Construction And Characterization Of A Nonencapsulated Mutant Of Streptococcus Suis Serotype 2

Streptocossus suis serotype 2(S.suis 2) is an important zoontic pathogen. So far, it has caused more than 200 cases of severe human infections worldwide including Europe, America, Australia and China. Specially, two major emerging infections disease outbreaks of S.suis 2 in China (one in Jiangsu Province, 1998, and the other in Sichuan Province, 2005), appeared to be a more invasive deep-tissue infection with STSS(Streptococcal toxic shock syndrome).Our joint research group completed a comprehensive study of comparative genomics, decoding the whole genome sequences of two virulent S.suis 2 strains, 98HAH12 and 05ZYH33(isolated from Chinese STSS patients) and an avirulent strain 05JY68(isolated from healthy pig). Results showed that 05ZYH33 contained capsule synthetic gene cps2B. Homology comparison revealed that the amino acid sequence similarity between cps2B gene encoded protein and Streptococcus pneumoniae capsular polysaccharide synthesis protein Wzd was 58%. A large number of studies have shown that capsular polysaccharide (capsular polysaccharide, CPS) plays an important role in the process of bacterial pathogenesis and is a virulence of a wide range of pathogens. To reveal the relationship between the candidate CPS and the high pathogenicity of S. suis 05ZYH33, the following experiments are conducted:1. Construction of a knockout mutant of cps2B: The flanking DNA sequences to cps2B were amplified from the chromosomal DNA of S. suis 05ZYH33 and cloned into pUC18 vector. Then, the Spc^R gene cassette amplified from the E. coli-S. suis shuttle vector pSET2 was inserted to generate the cps2B knockout vector pUC::cps2B. To obtain the isogenic mutantΔcps2B, the competent cells of 05ZYH33 were subjected to electrotransformation with pUC::cps2B. For all the Spc^R transformants, colony PCR assay was used to examine them with a series of specific primers. The suspected mutant was further confirmed by Southern blotting analysis to ensure that an isogenic knockout mutant of cps2B (namelyΔcps2B) was successfully constructed.2. Effect of cps2B deletion on the general biological characteristics of S. suis 05ZYH33: The general biological characteristics of the wild type strain 05ZYH33 and theΔcps2B mutant were compared under the same conditions. Results showed that theΔcps2B could grow steadily in the blood agar plate, there were no significant differences between knockout strains and wild strains in hemolytic activity and bacterial morphology. After deletion of the cps2B gene,Δcps2B were found to have reduced ability to form long chain. Electron microscopy showed that the mutant cannot synthesis capsule, the non-encapsulated mutant strain was successfully constructed. The mutantΔcps2B no longer agglutined with the specific anti-capsular serum in the agglutination experiment.3. Effect of cps2B gene deletion on the biological function of bacteria: In the same condition,Δcps2B could be cleared more easily by the whole blood of human and piglet, while the capacity of adhesion to epithelial cells increased greatly.4. Impact of cps2B mutation on the virulence of S. suis 05ZYH33: To evaluate the impact of cps2B deletion on the virulence of 05ZYH33, thirty Balb/c mice of 28-day-old were equally distributed into three groups. In murine modle, the first group's mice were inoculated intraperitoneally with 1ml volume of Streptococcus suis type 2 virulent bacterial suspension corresponding to 10⁸ CFU of bacteria, while the second group withΔcps2B. The last group's mice were inoculated with equal volume of THS served as negative control. Eight of ten the first group's mice dead in 2 days while no mice dead in the second group, though some of the inoculated mice were infected slightly.5. Protective evaluation of non-encapsuled mutant in mouse infection model. MutantΔcps2B were used to immunize mice, and the antibody titer was determined by ELISA. The antibody titers generated by immunization with mutantΔcps2B were at least 1∶25600. One week after the last time of immunization, 5 times of LD₅₀ of 05ZYH33 was used to challenge mice. Mice were observed for mortality in control group and immunization groups, of which survival rates were calculated. Differences of the overall survival rates between mice in control group and that in immunized groups were analyzed by Statistics software. Results indicated that the non-encapsuled mutant was capable of effectively stimulating Balb/c mice to produce protection against the lethal challenge with high virulent strain 05ZYH33 of S.suis 2.