Study On The Function Of Gene SSU05₁000 Of S.Suis Serotype 2 And The Mechanism Of Anti-phagocytosis

Streptococcus suis serotype 2（SS2） is a zoonosis pathogen with high infectivity. SS2 causes human Bacteremia, Streptoccuss Toxic Shock Syndrome（STSS）, Meningitis and so on. Two outbreaks, which had made 14 and 38 deaths seperatly, have occurred in humans in China at 1998 and 2005. Different from other contries, STSS was a major etiological and mortality factor in these outbreaks in China.Recent research mainly focused on selection of virulence factors and protective antigens, pathogenic mechanism, mechanism of anti phagocytosis. Many virulence factors had been revealed such as CPS, Sly and so on. But the virulence of these factors were not absolutely, for some non-virulent isolates was also found to preserve one or more virulence factors. This uncertainty makes it difficult to find out a general mechanism of pathogenesis. As a whole, it was becoming more and more important to find out new virulence factors and investigate there roles in pathogenic mechanism.SSU05₁000 was a newly found cell wall protein which has not been researched overseas or interiorly. This protein was presumed to play an important role in infection based on the high immunogenicity itself. We investigate the virulence of SSU05₁000, together with its role in anti-phagocytosis. Results are listed below:1 Construction, verification and biological phenomenon analysis ofΔ1000SS and CA1000SS05ZYH33 serves as starting strain, homologous recombination was performed through pSET-4S vector to constructΔ1000SS. Then pAT18 was used for complementation.05ZYH33,Δ1000SS and CΔ1000SS were employed for CD1 mouse competitive infection assay, HEP-2 and HUVEC adhesion assay, whole blood killing bacteria and expansion assay, PMN killing bacteria assay. The Competition Indices（CI） was 0.531 revealed thatΔ1000SS was much easier to be cleaned than 05ZYH33 in CD1 mouse. Compared to the result 8.23% and 5.35% of 05ZYH33,Δ1000SS has a significant low adhesion rate of 5.37% and 1.88% with HEP-2 and HUVEC respectively. Survival and expansion rate ofΔ1000SS were 16.35% and 2.98 respectively, meanwhile 05ZYH33 were 26.25% and 4.30.Δ1000SS performed a survival rate of 37.47% in PMN killing bacteria assay compared to 56.31% of 05ZYH33. These result revealed that SSU051000 is a virulence factor and it plays an important role in bacteria colonization and anti phagocytosis.2 Investigation on the role of SSU051000 in anti PMN phagocytosis2.1 investigation on nucleotidase activitySSU051000 protein with the purity of above 75% was purified after optimizing purification condition. SSU051000 was labled as nucleotidase by SANGER Institute（www.sanger.ac.uk）. But no evidence was found in DNA degradation assay, it can be inferred that SSU05 1000 was not a nucleotidase so as it can not affect NETs.2.2 His Pull-Down plasma protein inteact with SSU051000 and verify interactionWe got Fibrinogen（Fg） in his Pull-Down experiment which SSU051000 protein served as bait protein. ELISA, Far WesternBlot and FACS were performed to verify SSU051000 bind to Fg, Subsequently. PMN killing bacteria assay was performed to verified interaction between SSU051000 and Fg. In this experiment we found that survival rate of 05ZYH33 rise from 60.23% to 72.61% and 78.67% when add Fg exogenously or plasma, and the survival rate ofΔ1000SS shows Significant difference between serum（40.21%） and serum+Fg（63.03%）, plasma（70.88%）. This result proved a limited interaction between SSU05₁000 and Fg which was not the main cause of anti PMN phagocytosis.2.3 investigation on the role of SSU051000 in anti-phagocytosis by complement deposition assayWe examined complement deposition on 05ZYH33,Δ1000SS and CA1000SS under different circumstances through FACS method. We found that complement deposition rise from 64.74 to 139.16 when SSU051000 was knock out. Complement deposition on A1000SS was only 22.90% more than 05ZYH33 when specific IgG was added to serum. When C3b free serum was used for experiment, complement deposition on 05ZYH33 and A1000SS drops from 139.16 and 64.74 to 4.75 and 5.17 （without significance to negative control 2.07）, seperatly. A conclusion can be draw that SSU05₁000 can avoid C3b deposition on SS2, PMN killing SS2 was activated through alternative pathway when lacking specific IgG.In this research, we studied the function of gene SSU05₁000 and confirmed it is a virulence factor. We captured an important protein-fibrinogen which can interact with Streptococcus suis surface proteins by using Pull-Down method. We examined the interaction between SSU05₁000 and fibrinogen and the influence of SSU05₁000 on complement deposition on SS2. The result revealed the anti-phagocytosis mechanism of SS2 preliminary. These achievements laid the foundation for the laboratory to future research.