Structural And Functional Study Of Virulence-Associated Protein Hp0197, And The Study Of Quinolone-Resistance Associated Gene Mutations

Streptococcus suis (S. suis) is an important swine pathogen which causes an economical problem in the swine industry. The manifestations of S. suis infection in swine are meningitis, arthritis, endocarditis and septicemia. Thirty-three serotypes (types1-31,33and1/2) have been described on the basis of the capsular polys acchar ides, and S. suis serotype2(SS2) is most commonly associated with diseases in pigs in worldwide. Moreover, it’s also the causative agent of serious infections in humans. From the first infection case reported in Demark, more than700infection cases were reported at least20countries all over the world. Although S. suis infection has attracted great attention from the scientific community and the popular press, our current understanding of the S. suis pathogenesis remains limited. The attachment of bacteria to host cells and tissues and their subsequent invasion and spreading are key processes during pathogenesis. Interactions between the bacteria and host cells are responsible for these processes. Further study o is necessary to uncover the pathogenesis of S. suis.HP0197is an SS surface antigen that was previously identified by immunoproteomics.It is also a virulence-associated protein which function is still unknown. In this study, we tried to uncover its function via structure analysis. The antibiotics-resistance mechanisms of the pathogens have aroused more and more concern since the "superbug" has been discovered. We conducted a study on the novel quinolone-resistance associated mutations of S. suis DNA gyrase and topoisomerase Ⅳ. The mutations are characterized from the previous clinical isolates1. The interactions between HP0197and host cells surface glycosaminoglycans (GAGs)Here, we investigated the interaction between HP0197and host cell surface glycosaminoglycans (GAGs) using indirect immunofluorescence and cell adhesion inhibition assays. In addition, we determined that a novel18-kDa domain in the N-terminal region of HP0197functions as the GAG-binding domain. We then solved the three-dimensional structures of the N-terminal18-kDa and C-terminal G5domains using X-ray crystallography. Based on this structural information, the GAG-binding sites in HP0197were predicted, and they were then confirmed using site-directed mutagenesis and indirect immunofluorescence. The results indicate that the positively charged residues on the exposed surface of the18-kDa domain, mostly lysines, play a critical role in the HP0197-heparin interaction and it mediated bacteria-host cell adhesion. Understanding this molecular mechanism could provide a basis for developing effective medicines and therapeutic strategies for treating streptococcal infections.2. The quinolone-resistance associated mutations of S. suis DNA gyrase and topoisomerase IV.Analyed the amino acids sequences of gyrA, parC, gyrB and parE in fluoroquinolone-susceptible and-resistant Streptococcus suis clinical isolates,5high-frequent mutations are discovered. To evaluate the contribution of the mutations to the quinolone-resistance of Streptococcus suis, we cloned gyrA, parC, gyrB and parE with5mutations to vectors. These vectors were then transformed to E. coli. We found that the MICs of E. coli to quinolones are significantly increased when the mutants are transformed. This result demonstrates that the mutations of S. suis DNA gyrase and topoisomerase IV directly contribute to the quinolone-resistance of S. suis.