Screening And Identification Of Putative Virulent Genes Of Streptococcus Suis Type9Strains

Streptococcus suis (S. suis) is a major swine pathogen responsible for a wide range of diseases,including septicaemia, meningitis, endocarditis, arthritis and even acute death. In addition to causing disease in pigs, it is also an important zoonotic agent that afflicts people in close contact with infected pigs or pork-derived products. Thirty-three types (1-31,33, and1/2) have been described based on capsular polysaccharides, type2(SS2) is the most frequently isolated and associated with disease, while S. suis type9(SS9) is also a prevalent type that is frequently isolated from diseased pigs in countries such as Australia, Holland, Belgium, and Germany. Most studies about virulence factors are based on SS2.Unlike those of SS2, little is known about SS9virulence factors.Suppression subtractive hybridization (SSH) was used between virulent strain GZ0565and avirulent strain SH040917. Thirty fragments of putative virulent genes were found by this method. The distributions of these fragments in other S. suis types isolates were detected. The role of virulence genes in SS9pathogenesis were investigated by using isogenic mutant.1Suppression subtractive hybridization (SSH).Suppression subtractive hybridization was carried out between virulent SS9strain GZ0565and avirulent strain SH040917in order to identify gene sequences unique to the virulent strains. The tester (GZ0565) and driver (SH040917) genomic DNAs were digested with Rsal. 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 TOP10competent cells. Three hundred and four subtractive clones were randomly picked.286colonies were confirmed as positive clones, but with no inserts in18colonies in total304colonies. 2Selection and identification of putative virulent genesThe inserts of286positive 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 GZ0565were identified. These DNA fragments, containing putative virulence genes, encoded subtilisin-like serine protease, surface-anchored DNA nuclease, serine/threonine protein phosphatase, Hemolysins and related proteins containing CBS domains, GntR family regulatory protein, ABC transporter permease protein so on.3Distribution of putative virulent genes in Streptococcus suis isolatesAccording to the results of DNA sequencing of postive clones and published SS2strain genomic sequence, PCR primers for14significant DNA fragments were designed and used for detection of the distribution of these fragments in S. suis isolates from different sources, types, regions.The results showed that these14DNA fragments were widely distributed in most SS9strains, specially those isolated from diseased pigs,yet were absent among the avirulent strain SH040917.However, other slaughter-house isolates possessed few the genes. Moreover these fragments could be detected in all virulent SS2islotates.In other types of S. suis, each type had a different distribution.4.Construction of isogenic stp mutant strain and bionomics research of SS9WT and its derivativesSerine/threonine phosphatases (STP) are the second largest class of enzymes that catalyze dephosphorylation of proteins, after protein tyrosine phosphatases. Serine/threonine phosphorylation/dephosphorylation is intimately linked with signaling events inside the cell. STP affects both the virulence and morphology of the bacteria. To investigate the role of stp in pathogenesis of SS9, an isogenic stp mutant (△stp) was constructed by allelic replacement using a temperature-sensitive S. suis-E. coli shuttle vector, pSET4s.The inact stp was cloned into shuttle vector pSET2to construct the recombinat plasmid pSET2-STP. Then the plasmid pSET2-STP transformed by electroporation into the△stp strain.The complementation strain designed C△stp. The adherence assy showed that the adherence to△stp strain to HEp-2cell was68.8%of WT, The adherence of complentation strain was86.3%of WT. The percent survival rate of the wild type parent strain was45.7%in whole blood. In contrast, the mutant was much more sensitive, with a percent survival rate of only 23.4%. The complentation strain C△stp was with a percent survival rate of36.8%. LD50values was2.69×106CFU per mouse for WT strain,1.93×107CFU per mouse for the mutant strain,6.8×106CFU per mouse for complentation strain C△stp.The LD50values of△stp was higher seven fold than that of wild type strain.The virulence of complementation strain C△stp was restored. Our data suggest that stp is involved in the pathogenesis of SS9.5Construction of isogenic hlyC mutant strain and bionomics research of SS9WT and its derivativesTo investigate the role of hlyC in pathogenesis of SS9,an isogenic hlyC mutant(△hlyC) was constructed by allelic replacement using a temperature-sensitive S. suis-E. coli shuttle vector, pSET4s.The inact hlyC was cloned into shuttle vector pSET2to construct the recombinat plasmid pSET2-HlyC.Then the plasmid pSET2-HlyC transformed by electroporation into the△hlyC strain.The complementation strain designed pCHlyC. The adherence assy showed that the adherence to△hlyC strain to HEp-2cell was30.4%of WT, The adherence of complentation strain was88%of WT. The percent survival rate of the wild type strain was45.7%in whole blood,the mutant was with a percent survival rate of60.5%. The complentation strain was with a percent survival rate of48.2%.LD5o values was2.69×106CFU per mouse for WT strain,3.79×105CFU per mouse for the mutant strain,1.49×106CFU per mouse for complentation strain pCHlyC. The LD50values of mutant strain△hlyC was1/7of the wild type strain. The virulence of△hlyC was enhanced Complementation strain was restored. Our data suggest that hlyC is involved in the pathogenesis of SS9.6The immunologic properties of streptococcus suis endothelin-converting enzymeStreptococcus suis type2is a pathogen responsible for causing disease in both pigs and humans. Endothelin-converting enzyme (ECE) is an immunogenic protein of Streptococcus suis as well as a putative virulence factor. We expressed and purified ECE, which we demonstrate by Western blot is immunoreactive. In mice, ECE demonstrated a protective effect against S. suis infection. We demonstrate using ELISA assays that purified ECE can adhere to fibronectin and fibrinogen. We further show that ECE can bind to the surface of HEp-2cells via indirect immunofluorescent and adherence inhibition assays. Our results suggest that in addition to being a potential candidate subunit vaccine, ECE may also be important to S. suis pathogenesis.