Innate immune components in fish epidermal mucus

The mucus layer on the epidermis of fish consists of several innate immune components that provide the first line of defense against invading pathogens. Little is known about the mucosal immunity of Arctic char, brook trout, striped bass, koi carp, haddock, cod and hagfish. This study demonstrates the presence, isolation and characterization of constitutively produced antimicrobial substances from the epidermal mucus of these evolutionary diverse and economically important fish from North Atlantic region. The epidermal mucus of the examined fish were shown to contain various levels of innate immune enzymes including lysozyme, alkaline phosphates, cathepsin and proteases. The antimicrobial compounds that were extracted from the epidermal mucus of these fish, using acidic, organic and aqueous solvents, showed differences in activity against various pathogens. Of the studied species, the hagfish acidic epidermal mucus extract, which mainly contained low molecular mass proteins, displayed potent antimicrobial activity at minimum bactericidal concentrations (MBC) in the range of 6--82 microg/mL against all screened pathogens. Bioassay guided fractionation of the hagfish acidic epidermal mucus extract resulted in the isolation and structural characterization of a novel 12 amino acid antimicrobial peptide, myxinidin that had a molecular mass of 1327.68 [M+H]+. Myxinidin exhibited antimicrobial activity against a broad spectrum of pathogens at MBC's ranging from 1--10 microg/mL with no hemolytic activity against mammalian red blood cells. The activity of myxinidin was retained in the presence of NaCl concentrations up to 0.3 M. Hagfish extrude a large volume of slime when caught, during feeding and when stressed or disturbed. The observation of 2--5 fold elevated levels of innate immune enzymes than the normal epidermal mucus and the high levels of antimicrobial protein/peptide precursors including globin, histone H2B and H3-like suggest the involvement of extruded slime in hagfish immune defense. The presence of myxinidin and other possible unknown antimicrobial components in the extruded slime suggests that the extruded slime could be a novel source of antimicrobial components. This study provides a better understanding of mucosal innate immunity and highlights the presence of biologically active substances in fish epidermal secretions for fish and human health related applications.