Isolation and identification of antimicrobial components from the epidermal mucus of Atlantic cod (Gadus morhua)

Abstract

The epidermal mucus of fish species has been found to contain antimicrobial proteins and peptides, which is of interest in regard to fish immunity. An acidic extract from the epidermal mucus of the Atlantic cod (Gadus morhua) was found to exhibit antimicrobial activity against Bacillus megaterium, Escherichia coli and Candida albicans. This activity varied significantly when salt was added to the antimicrobial assay, and was eliminated by pepsin digestion. No lysozyme activity was detected in the extract. By using weak cationic exchange chromatography together with reversed‐phase chromatography, and monitoring the antimicrobial activity, we have isolated four cationic proteins from the mucus extract. Using N‐terminal and C‐terminal amino acid sequence analysis, together with MS, the antimicrobial proteins were identified as histone H2B (13 565 Da), ribosomal protein L40 (6397 Da), ribosomal protein L36A (12 340 Da) and ribosomal protein L35 (14 215 Da). The broad spectra of antimicrobial activities in the cod mucus and the characterization of four antimicrobial polypeptides suggest that mucus compounds contribute to the innate host defence of cod.

An Overview of the Immunological Defenses in Fish Skin

“…Many substances with biostatic and biocidal activity (e.g., complement, C-reactive proteins, proteases, lectins, lysozyme, haemolysins, agglutinin, proteolytic enzymes, antimicrobial peptides, antibodies, immunoglobulins) are present and have been identified in the fish epidermis and/or skin mucus [9,18,25,85,96,120]. Although the protective role of the epidermal mucus of fishes has been known for many years [8,100], of great interest at the present is to see the skin mucus as a source for isolation of new and potent antimicrobial components [121]. A brief overview of the most studied immune components of fish mucus is now presented.…”

“…Lysozyme (N-acetylmuramide glucanohydrolase or muramidase) is a ubiquitous bactericidal enzyme identified in a wide range of organisms including fishes. Lysozyme is present in mucus, lymphoid tissue, and serum of most fish species, but not in others (such as cod and wolfish) [119,121,122]. The bacteriolytic activity of lysozyme in fish skin mucus and other tissues contributes to its host defence mechanism against bacterial infection [80,123,124].…”

“…Low levels of antimicrobial peptides from healthy specimens or other factors such as pH could be the reason for difficulty in isolating antimicrobial peptides/polypeptides from mucus [121]. One of the scarce studies available demonstrated that the challenge of channel catfish (Ictalurus punctatus) with ich resulted in potent upregulation of a suite of AMPs in the skin (the total antibacterial activity response peaked at seven and fourteen days after challenge), including at least one polypeptide (HbbP-1) that is highly lethal to not only ich [187], but also to Tetrahymena pyriformis (a parasitic ciliate) and the important marine ectoparasite Aphyosemion ocellatum [188].…”

Isolation and Identification of a Novel Inducible Antibacterial Peptide from the Skin Mucus of Japanese Eel, Anguilla japonica

“…The majority of these peptides were amphipathic cation peptides, while few were anionic. In fish, there were also some AMPs whose MWs were greater than 10 kDa such as the histone 2B-like proteins isolated from the skin mucus of Ictalurus punctatu [34] and Gadus morhua [6], the histone 2A-like protein from Oncorhynchus mykiss [15], the glycoprotein-like antibacterial proteins from Cyprinus carpio [26], Tinca tinca, Oncorhynchus mykiss [12], Anguilla japonica [43,45] and Fugu rubripes [44].…”

Purification and Antimicrobial Activity of Antimicrobial Protein from Brown-spotted Grouper, <I>Epinephelus fario</I>

“…Compounds with broad antimicrobial activities are thought to be especially important for fish, as their adaptive immune system is structurally simpler than that of mammals or amphibians, and is not fully effective in young fry or at low environmental temperature (Silphaduang & Noga, 2001). The skin epithelium and other mucosal surfaces of fish are rich in anti-microbial peptides (Bergsson et al, 2005).…”