Isolation, Characterization And Mechanism Of Anti-Bacterial Activity Of Three Novel Goose Avian Beta-Defensins

Every year, many people throughout the world die of microbial diseases. The consumption of poultry derived products contaminated by bacteria or viruses is a major cause of food poisoning. Food safety has become a major concern of consumers and is reflected in food animal production and processing. Included in this new consumer advocacy is the call for the reduced use of antibiotics in preharvest pathogen control because of concerns about the possible consumption of antibiotic residues and the emergence of antibiotic-resistant bacteria as a result of subtherapeutic use of antibiotics to control bacteria. As a consequence, other methods to suppress microbial growth and infection of food animals are needed. One of the alternatives may be to stimulate the innate immune system of the animal by dietary modulation. Therefore, identification and characterization of new innate immune effector molecules is required. Innate immunity in animals depends in large part on the activity of non-specific effector molecules. Of these, animicrobial peptides displaying activities against microbial pathogens have been proposed as a novel control strategy to combat infections.Three novel avian β-defensin (AvBD) orthologues were isolated from spleen, bone marrow and Bursa of fabricius of geese by RT-PCR. Homology among the3goose AvBD genes and the other known AvBDs from other avian species were analyzed. The result showed that they were goose AvBD1, goose AvBD3, and goose AvBD6. Sequence analysis showed that goose AvBD1consisted of198bp, encoding65amino acids; the cDNA of goose AvBD3consisted of182bp encoding60amino acids; goose AvBD6consisted of204bp encoding67amino acids.In addition, phylogenetic relationships among the three AvBD genes, AvBDs from other avian species, and some mammalian beta-defensins were analyzed by Clustal Ⅴ of DNA Star software.The result demonstrated that goose AvBD1shared the highest amino acid homology (70.7%) with Ostrich AvBD1. Both goose AvBD3and AvBD6shared the highest amino acid homology (100%) with its chicken analogs,respectively. However, all of these genes shared low homology with mammalian β-defensins.The cDNA of goose AvBD1,3and6were cloned into pGEX-6p-1vector to construct recombinant plasmid pGEX-goose AvBD1,3and6which were translated into E. coli BL21and the bacteria were induced with IPTG, respetively. At last, the recombinant protein were expressed and purified. High levels of recombinant goose AvBD1,3and6(molecular weight,31-32ku) expression were noted after induction with IPTG, respectively. We also found that the recombinant proteins were produced as insoluble bodies in the cells. Additionally, putative mature goose AvBDs (1,3and6) were synthesized, and named rAvBDs.Twelve pathogenic bacterial strains were used to investigate the antibacterial activities of GST, rAvBDs and sAvBDs. It was showed that GST has no antibacterial activities against all of bacteria investigated. However, all of the3rAvBDs and3sAvBDs have high antibacterial activities including Gram-positive and Gram-negative strains investigated. Furthermore, It is found that both rAvBDs and sAvBDs were sensitive to salt concentration. The bacterial survival was decreased with the salt concentration elevation (0～150mM). In addition, both rAvBDs and sAvBDs have no haemolysis.In order to investigate whether the expression of AvBD mRNAs in tissues from geese was constitutive or inducible in response to S. enteritidis infection, geese were infected orally with S. enteritidis. Eight tissues, namely bone marrow, bursa of Fabricius, Harderian glands, thymus, cecal tonsil, spleen, large and small intestine, were collected from geese on6,24,48, and72h after infection to measure the levels of AvBD mRNAs. The results showed that highly significant upregulation of the level of expression of AvBD3in both bone morrow, thymus and large intestinewas observed when the geese were infected with S. enteritidis. Similarly, the level of expression of AvBD1in bone morrow of geese was increased significantly after infection. Furthermore, significant induction was observed for AvBD6expression in the Immunohistochemistry. These results suggested that AvBDs could be upregulated or induced by S. enteritidis. and also show that:when the geese bodys are infected by S. enteritidis, goose Toll-like receptor4activation of the innate immune response, but also provide stimulatory molecules, two goose cell factor produced by T cells may be induced through the activation of cellular signal transduction pathways (leukocyte interleukin2and18), and goose interferon gene (IFN-gamma) and three goose beta-defensin gene expression, together to build the acquired immune defense, to jointly resist Salmonella enteritis of goose body’s attack..