bassianolide and Escherichia-coli-Infections

Serum amyloid A (SAA) is an acute-phase protein that plays a crucial role in the inflammatory response. In this study, we identified an SAA homolog from Epinephelus lanceolatus (ElSAA). Molecular characterization revealed that ElSAA contains a fibronectin-like motif that is typical of SAAs. Recombinant ElSAA protein (rElSAA) was produced in E. coli BL21 (DE3) cells and purified as a soluble protein. To analyze its biological activity, mouse Raw264.7 macrophage cells were treated with various concentrations of rElSAA. Expression of several inflammation-related cytokines, including tumor necrosis factor alpha (TNF-α), interleukin (IL)-1β, IL-6, and IL-10, was induced by rElSAA. This protein also triggered macrophage differentiation, as evidenced by increases in cell size and complexity. To determine whether rElSAA regulates macrophage polarization, we assessed gene expression of M1 and M2 markers. The results demonstrated that rElSAA induced the expression of both M1 and M2 markers, suggesting that it promotes the differentiation of macrophages into a mixed M1/M2 phenotype. To evaluate whether rElSAA enhances phagocytosis via an opsonization-dependent mechanism, GFP-labeled E. coli cells were pretreated with rElSAA, followed by incubation with Raw264.7 cells. Flow cytometry was used to monitor the phagocytic uptake of GFP-labeled E. coli by macrophages. Surprisingly, incubating E. coli with rElSAA did not enhance bacterial uptake by macrophages. However, preincubating Raw264.7 cells with various concentrations of rElSAA, followed by infection with E. coli (multiplicity of infection = 20 or 40), resulted in a clear enhancement of macrophage phagocytic capacity. In conclusion, we have identified SAA from E. lanceolatus and have demonstrated that rElSAA promotes inflammatory cytokine production and macrophage differentiation. In addition, rElSAA enhances phagocytosis of bacteria by macrophages via an opsonization-independent mechanism.

Topics: Amino Acid Sequence; Animals; Bass; Cell Differentiation; Cloning, Molecular; Cytokines; DNA, Complementary; Escherichia coli; Escherichia coli Infections; Fish Diseases; Fish Proteins; Macrophages; Phagocytosis; Phylogeny; Recombinant Proteins; RNA, Messenger; Sequence Alignment; Serum Amyloid A Protein

Two isoforms of piscidin from Malabar grouper (Epinephelus malabaricus), EmPis-1 and EmPis-2, were cloned and studied. EmPis-1 and EmPis-2 showed the different in the 3'UTR features of mRNA and gene expression patterns. AUUUA-motif-containing ARE was found in mRNA of EmPis-1, but not in that of EmPis-2. EmPis-1 and EmPis-2 expressed not only in the potential sites of pathogen entry, but also in grouper's immune-related tissues such as head kidney (HD), peripheral blood leukocytes (PBL) and spleen. The expression level of EmPis-1 was higher than that of EmPis-2 in most fish tissues. Expression of both EmPis-1 and EmPis-2 were upregulated by V. parahaemolyticus significantly in the PBL, HD and spleen. Besides, expression of EmPis-1 was upregulated in gills. The putative mature peptides of EmPis-1 and EmPis-2, which were predicted to adopt an amphipathic α-helical conformation, posessed excellent microbicidal activities against both gram-negative and -positive bacteria. The hemolytic activity of the putative mature peptides of EmPis-1 and EmPis-2 increased in a dose-dependent manner to both grouper erythrocytes and rabbit erythrocytes. Interestingly, grouper erythrocytes were less vulnerable than rabbit erythrocytes to the peptides. Grouper piscidins excluded the signal peptide were not the inactive precursors but possessed high microbicidal activity evidenced by minimum bactericidal concentration (MBC) assay and by the scanning electron microscope (SEM) observation. The present phylogenetic analysis did not support the suggestion that piscidins are ancient AMPs widespread across invertebrate and vertebrate taxa, and that piscidins are included in the cecropin superfamily. Collectively, the present data improve our understanding of the piscidin family, and give greater insights into EmPis-1 and EmPis-2 of the grouper immune system.

Topics: Amino Acid Sequence; Animals; Base Sequence; Bass; DNA, Complementary; Escherichia coli; Escherichia coli Infections; Fish Diseases; Fish Proteins; Gene Expression Regulation; Phylogeny; Protein Conformation; Protein Isoforms; Sequence Alignment; Staphylococcal Infections; Staphylococcus aureus; Tissue Distribution; Vibrio Infections; Vibrio parahaemolyticus