| Streptococcus suis is one of the most important and prevalent pathogens in swine. It is also considered an important zoonotic agent. There are currently33recognized serotypes. Streptococcus suis type2(SS2) is the most prevalent and virulent in pigs and humans. There were two outbreaks of human SS2infections with clinical manifestations of toxic shock syndrome in China in1998and2005due to direct contact with pigs or carcasses infected with SS2. S. suis type2differs in virulence among isolates from different sources or even from different continents There are a number of findings be showed have different virulence. MLST has been used for typing of more than400SS2isolates and those belonging to ST1and ST27complexes are considered as more pathogenic. The pathogenesis of S. suis type2infections is not well understood. It is believed that the bacterium enters through the respiratory route to colonize tonsils, subsequently escapes from the immune system and reaches the blood circulation system, so causing meningoencephalitis, arthritis, septicemia and even sudden death.1. Genetic and virulence characterization of Streptococcus suis type2isolated from swine in Provinces of Zhejiang and Henan, China:This study was aimed to examine the genetic characteristics of44Streptococcus suis type2(SS2) isolates and the virulence attributes of23representative isolates. Multilocus sequence typing revealed five sequence types (ST1, ST7, ST28, ST86and ST162) with19isolates assigned to ST7(43.2%),14to ST1(31.8%) and9to ST28(20.5%). PCR typing of the pilus gene clusters classified the isolates into three types A (72.7%), B (22.7%) and N (4.5%). All isolates of pilus types A and N were assigned to the ST1complex containing ST1, ST7and ST86, while those of type B belonged to the ST27complex comprised of ST28and ST162. Only two strains had the putative pathogenicity island89kb cluster (89K) and were of type N. The type B strains had significantly lower adhesion, were more readily killed by macrophage, and had lower virulence to mice than those of types A and N. We conclude that SS2strains of both ST1and ST27complexes, parallel to the pilus types A and B, were prevalent in the pig populations in Zhejiang province, and ST7and ST1strains were the predominant genotypes in the diseased pigs with pneumonia.2. Sortase gene and virulence:The sortase is responsible for covalently linked many surface proteins with C-terminal motif (LPXTG) to cell wall of gram-positive bacteria and also plays key role in full virulence of pathogenic bacteria. Streptococcus suis type2(SS2) have been found five gene encoding proteins that were homologous to sortases of other bacteria. In order to characterized the role of a sortase homologue of streptococcus suis type2(SS2) in pathogenicity. we constructed a five mutants of SS2by allelic exchange. Analysis revealed that ASrtA, ASrtE and ASrtG displayed reduction in adhesion and invasion to endothelial cells. Furthermore, we investigated the ability of five mutants to transmigate through monolayers of microvascular endothelial cell. Significant reduction were also found in the ASrtA, ASrtE and ASrtG mutants. Mouse infection experiments showed that deletion of SrtA, SrtE and SrtG attenuated the full virulence of wild type. These results provide evidence that SrtA, SrtE and SrtG was involved in the virulence of SS2.3. CcpA gene and virulence:Catabolite control protein A (CcpA) is the major transcriptional regulator in carbon catabolite repression in several Gram-positive bacteria. We attempted to characterize the role of a CcpA homologue of streptococcus suis type2in sugar metabolism and virulence. Addition of glucose or sucrose in the defined medium significantly reduced the activity of raffinose-inducible a-galactosidase. cellobiose-inducible β-glucosidase and maltose-inducible a-glucosidase of the wild-type strain by about9,4and3folds respectively. Deletion of ccpA substantially derepressed the effects on a-galactosidase and β-glucosidase by repressing sugars. The ccpA deletion mutant showed reduced expression of virulence genes sly and eno, decreased adhesion to and invasion into endothelial cells by about40%, and attenuated virulence to mice with significant reduction of death rate and bacterial burdens in organs, as compared to the wild-type strain. Both in vitro and in vivo defects were restorable by ccpA complementation. Thus, this study shows that CcpA of S. suis type2plays an important role in carbon catabolite repression and virulence.4. SodA gene and virulence:Superoxide dismutase (Sod) is a virulence factor of certain pathogenic bacteria by diminishing the effect of oxidative burst of phagocytic cells. Earlier reports indicated the presence of manganese-cofactored Sod in Streptococcus suis type2(SS2). However, the biological role of Sod and its coding sequence in SS2has not yet been characterized. The SSU1356-ORF of a clinical SS2strain ZJ081101encodes a protein of201amino acids with81%-88%identity to SodA of other streptococcus spp. A sod deletion mutant (△SodA) from the clinical strain was constructed. Sod activity was absent in the cell extract from the△sodA mutant, but present in that from the wild-type or the sod-complemented (CASodA) strain. The△sodA mutant was more susceptible to oxidative stresses induced by hydrogen peroxide or paraquat. Survival of the sod deletion mutant in RAW264.7macrophages was only half of that of the wild-type strain. Deletion of sod significantly attenuated virulence of SS2to mice. Effects of such genetic deletion were complementable using the strain CASodA. The co-inoculation experiment in mice revealed that the AsodA mutant was far more easily cleared from the body than the wild-type strain as shown by about3-log reduction of its infection potential in blood and tissues. In summary, we reveal an important role of SodA in pathogenesis of S suis type2, most probably by scavenging reactive oxygen species from macrophages.5. Analysis of the immunoprotective activities of pilus subunit:SS2is the causative agent of several diseases in both pigs and humans. Pili of gram-positive bacteria are key virulence factors and their subunits are considered excellent vaccine candidates. In this study, we first identified a surface protein of SS2showing features of an ancillary pilus subunit, as evidenced by ELISA, Western blot and immunoelectron microscopy. The immunogenicity and protective efficacy of recombinant Sgpl, Sgp2a, and Sgp2b belong to SrtG pilus subunit from SS2was evaluated in mice. Immunization with recombinant SrtG pilus proteins induced a vigorous antiboby response, immunization of mice with Sgp1+Sgp2a+Sgp2b yield80%survival compared to the PBS (negative control) yield20%survival. The immunization with single protein (Sgpl, Sgp2a, and Sgp2b) were below to combination protein, and yield50%,50%, and40%respectively survival. These result suggest that SrtG pilus proteins can confer partial protection against SS2infection and could be useful for the development of subunit vaccines against SS2.Our study demonstrated that SS2strains, pilus types A and B, were prevalent in the pig populations in Zhejiang province, and pilus types A showed more virulence than pilus types B; deletion of SrtA, SrtE and SrtG attenuated the full virulence of wild type by decreaseing the ability to adhesion, invasion and transmigrate to BMEC; CcpA of S. suis type2plays an important role in carbon catabolite repression and virulence; SodA of S suis type2could rise the survival rates in macrophages most probably by scavenging reactive oxygen species from macrophages. These studies provided a base on further to research the pathogenesis of SS2. |
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