Antimicrobial peptides from the skin of the Japanese mountain brown frog, Rana ornativentris

Six peptides with antimicrobial activity were isolated from an extract of f reeze-dried skin of the Japanese mountain brown frog Rana ornativentris. Tw o structurally related peptides (brevinin-20a GLFNVFKGALKTAGKHVAGSLLNQLKCKV SGGC, 11 nmol/g dried tissue, and brevinin-20b GIFNVFKGALKTAGKHVAGSLLNQLKCK VSGEC, 170 nmol/g) belong to the brevinin-2 family, previously identified i n Asian and European, but not North American, Ranid frogs. Four peptides (t emporin-1Oa FLPLLASLFSRLL.NH2, 13 nmol/g; temporin-1Ob FLPLIGKILGTI L.NH2, 350 nmol/g; temporin-1Oc FLPLLASLFSRLF.NH2, 14 nmol/g; and temporin-1Od FLP LLASLFSGLF.NH2, 8 nmol/g) are members of the temporin family first identifi ed in the European common frog Rana temporaria but also found in the skins of North American Ranids. The brevinin-2 peptides showed broad-spectrum act ivity against the gram-positive bacterium, Staphylococcus aureus, the gram- negative bacterium, Escherichia coli and the yeast Candida albicans, wherea s the temporins showed potent activity only against S. aureus. The brevinin s and temporins belong to the class of cationic antimicrobial peptides that adopt an amphipathic alpha -helical conformation but it is significant tha t: temporin-1Od, which lacks a basic amino acid residue, is still active ag ainst S. aureus (minimum inhibitory concentration = 13 muM compared with 2 muM for temporin-1Oa). This suggests that strong electrostatic interaction between the peptide and the negatively charged phospholipids of the cell me mbrane is not an absolute prerequisite for antimicrobial activity.