in the interaction between bacteria and host. Some novel immunogenic proteins were identified by immunoproteomics in our research group and one of proteins ?encoded by ssu05₁815? was found to be located in cell walls of SS2 by bioinformatics analysis, which suggested maybe interact with bacteria and host. Therefore, we chose one gene which encode cell walls immunogenic protein through immunoproteomics and two genes which encode Type?secretion system ?T4SS? in 89K as a putative virulence factors for studying.1. To construct gene knock-out mutant and complementary strain of ssu05₁815.The flanking DNA sequence of ssu05₁815 is amplified from the chromosomal DNA of S. suis 05ZYH33, the chloromycetin ?Cm? gene cassette are amplified from the E.coli-S. suis shuttle vector pSET1, the fused fragment was amplified to three fragments using overlap PCR technique. Followed the DNA fused fragment was cloned at the pSET4s vector directionally to generate the 1815 knockout vector pSET4s-1815 consisting of Cm cassette. Then isogenic ssu05-1815 deficient mutant are screened by allelic replacement. PCR analysis and sequencing indicate that the coding gene is replaced completely by Cm cassette. The fragment containing ssu05₁815 gene open reading frame and its upstream promoter is amplified from the chromosomal DNA of S.suis 05ZYH33. The resulting product is digested and cloned into the corresponding sites in the pAT18 to create successfully complementary plasmid pAT18-C-1815. The complementary plasmid is electro-transformed into the competent cells of?1815 and obtained the complementary strain C?1815, which are sensitive for Cm and erythromycin ?Em?.2. The study of virulence of mutant?1815The mutants?1815?C?1815 and wild type strain are plated on columbia agar containing 5%?vol/vol? sheep blood,37?5% CO2 for 24h, to observe colonial morphology. And three different strains were cultured in THB at 37?5% CO2, to determine the growth curve through OD600nm each hour. Experiments show colony form, characterization of producing zone of hemolysis and grow rate of the mutant strains and complementary strain don't change significantly by comparing with wild type strain.The adhesion of wild type strain and mutants with Human larynx epithelial cell ?Hep2? are evaluated, the results subsequently reveal that?1815 presented lower adherent ability than the wild type strain, the adherence of C?1815 is restored, and is not significant different with the wild type strain. In further virulence analysis, whole blood bactericidal experiment of?1815 show, the mutant?1815 was killed easily comparing with wild type strain after one hour incubation in whole blood, a bactericidal percent rate is obviously higher than 05ZYH33, bactericidal percent rate of CA1815 is restored. The combined infection experiment of CD1 mice and piglets show that, the proliferation of?1815 is inhibited in the process of combined infection, competitive index ?CI? is significantly lower than 1.0. CD1 mice meningitis experimental showed that the CFU of mutant A1815 in the brain is lower than the wild type strains, the gene may be related to the strain passing through the blood-brain barrier. This suggested that the gene ssu05₁815 is related to virulence, which lay the foundation for the further SS2 pathogenesis.3. To construct gene knock-out mutants of ssu05₀969 and ssu05₀973 and analysis their virulence.To obtain gene knock-out mutants of ssu05₀969 and ssu05₀973 through homologous recombination, PCR analysis and sequencing indicate that two coding genes are replaced completely by Cm cassette.The growth curve and colonial morphology on the columbia agar show that, colony form, characterization of producing zone of hemolysis and its size and grow rate of mutant strains?0969 and?0973 don't change significantly by comparing with wild type strain. The adhesive experiment of Human larynx epithelial cells?Hep2? reveal that, the adhesion of A0969 and?0973 are not significant with wild type strain.In further virulence analysis, whole blood bactericidal experiment show, the mutants?0969 and?0973 are killed easily comparing with wild type strain, after one hour incubation in whole blood, a bactericidal percent rate is obviously higher than 05ZYH33. The virulence of two mutants?0969 and?0973 are decreased in the combined infection model of CD1 mice, competitive index ?CI? are lower than 1.0, their proliferation are inhibited in the process of combined infection. The results of the combined infection model of piglets are the same as model of CD1 mice, the virulence of the mutant A0973 is decreased, competitive index ?CI? are lower than 1.0, their proliferation are inhibited in the process of combined infection. This suggested that the genes ssu05₀969 and ssu05₀973 might be related to virulence, which lay the foundation for the further SS2 pathogenesis.