Adherence And Influence Of Inflammation-associated Cytokines Transcription Of Streptococcus Suis Serotype2to The Cells

Streptococcus suis serotype2(S. suis2, SS2) is a worldwide causative agent of many forms of swine infection and is also recognized as a zoonotic agent causing human disease, including meningitis. It is seriously harmful to the pig industry and related employees. The pathogenesis of S. suis infection has not been completely defined. But, it has been proposed that the bacterial adherence and inflammation played an important role in infection. Once S. suis reaches deep tissues and/or the bloodstream, it is subject to the action of phagocytic cells of the innate immune system. However, S. suis is able to resist phagocytosis and persist in blood at high concentrations, with inflammatory consequences. Adherence capacity is whether as a maker for bacterial virulence, and the molecular mechanisms which the adherence capacity of different SS2strains is different have not been well clarified. When mononuclear phagocytes interacted with SS2, the cells could release inflammation-associated cytokines. By studying the cytokines change, we could elucidate how SS2mediated and regulated the inflammatory reaction.In this study, we chose five SS2strains which had different virulence factors and were isolated from different places to incubate with PK-15, HEp-2, and PIEC cells for3h, and then observed the ability of adherent cells by plate counting. Also, by developing real-time PCR assays, we detected the mRNA transcriptional level of adhesive factors of SS2, and wanted to determine the transcriptional level of adhesion factors wheather affect the bacterial adhesion to cells. Besides, by establishment of porcine IL-1β, IL-18, IL-8, IL-12and IL-10real-time PCR assays, we detected the changes of the cytokine mRNA transcriptional level, when parts of the strains selected to stimulate the3D4porcine alveolar macrophages.1. Adherence of SS2to PK-15/HEp-2and PIEC cellsAfter1.0×107CFU SS2incubated with the PK-15/HEp-2and PIEC cells for3h, and then plate counted. Results showed individual variations of adhesion to the cell were observed among different S. suis strains, while adhesion of the same strain was also different among the three cells. When the strains were treated with some proteases, they could reduce adhesion level. In this study, we demonstrated that S. suis2has adhesion to epithelial cells and vascular endothelial cells. Besides, adherence ability is associated with the S. suis2strains and cell types. There is no necessary connection between the adherence capacity and bacterial virulence.2. Development and mRNA transcriptional level in vitro of SYBR Green Ⅰ-based real-time PCR assays for detection of adhesive factors of SS2The specific primers were designed and synthesized according to the nucleotide sequence of the different adhesive factors and housekeeping gene aroA of SS2. The fragments of adhesive factors were amplified by RT-PCR from SS2strain. The recombinant plasmids containing the target gene were constructed, and then used as the real-time PCR standard templates. real-time PCR assays based on SYBR Green Ⅰ for detection of the adhesive factors and aroA were established. By detecting mRNA transcriptional level of the adhesive factors of the strains which is used in adhesion assays, we elucidated the differences of adhesion ability in molecular level, and we indeed found mRNA transcriptional level of adhesive factors influenced the adhesion level of S. suis strains. In this study, we preliminarily obtained molecular mechanisms of cell adherence of SS2strains, and provided a platform for deep research of the pathogenesis of SS2infection in future.3. Development of SYBR Green Ⅰ-based real-time PCR assays for detection of porcine IL-1β, IL-18, IL-8, IL-10, IL-12and β-actin, and mRNA transcriptional level analysis of inflammation-associated cytokines of3D4cells stimulated by SS2The IL-1β, IL-18and β-actin fragments were amplified by RT-PCR from3D4porcine alveolar macrophages, and then the fragments were cloned and sequenced. The3recombinant plasmids containing the target gene were constructed,and then used as the real-time PCR standard templates, while the recombinant plasmids of IL-8, IL-10and IL-12had been constructed and conserved in the lab. Real-time PCR assays based on SYBR Green Ⅰ for detection of IL-1β, IL-18, IL-8, IL-10, IL-12and β-actin were established. Using the established method for detection of porcine cytokines, S. suis2strains could induce the mRNA transcriptional level of different inflammation-associated cytokines by3D4porcine alveolar macrophages. Our results indicated that different virulence and genotype strains of SS2seemed to influence the mRNA transcriptional level of the cytokines.