Dybowskin-4

Nine peptides displaying varying degrees of antimicrobial activity were extracted from the skin of the Hokkaido frog, Rana pirica. Five structurally related peptides were identified as members of the brevinin-2 family. These peptides were active against reference strains of Gram-negative (Escherichia coli, Pseudomonas aeruginosa, Enterobacter cloacae, Klebsiella pneumoniae) and Gram-positive (Staphlococcus aureus) bacteria but displayed relatively low hemolytic activity. The most abundant peptide, brevinin-2PRa (680 nmol/g weight of dry skin) showed high potency [minimal inhibitory concentration (MIC) values between 6 and 12 microM] against a range of clinical isolates of P. aeruginosa. In addition, activity was unaffected by NaCl concentrations up to 200 mM. Cladistic analysis based on the primary structures of brevinin-2 peptides supports a close phylogenetic relationship between R. pirica and Japanese mountain brown frog Rana ornativentris. One peptide of the ranatuerin-2 family and one strongly hemolytic peptide of the brevinin-1 family were also isolated from the extract along with two members of the temporin family, temporin-1PRa (ILPILGNLLNGLL.NH(2)) and temporin-1PRb (ILPILGNLLNSLL.NH(2)) that atypically lacked basic amino acid residues and showed only very weak antimicrobial and hemolytic activity.

The Tsushima brown frog Rana tsushimensis Stejneger, 1907 exists in reproductive isolation on the island of Tsushima, Japan. Six peptides with antimicrobial activity were isolated in pure form from an extract of the skin of this species and their amino acid sequences identified them as members of the brevinin-1 (one peptide), brevinin-2 (one peptide) and temporin (four peptides) families. The C-terminally alpha-amidated brevinin-1 peptide (FLGSIVGALASALPSLISKIRN.NH2) lacks the cyclic heptapeptide domain Cys18-(Xaa)4-Lys-Cys24 at the COOH-terminus of the molecule that characterizes other members of that family. A structurally similar brevinin-1 peptide, also lacking the cyclic domain, was previously isolated from the skin of the Ryukyu brown frog Rana okinavana, indicative of a close phylogenetic relationship between the species. Brevinin-2TSa (GIMSLFKGVLKTAGKHVAGSLVDQLKCKITGGC) showed broad-spectrum growth inhibitory activity against a range of Gram-negative and Gram-positive bacteria (including methicillin-resistant Staphylococcus aureus) (minimum inhibitory concentrations< or =25 microM) and relatively low hemolytic activity against human erythrocytes (LD50=100 microM). The peptide therefore represents a candidate for drug development.

The skins of amphibians secrete small antimicrobial peptides that fight infection and are being explored as potential alternatives to conventional antibiotics. In this study we combined mass spectrometry with cDNA sequencing to examine antimicrobial peptides in skin secretions from the Chinese frog Rana dybowskii. Thirteen peptides having precursor sequences that resemble known antimicrobial peptides from this genus were identified, ten of which were members of previously described peptide families based on their primary structures; i.e., brevinin-1, Japonicin-1, brevinin-2 and temporin. The other three peptides from R. dybowskii, which were named dybowskin-1CDYa, dybowskin-2 CDYa and dybowskin-2CDYb, had different amino acid compositions and little sequence similarity to known antimicrobial peptides. The carboxyl terminus of dybowskin-1CDY lacked amidation and is therefore clearly distinct from temporin peptides, whereas dybowskin-2CDYa and dybowskin-2CDYb consisted of 18 amino acids and were rich in Arg residues. Chemically synthesized peptides corresponding to mature dybowskin-1CDYa and dybowskin-2CDYa had strong antimicrobial activity and caused little hemolysis of human erythrocytes, suggesting they may serve as interesting templates for the development of novel antibiotics.