Multipurpose peptides: The venoms of Amazonian stinging ants contain anthelmintic ponericins with diverse predatory and defensive activities

Abstract

In the face of increasing drug resistance, the development of new anthelmintics is critical for controlling nematodes that parasitise livestock. Although hymenopteran venom toxins have attracted attention for applications in agriculture and medicine, few studies have explored their potential as anthelmintics. Here we assessed hymenopteran venoms as a possible source of new anthelmintic compounds by screening a panel of ten hymenopteran venoms against Haemonchus contortus, a major pathogenic nematode of ruminants. Using bioassay-guided fractionation coupled with liquid chromatography-tandem mass spectrometry, we identified four novel anthelmintic peptides (ponericins) from the venom of the neotropical ant Neoponera commutata and the previously described ponericin M-PONTX-Na1b from Neoponera apicalis venom. These peptides inhibit H. contortus development with IC₅₀ values of 2.8-5.6 μM. Circular dichroism spectropolarimetry indicated that the ponericins are unstructured in aqueous solution but adopt α-helical conformations in lipid mimetic environments. We show that the ponericins induce non-specific membrane perturbation, which confers broad-spectrum antimicrobial, insecticidal, cytotoxic, hemolytic, and algogenic activities, with activity across all assays typically correlated. We also show for the first time that ponericins induce spontaneous pain behaviour when injected in mice. We propose that the broad-spectrum activity of the ponericins enables them to play both a predatory and defensive role in neoponeran ants, consistent with their high abundance in venom. This study reveals a broader functionality for ponericins than previously assumed, and highlights both the opportunities and challenges in pursuing ant venom peptides as potential therapeutics.

Keywords: Ant; Anthelmintic; Antimicrobial; Pain; Ponericin; Venom peptide.

Fig. 1.. Screen of crude hymenopteran venoms against drug-susceptible H. contortus.

Bars s how inhibition of la1val development (relative to negative control) calculated from triplicate assays at each of the three indicated concentrations. Data are mean ± SEM.

Fig. 2.. Isolation of ponericins with anthelmintic activity from the venoms of N. apicalis and N. commutata.

Chromatograms showing analytical RP-HPLC fractionation of crude venom from (A) N. commutata and (D) N. apicalis, with peaks containing anthelmintic activity highlighted. Insets show N. commutata and N. apicalis worker ants (photographs by Dr Alex Wild, The University of Texas at Austin, USA). (B, C, E–G) Successive purification by analytical RP-HPLC identified five anthelmintic ponericins. Insets are MALDI TOF MS spectra showing the monoisotopic mass of each peptide. Amino acid sequences were determined using LC-MS/MS and compared against a draft venom-gland transcriptome for N. commutata and previously identified ponericins for N. apicalis. (H) An example mass spectrum showing ions leading to identification of the amino acid sequence for ponericin Nc2a.

Fig. 3.. Sequence alignments of anthelmintic and canonical ponericins.

Nc1a, Nc2a, Nc3a and Nc3b, and Nal b show homology to the three described subfamilies of ponericins: W, L, and G. Ponericins identified in this study are shown in bold. Identity (%ID) and similarity (%Sim) are shown compared to canonical ponericins at the top of the alignment for each subfamily. Identical residues are highlighted in yellow, positively-charged residues (K and R) are coloured blue, and conservative substitutions are shown in red. Additional species included in the alignments are: Apis flora, A. flora; Dinoponera quadriceps, D. quadriceps; Ectatomma brumneum, E. brumneum; Phasmahyla jandaia, P. jandaia; Antheraea pernyi, A. pernyi.

Fig. 4.

Analytical RP-HPLC chromatograms and ESI-MS spectra of purified synthetic peptides. Calculated masses are shown adjacent to each mass spectrum.

Fig. 5.. Ponericins are active against parasitic nematodes and sheep blowflies.

(A) Concentration response ctuves for inhibition of the la1val development of H. contortus by synthetic ponericins. Each data point represents mean ± SEM based on three experiments of triplicate assays. (B) Motility of adult male B. malayi (movements per minute normalised to control, MMU) after addition of synthetic ponelicins over a 96 h timeframe. Points represent mean ± SEM from three independent experiments using four worms per dose (n = 12). Asterisks indicate significant differences, detennined by one-way ANOVA with Dunnett’s correction comparing ponericin-treated worms with untreated worms. (C–D) Dose response ctuves for insecticidal effects ofponericins following microinjection into adult sheep blowflies (L. cuprina). Dara are shown for paralysis at 1 h (C) and lethality at 24 h (D). Each data point represents the mean± SEM for three experiments (n = 10 flies per dose, per experiment).

Fig. 6.. Ponericins perturb cell membranes in vitro and induce spontaneous pain behaviour in vivo.

(A,D) Photomicrographs (20 × ) of DRG cells before and at 90 s and 120 s after application of 10 μM Na1b (A) and Nc1a (D). (B,E) Changes in intracellular calcium were measured as an increase in fluorescence relative to baseline (ΔF/F₀), where each line represents an individual cell response. (C) Comparison of median effective concentration (EC₅₀ values) observed in FLIPR assays for F11 and HEK293 cells for each peptide, calculated from concentration-response curves shown in (G,I,K,M), and compared via Student’s t-tests with Welch’s correction. Melittin was used as a positive control. (F, H, J, L) Fluorescence responses of F11 cells after addition of peptides Na1b, Nc1a, Nc3a and Nc3b over a range of concentrations (0.01–100 μM) recorded using a FLIPR. The corresponding concentration response ctuve is plotted adjacent to each fluorescence trace and was calculated from the change in fluorescence (ΔF) from the maximal response less baseline (n = 3). (N–P) Nocifensive behaviour in mice after injection of ponericins (30 μM). Asterisks indicate statistical significance (p < 0.05) (Student’s t test with Welch’s correction relative to vehicle). Error bars throughout the figure indicate SEM.

Fig. 7.. Ponericins adopt an α-helical conformation in lipid-like environments.

CD spectra of synthetic ponericins (25 μM) dissolved in (A) ultrapure water, (B) 20% TFE, and (C) 20 mM SDS. Α-Helical wheel projections generated using NetWheels (35] for (D) Na1b; (E) Nc1a; (F) Nc2a; (G) Nc3a (N-tenninal, Gl-Pl8); (H) Nc3a CC-terminal El9-N30); and (I) Nc3b. Two wheels are shown for Nc3a representing the regions either side of a proline residue, which is likely to disrupt helicity.

Fig. 8.. Characterization of Nc3b analogues.

(A) Sequence alignments of Nc3a and Nc3b with analogues. Positively charged residues are shown in blue, negatively charged residues are shown in purple, and amino acid substitutions in analogues are highlighted in pink. (B) Concentration-response cu1ves for inhibition of H. contortus larval development by Nc3a, Nc3b, and active analogues. Each point represents mean ± SEM from three experiments of triplicate assays. (C) Dose-response cmves for paralytic effects of Nc3a, Nc3b, and active analogues at 1 h after microinjection into L. cuprina. Each point represents mean ± SEM for three experiments (n = 10 flies per dose, per experiment). (D-F) CD spectra for Nc3a, Nc3b and analogues in (D) water, (E) 20% TFE, and (F) 20 mM SDS. (G) Photo-micrograph (20 × ) of DRG cells before and at 90 s after application of 10 μM [T3W]Nc3b. (H) Corresponding increase in intracellular calcium in DRG cells after application of 10 μM [T3W]Nc3b measured as an increase in fluorescence relative to baseline (ΔF/F₀), where each line represents an individual cell response. (I) Concentration-response curves for activation of F11 cells in a FLIPR assay by Nc3a, Nc3b and selected analogues. (J) Selectivity index for Nc3a, Nc3b and analogues, calculated as EC₅₀ of mammalian cell line divided by the IC₅₀ for inhibition of H. contortus larval development. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)