Identification And Characterization Of Putative Virulent Genes Of Streptococcus Suis Type 2 Strains

Swine Streptococcusis caused by Streptococcus suis manifested as meningitis, arthritis, septicemia and sudden death. Streptococcus suis type 2 (SS2), the most prevanlent and virulent serotype among the 35 known serotypes, is also a zoonatic agent for people. In China, SS2 was first isolated in 1991, later a severe outbreak of SS2 infection in human and pigs occurred in Jiangsu Province in 1998 and in Sichuan Province in 2005. The mechanisms involved in the pathogenesis and virlence of SS2 are not completely understood. Suppression subtractive hybridization (SSH) was used between virulent strain HA9801 and avirulent strain 12. Thirty fragments of putative virulent genes were found by this method. The distributions of these fragments in other S. suis serotype strains were detected.1 Suppression subtractive hybridization (SSH).Suppression subtractive hybridization was carried out between virulent SS2 strain HA9801 and avirulent SS2 strain T15 in order to identify gene sequences unique to the virulent strains. The tester (HA9801) and driver (T15) genomic DNAs were digested with RsaI. The tester DNA was then subdivided into two portions, each of which was ligated with a different adaptor provided. Two hybridizations were performed. The entire population of molecules was then subjected to PCR to amplify the tester-specific sequences. The PCR amplification product was cloned into PMD-18T simple vetor and transformed into E. coli TOP10 competent cells. Four hundred and twenty subtractive clones were randomly picked. 408 colonies were confirmed as positive clones, but with no inserts in 12 colonies in total 420 colonies.2 Selection and identification of putative virulent genesThe inserts of 408 positive clones were amplified and arrayed on nylon membranes. Membranes were screened by hybridization with genomic DNA probe from tester and driver. Thirty gene sequences unique to virulent strain HA9801 were identified. These DNA fragments, containing putative virulence genes, encoded haemolysinâ…¢related membrane protein, typeâ…£secretory pathway VirB4 component, zeta toxin, DNA recombinase, transporter, transcriptional regulator, amino acid permease and so on. Align these sequences with 98HAH33 whole genomic sequence to learn about distribution of putative virulent genes in genome. Alignment results of these genes showed high homology to virulent strain 98HAH33 and can be located on 98HAH33 genome.3 Distribution of putative virulent genes in Streptococcus suis strainsAccording to the published SS2 strain 98HAH33 of human origin genomic sequence, PCR primers for 14 significant DNA fragments were designed and used for detection of the distribution of these fragments in S. suis strains from different sources, serotypes, regions, and other streptococcus. The results showed that these 14 DNA fragments were widely distributed in most SS2 strains, yet were absent among the avirulent strain T15. Moreover, these fragments could be detected in other serotypes of S. suis, but each serotype had a different distribution with most of the fragments being absent in Streptococcus equi ssp. zooepidemicus strains.4 Expression of TypeIV secretory pathway VirB4 component and DNA recombinase of Streptococcus suis type 2the TypeIV secretory pathway VirB4 component and DNA recombinase gene without signal peptide was amplified from genomic DNA of SS2 HA9801 strain by polymerase chain rection (PCR),then the amplified fragment was cloned in the proper orientation into the site between EcoR I and Xho I of pET-28a (+) via restriction endonuclease EcoR I and Xho I . The recombinant plasmid was verified by nucleotide sequencing and transformed into E. coli BL21 .Two fusion proteins were expressed in BL21 after induced by IPTG, SDS-PAGE and Western bloting analysis showed that recombinant proteins were about 30 kD, and positive reaction with the antiserum against ZY05719.