Multiple antimicrobial peptides and peptides related to bradykinin and neuromedin N isolated from skin secretions of the pickerel frog, Rana palustris.

The skin secretions of the North American pickerel frog Rana palustris are toxic to both microorganisms and predators. A total of 22 peptides with differential growth-inhibitory activity towards bacteria and yeast were isolated from the electrostimulated secretions of R. palustris skin and were characterized structurally. Thirteen of the antimicrobial peptides belong to five of the known families previously identified in the skins of other species of Ranid frogs: brevinin-1 (3 peptides), esculentin-1 (2 peptides), esculentin-2 (1 peptide), ranatuerin-2 (6 peptides), and temporin (1 peptide). Nine peptides show little structural similarity towards other known antimicrobial peptides and so are classified in new families: palustrin-1 (4 peptides) with 27-28 amino acid residues and a cystine-bridged heptapeptide ring; palustrin-2 (3 peptides) with 31 amino acids and a cyclic heptapeptide region and palustrin-3 (2 peptides) with 48 amino acids and a cyclic hexapeptide region. Peptides belonging to the esculentin-1, esculentin-2 and palustrin-3 families are the most potent (minimal inhibitory concentrations approximately 1 microM against Escherichia coli) whereas peptides of the brevinin-1 and esculentin-2 families show the broadest spectrum of activity. As well as bradykinin that is identical to the human peptide, a further 4 peptides structurally related to [Leu(8)]bradykinin and two peptides related to neuromedin-N (the hexapeptide KKPYIL and a larger, cystine-containing form HLRRCGKKPYILMACS) were purified from the skin secretions.

Purification and characterization of antimicrobial and vasorelaxant peptides from skin extracts and skin secretions of the North American pig frog Rana grylio.

Eight peptides with differential growth-inhibitory activity against the gram-positive bacterium Staphylococcus aureus, the gram-negative bacterium Escherichia coli and the yeast, Candida albicans were isolated from an extract of the skin of the North American pig frog Rana grylio. The primary structures of these antimicrobial peptides were different from previously characterized antimicrobial peptides from Ranid frogs but on the basis of sequence similarities, the peptides may be classified as belonged to four previously characterized peptide families: the ranatuerin-1, ranatuerin-2 and ranalexin families, first identified in the North American bullfrog, Rana catesbeiana, and the temporin family first identified in the European common frog Rana temporaria. Peptides belonging to the brevinin-1, brevinin-2, esculentin-1, and esculentin-2 families, previously isolated from the skins of other species of Ranid frogs, were not identified in the extracts. The ranatuerin-1 and ranalexin peptides showed broadest spectrum of antimicrobial activity whereas the temporins were active only against S. aureus. Synthetic replicates of temporin-1Gb (SILPTIVSFLSKFL.NH(2)) and temporin-1Gd (FILPLIASFLSKFL.NH(2)) produced concentration-dependent relaxation of preconstricted vascular rings from the rat thoracic aorta (EC(50) = 2.4+/-0.1 microM for temporin-1Gb and 2.3+/-0.2 microM for temporin-1Gd). The antimicrobial peptides that were isolated in extracts of the skin R. grylio were present in the same molecular forms in electrically-stimulated skin secretions of the animal demonstrating that the peptides are stored in the granular glands of the skin in their fully processed forms.

Purification and characterization of antimicrobial peptides from the skin of the North American green frog Rana clamitans.

Ten peptides with differential growth-inhibitory activity against the gram-positive bacterium, Staphylococcus aureus, the gram-negative bacterium, Escherichia coli, and the yeast Candida albicans were isolated from an extract of the skin of a North American frog, the green frog Rana clamitans. Ranatuerin-1C (SMLSVLKNLGKVGLGLVACKINKQC), ranalexin-1Ca (FLGGLMKAFPALICAVTKKC), ranalexin-1Cb (FLGGLMKAFPAIICAVTKKC), ranatuerin-2Ca (GLFLDTLKGAAKDVAGKLLEGLKCKIAGC KP), and ranatuerin-2Cb (GLFLDTLKGLAGKLLQGLKCIKAGCKP), are members of three previously characterized families of antimicrobial peptides, first identified in the North American bullfrog Rana catesbeiana. In addition, five structurally related peptides (temporin-1Ca, -1Cb, -1Cc, -1Cd, and -1Ce), comprising 13 amino acid residues and containing a C-terminally alpha-amidated residue, belong to the temporin family first identified in the European common frog Rana temporaria. Peptides belonging to the brevinin-1, brevinin-2, esculentin-1, and esculentin-2 families, previously isolated from the skins of Asian and European Ranid frogs, were not identified in the extract. The data support the hypothesis that the distribution and amino acid sequences of the skin antimicrobial peptides are valuable tools in the identification and classification of Ranid frogs.