ABSTRACT
Skin secretions of certain frog species represent a source of host-defense peptides (HDPs) with therapeutic potential and their primary structures provide insight into taxonomic and phylogenetic relationships. Peptidomic analysis was used to characterize the HDPs in norepinephrine-stimulated skin secretions from the Amazon River frog Lithobates palmipes (Ranidae) collected in Trinidad. A total of ten peptides were purified and identified on the basis of amino acid similarity as belonging to the ranatuerin-2 family (ranatuerin-2PMa, -2PMb, -2PMc, and-2PMd), the brevinin-1 family (brevinin-1PMa, -1PMb, -1PMc and des(8-14)brevinin-1PMa) and the temporin family (temporin-PMa in C-terminally amidated and non-amidated forms). Deletion of the sequence VAAKVLP from brevinin-1PMa (FLPLIAGVAAKVLPKIFCAISKKC) in des[(8-14)brevinin-1PMa resulted in a 10-fold decrease in potency against Staphylococcus aureus (MIC = 31 µM compared with 3 µM) and a > 50-fold decrease in hemolytic activity but potency against Echerichia coli was maintained (MIC = 62.5 µM compared with 50 µM). Temporin-PMa (FLPFLGKLLSGIF.NH2) inhibited growth of S. aureus (MIC = 16 µM) but the non-amidated form of the peptide lacked antimicrobial activity. Cladistic analysis based upon the primary structures of ranaturerin-2 peptides supports the division of New World frogs of the family Ranidae into the genera Lithobates and Rana. A sister-group relationship between L. palmipes and Warszewitsch's frog Lithobates warszewitschii is indicated within a clade that includes the Tarahumara frog Lithobates tarahumarae. The study has provided further evidence that peptidomic analysis of HDPs in frog skin secretions is a valuable approach to elucidation of the evolutionary history of species within a particular genus.
Subject(s)
Ranidae , Staphylococcus aureus , Animals , Amino Acid Sequence , Phylogeny , Staphylococcus aureus/metabolism , Ranidae/metabolism , Amphibian Proteins/metabolism , Skin/metabolismABSTRACT
Ocellatins are peptides produced in the skins of frogs belonging to the genus Leptodactylus that generally display weak antimicrobial activity against Gram-negative bacteria only. Peptidomic analysis of norepinephrine-stimulated skin secretions from Leptodactylus insularum Barbour 1906 and Leptodactylus nesiotus Heyer 1994, collected in the Icacos Peninsula, Trinidad, led to the purification and structural characterization of five ocellatin-related peptides from L. insularum (ocellatin-1I together with its (1-16) fragment, ocellatin-2I and its (1-16) fragment, and ocellatin-3I) and four ocellatins from L. nesiotus (ocellatin-1N, -2N, -3N, and -4N). While ocellatins-1I, -2I, and -1N showed a typically low antimicrobial potency against Gram-negative bacteria, ocellatin-3N (GIFDVLKNLAKGVITSLAS.NH2) was active against an antibiotic-resistant strain of Klebsiella pneumoniae and reference strains of Escherichia coli, K. pneumoniae, Pseudomonas aeruginosa, and Salmonella typhimurium (minimum inhibitory concentrations (MICs) in the range 31.25-62.5 µM), and was the only peptide active against Gram-positive Staphylococcus aureus (MIC = 31.25 µM) and Enterococcus faecium (MIC = 62.5 µM). The therapeutic potential of ocellatin-3N is limited by its moderate hemolytic activity (LC50 = 98 µM) against mouse erythrocytes. The peptide represents a template for the design of long-acting, non-toxic, and broad-spectrum antimicrobial agents for targeting multidrug-resistant pathogens.