Pathogenic Roles Of OMP40of SS2and Identification Of Proteins Interaction Between MRP And Swine Brain Tissue

Streptococcus suis is an important swine pathogen that causes many pathological conditions, such as arthritis, endocarditis, meningitis, pneumonia, and septicemia. It is also an important zoonotic agent for humans. A total of33capsular types have been identified. There are many differences in virulence between different strains. Among them, the serotype2has always been considered the most virulent and the most frequently isolated serotype from diseased animals.Microbial pathogenicity is a complex phenomenon encompassing diverse mechanisms. There are, however, several common strategies that pathogenic organisms use to sustain themselves and overcome host barriers, one of them being the firm adhesion of the microorganism to host cells. Colonization is crucial to pathogenesis of bacteria, being the earliest stage during onset of the disease and the ability to adhere to host surfaces is by far the most vital step in the successful colonization by microbial pathogens. Streptococcal adhesions fall into two major binding categories. One large group of adhesions is the so-called MSCRAMMs (microbial surface cell recognition adhesion matrix molecule) that bind to extracellular matrix proteins and to cell-associated integrins. Many MSCRAMMs are cell wall-anchored LPxTG proteins. omp40and MRP belong to this protein in structural. In this study, Western affinity blot and gene knock out technology were used to explore the role of omp40in SS2pathogenesis. cDNA library was constructed using mRNA of swine brain. Screen the host protein interact with MRP using Y2H technology.1Distribution and expression of omp40gene of SS2omp40gene was identified using Suppression Subtractive Hybridization(SSH)in our previous study, whereas the role of omp40was not clear. Thus, the sequence and distribution of omp40in31different SS were analyzed, which contribute to study the pathogenic mechanism of omp40in SS2. The open reading frame (ORF) of omp40gene is3000bp, which encoded1000aa protein. Based on predicted protein features sharing same conserved domain with the collagen-binding protein Cna of Staphylococcus aureus, omp40is likely to function as a direct mediator of collagen adhesion. Western blotting using swine convalescent sera and omp40-specif\c antiserum confirmed its role as an immunogenic cell wall protein. Collagen binding activity can be detected by western affinity blot. Using PCR detect the distribution of the omp40in S.suis strains from different sources, serotypes, regions. The results showed that the omp40can only be found in SS2which were isolated from diseased pigs, which means this gene is related to the virulence.2. Construction and Characterization of omp40gene mutant strains and complementation strains in ZY05719To research the function of omp40gene, the isogenic omp40mutant (△.omp40) was constructed by allelic replacement using a temperature-sensitive S. suis-E. coli shuttle vector, pSET4s. Deletion of the omp40gene in SS2reduced its adhesion to collagen and Hep-2cells, capacity for biofilm formation, and its virulence in a zebrafish infection model. Our data suggest that omp40is involved in the pathogenesis of SS2.3. Microarray analysis of omp40-mediated bEnd.3infectionTo increase our knowledge of the mechanism of omp40in SS2infection, we profiled the response of bEnd.3to infection with SS2strain ZY05719and omp40-knockout strain using the Roche NimbleGen Porcine Genome Expression Array. Found omp40contributed to differential expression of30genes. Gene Ontology category and KEGG pathway were analyzed for relationships among differentially expressed genes. These genes were represented in a variety of functional categories, including genes involved in immune response, regulation of chemokine production, signal transduction and regulation of apoptosis. The data showed that omp40have an effect on SS2infection CNS.4. Identification of Collagen and Fibronectin-binding proteins of SS2Collagen and Fibronectin belong to ECM proteins and mediate bacteria adhesion to the host as substrates.2-DE gels, Western affinity blot and MS were combined to screen the Collagen and Fibronectin-binding proteins of SS2. The results showed that there were8Collagen-binding proteins and15Fn-binding proteins of SS2. Among them,7proteins showed the activity to bind Collagen and Fn simultaneously.5. Identification of proteins interaction between MRP and swine brain tissueThree-frame cDNA expression library was constructed using mRNA of brain tissue of swine. cDNA were synthesized using biotin-conjugated Oligo (dT) primer in the5’end. Double-strand cDNA was ligated to three-frame adapter and passed the cDNA Size Fractionation Columns. cDNA entry libraries were constructed by BP recombination. The libraries have a high titer of1.662×106CFU/mL, and contains a total clones of6.648×106CFU with an average inserts size of>1kb. The constructed cDNA expression library by LR recombination has a titer of1.78×106CFU/mL and contains total clones of7.12×106CFU, with an average inserts size of>1kb.MRP gene was directional cloned into pDHB1vector after the amplified PCR product and pDHB1vector were digested with sfi I, yielding the bait plasmid pDHBl-mrp. The library was screened by transformation into a yeast strain contains bait protein. After plasmid isolation, confirmations assay, sequencing, BLAST analysis, two positive target plasmid received from the swine brain cDNA library. One of these genes is identical to the Homo sapiens claudin5. The other showed homology to Homo sapiens VAMP (vesicle-associated membrane protein)-associated protein A.