Construction And Characterization Of Cps2E Deleted Mutants Of Streptococcus Suis Serotype2

Streptococcus suis （S. suis） is a major swine pathogen responsible for huge economic losses to the swine industry worldwide, and an emerging zoonotic agent serious threat to human public health. Since deadly human S. suis outbreaks have occurred in China in1998and2005, more and more attentions have been paied on S. suis. S. suis can destruct the host epithelial barrier into the blood, and then invading different organs. The pathogen can cause a porcine acute septicemia, meningitis, endocarditis, arthritis and human meningitis, streptococcal toxic shock syndrome （STSS）. Streptococcus suis serotype2（or S. suis2） is the major pathogen among all serotypes with strong pathogenicity, broad popular and high clinical isolate rate.In recent years, the studies on the pathogenesis and virulence factors of S. suis2indicate that the capsular polysaccharide （CPS） is recognized as one of the important virulence factors. Smith provided S. suis2CPS biosynthetic gene cluster, Van Calsteren initially established the correlation between S. suis2CPS repeating unit sequence and the cps cluster encoding enzymes involved in the biosynthesis. Nevertheless, the understanding of the role CPS played in pathogenesis of S. suis2, the diversity and evolution is still very limited. In order to clarify these questions, further studies on the genes involved in S. suis2CPS biosynthesis is required.Yother predicted Cps2E from Streptococcus pneumoniae serotype2was an initial glycosyltransferase in its Wzx/Wzy dependent CPS biosynthetic pathway. In addition, Smith found that S. suis2cps2E gene had a high specificity of serotype2. Therefore, we choose S. suis2cps2E gene for study, mainly on its biological characteristics and pathogenicity, in order to determine its function in the CPS biosynthesis and make foundation of studies on other genes related to S. suis2CPS biosynthesis. The reaserch main contents are as follow:1. Bioinformatics analysis of S. suis2cps2EHomology and phylogenetic analysis among CpsE protein sequences from S. suis SC84,05ZYH33, P1/7strains and corresponding protein sequences from different bacteria strains have showed that:cps2E from the three S. suis strains are highly conserved; Meanwhile, cps2E gene has high similarity with the gene from Streptococcus pneumoniae and Streptococcus agalactiae.2. Construction of gene deletion mutant△cps2EPrimers have been designed according to genome sequence of S. suis205ZYH33strain （Accession No.:NC₀09442） whose cps2E loci is SSU05₀568. Amplify the upstream and downstream homology arm of cps2E from S. suis2SC19, and the ligation products obtained by SOE PCR. The amplification products have been connected to pSET4s temperature-sensitive shuttle plasmid. The recombinant plasmid pSET4s-Acps2E was identified by restriction enzyme digestion and PCR.3. Analysis of△cps2E biological traitsResearch the growth characteristics of Acps2E and WT at37℃. After inoculation with4weeks old BALB/c mice respectively, Acps2E and WT were isolated and purified from the blood. Then we analyzed the stains by Gram stain, transmission electron microscopy and determination of CPS sugar content. The results have showed that: compared with WT, the growth rate of Acps2E is significantly faster, and has a larger number of cells at stable phase; different from WT, Acps2E mostly be in single order; notedly, the capsul of Acps2E is different from WT; Acps2E CPS extraction rate is only34.52%of WT.4. Analysis of Acps2E biological fuctionConduct cell infection experiment with Acps2E and WT at37℃,5%CO2. On the one hand, Hela experiments have showed that the adhesion by Acps2E is not significantly different from WT, and the invasion capacity of Acps2E is enhanced by1.65times; on the other hand, RAW264.7phagocytosis experiments have showed that the anti-phagocytic ability of Acps2E is significantly declined than WT.Acps2E and SC19intraperitoneal injected4weeks old BALB/c mice respectively, and LD50were tested. The results have showed that:the virulence of Acps2E decreased by4.47times compared with WT, indicating that cps2E gene has some influence on S. suis2virulence. Then the survival rate test by BALB/c mice have showed that:the survival rate of Acps2E group was100%, while the survival rate of WT group was only30%, Therefore we conclude that Acps2E weakened pathogenicity. According to the dynamic changes of bacteria content in blood from infected mice, Acps2E showed a trend of gradual collapse, were wiped out by the body in the5th day after infection, while the WT still maintaind a certain amount of bacteria in blood.