A New Family of Diverse Skin Peptides from the Microhylid Frog Genus Phrynomantis.

A wide range of frogs produce skin poisons composed of bioactive peptides for defence against pathogens, parasites and predators. While several frog families have been thoroughly screened for skin-secreted peptides, others, like the Microhylidae, have remained mostly unexplored. Previous studies of microhylids found no evidence of peptide secretion, suggesting that this defence adaptation was evolutionarily lost. We conducted transcriptome analyses of the skins of Phrynomantis bifasciatus and Phrynomantis microps, two African microhylid species long suspected to be poisonous. Our analyses reveal 17 evolutionary related transcripts that diversified from to those of cytolytic peptides found in other frog families. The 19 peptides predicted to be processed from these transcripts, named phrynomantins, show a striking structural diversity that is distinct from any previously identified frog skin peptide. Functional analyses of five phrynomantins confirm the loss of a cytolytic function and the absence of insecticidal or proinflammatory activity, suggesting that they represent an evolutionary transition to a new, yet unknown function. Our study shows that peptides have been retained in the defence poison of at least one microhylid lineage and encourages research on similarly understudied taxa to further elucidate the diversity and evolution of skin defence molecules.

The anuran skin peptide bradykinin mediates its own absorption across epithelial barriers of the digestive tract.

When faced with a potential predator, a wide range of frog species secrete a mixture of peptide toxins from their skin to defend themselves. We have recently shown that antimicrobial peptides (AMPs) in a frog's defensive poison enhance the uptake of these peptides across epithelia, thereby speeding up the process of predator intoxication. This study provides evidence that bradykinin, a widespread peptide toxin in anurans (frogs), is capable to pass through epithelial barriers independent of this delivery system. We quantified bradykinin peptides secreted by Bombina orientalis during acute stress, and found that at biologically relevant concentrations, bradykinin passage across model epithelia occurs even in the absence of AMPs. Monitoring of transepithelial electric resistance showed that bradykinin treatment caused a subtle yet prolonged reduction in barrier function, indicating that the peptide itself is capable to increase the permeability of epithelia. Yet, bradykinin does not cause cells to leak lactate dehydrogenase, suggesting that it does not damage cell membranes. Moreover, imaging of bradykinin-treated monolayers shows no endocytosis of fluorescent propidium iodide, indicating that the peptide does not perforate cell membranes at smaller scale and therefore is unlikely to cross epithelia via a transcellular passage. Together, these observations suggest that bradykinin, unlike other amphibian neuropeptide toxins, mediates its own passage across mucosal barriers, possibly through a paracellular route. This "self-administering" property, combined with the fact that bradykinins can potently disturb multiple physiological processes, could explain why these peptides are one of the most widespread antipredator peptides in the defensive secretions of frogs.

Antimicrobial peptides in frog poisons constitute a molecular toxin delivery system against predators.

Animals using toxic peptides and proteins for predation or defense typically depend on specialized morphological structures, like fangs, spines, or a stinger, for effective intoxication. Here we show that amphibian poisons instead incorporate their own molecular system for toxin delivery to attacking predators. Skin-secreted peptides, generally considered part of the amphibian immune system, permeabilize oral epithelial tissue and enable fast access of cosecreted toxins to the predator's bloodstream and organs. This absorption-enhancing system exists in at least three distantly related frog lineages and is likely to be a widespread adaptation, determining the outcome of predator-prey encounters in hundreds of species.