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

Abstract

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.

Fig. 1

A frog antimicrobial peptide promotes the transepithelial passage of a cosecreted toxin. a Litoria caerulea (an Australian tree frog) attacked by the snake Dendrelaphis punctulatus. Note the defensive skin secretion (black arrow), known to contain toxins (including caerulein) and AMPs (photograph taken by Jannie Smit). b Amino-acid sequences of the X. laevis skin toxin caerulein (Cae) and its cosecreted antimicrobial peptide, caerulein precursor fragment (CPF). c Lactate dehydrogenase (LDH) leakage indicates cell damage in a Caco-2 epithelial model exposed to a mixture of caerulein and CPF but not to caerulein alone (n = 7; 100% corresponds to the LDH leakage caused by complete cell lysis as induced by Triton-X). d Co-administration of CPF induces a rapid prolonged drop in transepithelial electrical resistance (TEER) of Caco-2 monolayers (n = 9, one-way ANOVA, F(4, 40) = 1345.6, p < 0.0005). e Caerulein alone does not damage Caco-2 monolayers, but co-administration of CPF results in intercellular ruptures (white arrow), as revealed by scanning electron microscopy. Scale bars represent 100 μm. f Co-administration of CPF at the apical side leads to higher caerulein levels after 60 min at the basolateral side, indicating enhanced transepithelial transport across Caco-2 monolayers (n = 9, t-test, t(8.2) = −7.3, p < 0.0005) and an oral multilayer model (n = 8, t-tests, t(9.1) = −9.6, p < 0.0005). All data are mean ± s.e.m., error bars not shown when covered by data symbols

Fig. 2

AMP co-administration accelerates toxin absorption in a live predator. a Investigated organs and tissues in the model predator Thamnophis eques; numbers are cross-referenced in b and c. b Caerulein (Cae) blood concentrations reach higher levels (left graph), which leads to higher systemic exposure (right graph) when orally co-administered with CPF at 1 mM (n = 3, linear mixed model, X ² (1) = 11.4, p < 0.001). c Caerulein levels in organs are higher in snakes when co-administered with CPF after 30 min (n = 3, linear mixed model, X ² (1) = 4.7, p = 0.029). Symbols represent individual data values. Linear mixed models are explained in the Methods section

Fig. 3

The role of frog AMPs in toxin absorption in other frog species. a Representatives of three major frog lineages cosecrete peptide toxins and AMPs. b Amino-acid sequences of the Bombina ortienalis toxin bombesin (Bom) and AMP bombinin-like peptide 1 (BLP), and of the Phyllomedusa sauvagii toxin dermorphin (Drm) and its AMP dermaseptin-S1 (Drs). c LDH leakage indicates cell damage in Caco-2 monolayers when exposed to the AMP + toxin mixtures, but not to the toxins alone (n = 6–7; 100% corresponds to the LDH leakage caused by complete cell lysis as induced by Triton-X). d BLP and Drs induce a dose-dependent rapid drop in transepithelial electrical resistance (TEER) of Caco-2 monolayers (n = 9, one-way ANOVA, F_DRS(4,40) = 1284, F_BLP(4,40) = 2054.1, p < 0.0005). e Co-administration of BLP and Drs at the apical side of Caco-2 monolayers leads to higher toxin levels after 60 min at the basolateral side, showing enhanced transepithelial transport of bombesin (n = 9, t-test, t(11.6) = −11.2, p < 0.0005) and dermorphin (n = 8–9, t-test, t(8.1) = −4.8, p < 0.0005). All data are mean ± s.e.m., error bars not shown when covered by data symbols