Identification And Characterisation A Surface-Associated Arginine Peptidase In Streptococcus Suis Serotype 2

Streptococcus suis is an emerging significant zoonotic agent which can infect swine and human beings, leading to a wide range of diseases such as septicemia, arthritis, endocarditis and meningitis. To date, it has been documented that the S.suis has affected over 1584 peoples among up to 20 countries. Thus far, thirty-three serotypes have been identified and described on the basis of the capsular polysaccharides, and of them, the S.suis serotype 2 is most often related to the clinical infection, following by the serotype of 14 and, far distanced,by other serotypes such as serotype 1,4,5,16 and 24. Although the growing attention from the academic community on S.suis in recent years, however, the critical putative pathogenesis of Streptococcus suis, especially the underlying mechanism by which the S.suis causes the meningitis and streptococcal toxic shock syndrome in humans still remains unclear.It is well known that the protease within pathogens plays a significant role in the host tissues degradation and escape and neutralization of the immune defense system. Therefore, in the present study, the primary objective was to investigate the role of surface-associated protease in the course of S.suis infection.Based on our previous work and online available data, we mainly focus on the putative arginine protease in Streptococcus suis serotype 2,designated amylase binding protein B in the genome annotation, and explore its role in the process of infection. The recombination protein was over-expressed in E.coli system and purified using fast protein liquid chromatography with a Ni-NTA column. To test its putative arginine protease attributes, the gelatin zymography assay was employed to verify the protease property and the H-Arg-p-nitro-aniline was used to determine its arginine peptide cleavage activity. In addition, the recombination protein was capable of trigging the release of high level cytokines in vitro in the RAW264.7 cell lines. To investigate the immunogenicity of Abpb protein, the Balb/C intraperitoneal immune mice model was exploited. The Elisa assay was conducted to determine the antibody titer against the Abpb in the serum, and the passive lethal infection experiment was utilized to test its protection effect. As to subcellular localization of Abpb, we ascertain its expression in the surface of S.suis by a variety of Western blot assays and flow cytometer. Additionally, the slight secretory form of Abpb was also detected. To explore the role of Abpb in the course of S.suis infection, we constructed an Abpb-defective mutant(△Abpb) from the wild type SC19 strain by homologous recombination with the aid of thermosensitive suicide vector p SET4 s.The abpb mutant showed an attenuated virulence compared to the wild strain in the mice intraperitoneal infection model. Given that the early effective attachment and invasion into the epithelial cells and subsequent escape and resistant to the elimination of immune cells are the pivotal steps for S.suis successful infection, therefore we compared the capability of wild SC19 and the △Abpb mutant to adhere to epithelial cells Hep-2. The result exhibited an decreased ability of the mutant strain to adhere to the epithelial cells. What’s more, in another experiment, the mutant could also be effectively eliminated by the primitive macrophages from the peritonea of mice accounting for 35%, while there is no obvious phagocytosis detected in the wild SC19 group. Intriguingly, when the macrophages were superseded by neutrophils, both of the SC19 and mutant cannot be cleared and killed. To further confirm aforementioned phagocytosis difference, an co-infection experiment was employed and conducted. After the infection of the equivalent amount of wild SC19 and △Abpb mutant in the common mice, the bacterial loads of wild SC19 and △Abpb in the same organs of spleen, lung, liver and brain and also the blood was isolated and analyzed. The results indicated that the △Abpb mutant has an impaired ability to clone in vivo and to escape from the elimination of the immune system.In conclusion, in this study, we identified the arginine protease expressed on the cell wall in Streptococcus suis serotype 2, which can degrade the host tissue and contribute to the in vivo infection. Furthermore, it was closely related to the elimination of S.suis by macrophages and was verified to be a novel important virulence factor within Streptococcus suis serotype 2. Additionally, the recombinant abpb can provide an excellent protection against the lethal challenge of S.suis 2 in mice model, suggesting an another ideal candidate for subunit vaccine development.