Peptidomic Analysis of Skin Secretions of the Caribbean Frogs Leptodactylus insularum and Leptodactylus nesiotus (Leptodactylidae) Identifies an Ocellatin with Broad Spectrum Antimicrobial Activity

Abstract

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.

Antimicrobial, cytotoxic, and insulin‐releasing activities of the amphibian host‐defense peptide ocellatin‐3N and its L‐lysine‐substituted analogs

“…16 A previous study identified multiple ocellatin peptides in norepinephrine-stimulated skin secretion from the Caribbean frogs Leptodactylus insularum and Leptodactylus nesiotus. 17 In common with ocellatins from other Leptodactylus species, these peptides displayed only relatively weak growth inhibitory activity against Gram-negative bacteria but ocellain-3N (GIFDVLKNLAKGVITSLAS.NH 2 ) from L. nesiotus was also active against Gram-positive bacteria. The aim of the present study was to determine whether ocellatin-3N and analogs with increased cationicity that maintain the amphipathic helical character of the peptide show therapeutic potential as antimicrobial agents for treatment of patients infected with antibiotic resistant microorganisms.…”

“…A previous study identified multiple ocellatin peptides in norepinephrine‐stimulated skin secretion from the Caribbean frogs Leptodactylus insularum and Leptodactylus nesiotus 17 . In common with ocellatins from other Leptodactylus species, these peptides displayed only relatively weak growth inhibitory activity against Gram‐negative bacteria but ocellain‐3N (GIFDVLKNLAKGVITSLAS.NH 2 ) from L. nesiotus was also active against Gram‐positive bacteria.…”

Manual mass spectrometry de novo sequencing of the anionic host defense peptides of the Cuban Treefrog Osteopilus septentrionalis

“…The array of skin peptides is unique for each amphibian species and may be treated as a reliable taxonomic characteristic, 36,37 which has been used to distinguish between closely related frog species 38 and even between populations 39 . Therefore, the study of the skin peptidome represents an important tool to discover phylogenetic connection inside Anurans, allowing the evolution pathways in the amphibian kingdom and of the whole planet to be revealed 40–42 …”

“…39 Therefore, the study of the skin peptidome represents an important tool to discover phylogenetic connection inside Anurans, allowing the evolution pathways in the amphibian kingdom and of the whole planet to be revealed. [40][41][42] Determination of the sequence of HDPs is the first step in the study of their physicochemical properties and related biological activity. Unfortunately, the "bottom up" sequencing approach involving trypsinolysis of the initial mixture of the skin peptides followed by automatic sequencing of the tryptic peptides and revealing the full peptide structures using databases is inefficient and often ineffective for the frog peptides.…”

Development of Antimicrobial Peptides from Amphibians

“…Among these, ocellatin-3N showed activity against antibiotic-resistant strains of E. coli and Klebsiella pneumoniae, and reference strains of P. aeruginosa and Salmonella typhimurium (as well as S. aureus and Enterococcus faecium with an MIC of 31.25-62.5 µM). Despite the therapeutic potential of ocellatin-3N is limited by its moderate hemolytic activity, it represents a template for the design of long-acting, non-toxic, broad-spectrum antimicrobial agents to target multidrug-resistant pathogens [24].…”