The Study On Virulence-associated Genes Of Streptococcus Suis Type 2

Streptococcus suis type 2(SS2)is an important zoonosis pathogenic bacteria, which caused the two recent large-scale outbreaks of human and pigs. SS2 can be divided into virulent strain, low virulent strain and avirulent strain. Current studies showed that the main virulence-related factors include the capsular polysaccharide (CPS), the extracellular factor (EF), the release of lysozyme protein (MRP) and the hemolysin (Sly), and so on, but their pathogenicity is still unclear. The compositions of the SS2 virulence factors are very complicated. Study found that some of SS2 isolates had highly pathogenic although without EF, MRP and SLY. In contrast, some isolates which possessed these virulence factors with weak pathogenicity. So that SS2 may have some other unknown virulence factors.In order to find other potential virulence factors in SS2, we tried to find out the difference between the genomes of virulent strains and avirulent strains. Finally we established difference library through suppression subtractive hybridization (SSH). We obtained 42 specific fragments of SS2 virulent strains. According to the analysis of the ORFs of SS2 genome, these fragments were parts of the genes which encode MRP, EF, Sly, drug-resistance, type I restriction-modification system, LPXTG-motif cell wall anchor domain protein and Virulence-associated protein E, and so on. All these data provided a basis for the biological characteristic analysis of these virulence factors. The genome difference library and its biological information analytic data provided a basis for the discovery of SS2 virulence factors.virulence-associated protein E (vapE) gene was found by SSH, which belongs to Virulence-associated protein family. Virulence-associated protein family contains many proteins and enzymes which have close relationship with pathogenicity. Up to now, there is no evidence for the role of the VapE in the process of the infection of SS2. So we constructed the vapE gene deletion mutant of SS2 chinese strain 458#. Using the rabbit SS2 infection model, and with the contrast of 458#, the pathogenicity of 458#?÷vapE was researched. The result showed that the infection of 458 # was lethal to rabbits while rabbits inoculated 458#?÷vapE abdominal with 109CFU survived. The result confirmed that VapE would be an important virulent factor of SS2.A candidate pathogenicity island (PAI4) was predicted, which is only present in the epidemic virulent strains of SS2 but not in other chinese avirulent strains isolated from health pigs. However, this bioinformatically predicted candidate PAI4 needs experimental validation and its linkage to SS2-related high pathogenicity retains unknown. The analysis of PCR and DNA sequencing showed PAI4 (11kb) contained the flank distinct genetic elements acquired by horizontal gene transfer. PAI4 was knockouted from chinese epidemic virulent strain 458# of SS2 by suicide vector pSET4s, to find out the phenotype difference between 458#?÷PAI4 and parent strain 458#. 4 rabbits inoculated abdominally with 109CFU of the PAI4-deletion mutant 458#?÷PAI4 of SS2 were survived with no clinical symptoms. The result showed the candidate pathogenicity island (PAI4) is requisite for the full virulence of chinese virulent isolates of SS2, thus providing experimental evidence for the validity of predicted PAI4.