Research On Function Of Gene SSU05₂036and SSU05₂039Regulated By Two-component Regulatory System CiaRH In Streptococcus Suis Serotype2

Streptococcus suis serotype2（SS2） is an important zoonotic pathogen causing pigs meningitis, endocarditis, serous inflammation, arthritis, pneumonia, sepsis, etc. An outbreak of SS2in Sichuan, China in2005has brought huge losses to pig industry, but caused great threat to the public health. Therefore, the pathogenesis of Streptococcus suis type2has become a research hotspot at home and abroad. Currently, there are some virulence factors of SS2have been reported: the capsular polysaccharide （CPS）, lysozyme release and extracellular protein （MRP）, factor EF, hemolysin Sly, fibronectin-binding protein FBPS, Serum Opacific Factor （OSF）,89K virulence Island and two-component regulatory system etc.It has been reported there are15pairs of two-component regulatory system in SS2including CiaRH, SalK/SaIR, RevS,2148hk/rr and so on. CiaRH is the first two-component regulatory system found in Streptococcus pneumoniae. It not only have a strong reaction to single signal, but maintain high levels of gene expression in a variety of conditions. CiaRH involves in various processes such as competence development, host colonization, autolysis, bacteriocin production and virulence. Meanwhile CiaRH also participate in biofilm formation, which plays an important role in the escape of antibiotics and bacteria immune tolerance.Dr. Li constructed a mutant strain of the CiaRH and measured its virulence in vitro and vivo in our group. Compared with the wild type strain SC19, the mutant strain exhibited a significant decrease in virulence and lethality of the mice and pigs. We found that CiaRH can affect biofilm formation of SS2. However, the effection of CiaRH on biofilm formation and its regulatory mechanism in SS2are unknown. Therefore, we conducted a series of inquiry on this issue to screen regulatory gene associated with biofilm of CiaRH, such as microarray data, realtime PCR and EMSA （electrophoretic mobility shift assays）. Then we constructed mutants and searched its biological characteristics, biofilm formation, effection to model animal and RAW264.7to investigate whether the deletion of its regulatory genes affect the biofilm formation and evasion from the host immune attack. Accordingly, we explore the pathogenesis of SS2. Specific experiments are as follows:1. Screening regulatory gene of CiaRH and expression of regulator CiaRWe filtered out SSU05₂036and SSU05₂039associated with biofilm by the microarray analysis result of CiaRH. We refered to05ZYH33genome as a template to amplify the sequence of regulator CiaR and ligated the amplified sequence into the prokaryotic expression vector pET-30a to construct pET-CiaR. The plasmid pET-CiaR was transformed into BL21, induced by IPTG. We got the regulator CiaR. Then we used realtime PCR and EMSA to verifier them. The results showed that SSU052036and SSU052039can regulated by regulator CiaR.2. Construction and identification of ΔSSU052036and ΔSSU052039in SS2Using05ZYH33（SC19） whole-genome sequences as template, we amplified the upstream and downstream fragments of ASSU05₂036and ASSU05₂039, and structured recombinant plasmid pSET4s-2039and pSET4s-2036by using the temperature-sensitive suicide plasmid pSET4s. Based on Spc-sensitive selection and two-step homologous method, we constructed ΔSSU05₂036and ASSU05₂039successfully.3. Research on the biological characteristics of deletion mutant ΔSSU05₂036and ΔSSU05₂039We had research on growth curve determination, hemolytic activity, biofilm formation, mice infection experiments（the median lethal dose calculation and organizations carrier experiment） and phagocytosis experiments. The results show that the deletion of SSU05₂036and SSU05₂039did not affect the growth and hemolytic activity of SS2, but increased the amount of biofilm formation. Experimental infection to the BALB/c mice and organizations carrier experiment showed that the deletion of SSU05₂036and SSU05₂039did not reduce virulence of SS2, but enhanced the bacteria colonization capacity in mice. And then slowed down the speed of removal by the body. Phagocytosis experiments showed that compared with SC19, the anti-phagocytic ability of deletion strains enhanced significantly. These results suggest that SSU05₂036and SSU05₂039can affect the biofilm formation of SS2, further affect the ability of colonization and resistance to phagocytosis of SS2in the BALB/c mice.