The Molecular Mechanism Of IFN-β Induction By The Major Antigen Bacterioferritin Of Salmonella Pullorum

Salmonella enterica serovar Pullorum (S. Pullorum) causes pullorum disease in poultry, and results in a high mortality rate in young chicks within 2-3 weeks, and it shows chronic carrier state without severe clinical symptoms in adult chickens. Because of its vertical and horizonal transmissions, it could cause great economic loss to poulty industry and harm to public health security. Although this disease has been successfully eradicated in many western countries, it still occurs here and there in China. Currently there are no effective control measures for pullorum disease except eradication. In particular, the pathogenesis of S. Pullorum infection is still not very clear. IFN-P plays an important role in the innate immune response, such as antiviral and intracellular bacteria activities and induction of apoptosis. In this study, the major antigen bacterioferritin of S. Pullorum was screened out, and the mechanism underlying bacterioferritin-induced IFN-P was investigated. Our findings provide new insights into the molecular mechanisms of pathogenesis of Salmowella-infection.The major antigens bacterioferritin (Bfr), GroEL, and Alcohol Dehydrogenase E (AdhE) of S. Pullorum were screened by pull-down assays and analysed by mass spectrometric and bioinformatics. The bfr gene was cloned and ligated into prokaryotic expression vector pET28a (+) and pGEX-6p-1 by standard molecular biotechnology. The recombinant protein His-Bfr and GST-Bfr were expressed by induction and purified via affinity chromatography. Recombinant protein His-Bfr could uptake irons in vitro, showing that it had biological activities. The BALB/c mice was immunized by the purified recombinant protein His-Bfr, and the splenocytes from the mice was fused with SP2/0 cells to produce hybridomas cell. The recombinant GST-Bfr protein was used as a coating antigen in ELISA to screen for positive clones of cells. After 3 rounds of sub-cloning, we obtained 3 clones of hybridomas that could secrete monoclonal antibodies (McAb) against Bfr. All of these McAb were indentified as isotype IgGl, recognizing the same epitope of Bfr, and then have high affinity and high specificity.Since Salmonella can invade the cell to survival and proliferate, we analyzed the biological functions of S. Pullorum Bfr within the host cells, we made a pEGFP-bfr-expression construct, and transfected DF-1 cells with pEGFP-bfr or pEGFP-Nl as control. The results showed that Bfr had the ability to induce IFN-β production. By analysis of the trucancated Bfr proteins, we found that the 1-50 amino acids play a key role in IFN-β expression. To dissect the signaling pathways involved in Bfr-induced IFN-P expression, we treated DF-1 cells with the inhibitors of the key signaling molecules including p38 MAPK and JNK MAPK before pEGFP-bfr transfection and real-time PCR was performed to examine the mRNA levels of IFN-β. The results showed that p38 MAPK inhibitor, but not JNK MAPK inhibitor, significantly inhibited IFN-β expression in cells and p38 phosphorylation was markedly enhanced in pEGFP-bfr transfected cells. Taken together, these results clearly show that the p38 MAPK pathway is essential for Bfr-induced IFN-β expression. To examine the role of Bfr in S. Pullorum infected cells, we generated Bfr-deficient S. Pullorum (KO) and the complemented S. Pullorum (RS) strains based on S. Pullorum wild type (WT) using 1-Red homologous recombination system and infected DF-1 cells with these strains. As a result, compared with S. Pullorum wild type and the complemented strains, the Bfr-deficient strain infection could markedly inhibit IFN-P expression in DF-1 cells. Since Bfr induced IFN-P expression, we examined the role of Bfr-induced apoptosis in cells using flow cytometry. The results show that Bfr in DF-1 cells could cause remarkable apoptosis. Furthermore, using transient transfection of the pEGFP-bfr vector in HEK 293T cells and Hela cells, we found that Bfr induced apoptosis was dependent IFN-β expression. These results clearly show that Bfr acts a major antigen of S. Pullorum and can induce IFN-β expression via the p38 MAPK signaling pathway. To examine the affect of Bfr on the functions of macrophages, we examined the phagocytosis and killing of S. Pullorum by macrophages and the expressions of proinflammatory cytokines by macrophages infected with WT, Bfr-KO and Bfr-RS S. Pullorum strains. We found that Bfr may inhibits the phagocytosis and killing of macrophages and the production of IL-12, TNF-a by macrophages.Taken together, the major antigens Bfr, GroEL, and AdhE of S. Pullorum were identified by pull-down assay in this study. Bfr was found to have the effect of inducing IFN-P expression via p38 MAPK signaling pathway and inducing the apoptosis. The results of this study would help to establish a better detection method for S. Pullorum, and provide a novel insight into understandings of the pathogenesis of 5. Pullorum infection.