Heterodimeric insecticidal peptide provides new insights into the molecular and functional diversity of ant venoms

Abstract

Ants use venom for predation, defence and communication, however, the molecular diversity, function and potential applications of ant venom remains understudied compared to other venomous lineages such as arachnids, snakes and cone snails. In this work, we used a multidisciplinary approach that encompassed field work, proteomics, sequencing, chemical synthesis, structural analysis, molecular modelling, stability studies, and a series of in vitro and in vivo bioassays to investigate the molecular diversity of the venom of the Amazonian Pseudomyrmex penetrator ants. We isolated a potent insecticidal heterodimeric peptide Δ-pseudomyrmecitoxin-Pp1a (Δ-PSDTX-Pp1a) composed of a 27-residue long A-chain and a 33-residue long B-chain crosslinked by two disulfide bonds in an antiparallel orientation. We chemically synthesised Δ-PSDTX-Pp1a, its corresponding parallel AA and BB homodimers, and its monomeric chains and demonstrated that Δ-PSDTX-Pp1a had the most potent insecticidal effects in blow fly assays (LD50 = 3 nM). Molecular modelling and circular dichroism studies revealed strong alpha-helical features, indicating its cytotoxic effects could derive from membrane disruption, which was further supported by insect cell calcium assays. The native heterodimer was also substantially more stable against proteolytic degradation (t1/2 =13 h) than its homodimers or monomers (t1/2 <20 min), indicating an evolutionary advantage of the more complex structure. The proteomic analysis of Pseudomyrmex penetrator venom and in-depth characterisation of Δ-PSDTX-Pp1a provide novel insights in the structural complexity of ant venom, and further exemplifies how nature exploits disulfide-bond formation and dimerization to gain an evolutionary advantage via improved stability; a concept that is also highly relevant for the design and development of peptide therapeutics, molecular probes and bioinsecticides.

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

“…Similarly, peptide PONTX-Ae1a from venom of the predatory ant Anochetus emarginatus [50] and venom peptides from Manica rubida induce rapid paralysis in blowflies but are not lethal [51]. The exception in this study was Nc3a, which had similar PD50 and LD50 values and was the most active peptide tested; its LD50 (3.5 ± 0.9 nmol g -1 ) is similar to peptide ∆-PSDTX-Pp1a isolated from the venom of Pseudomyrmex penetrator (LD50 3 nmol g -1 in L .cuprina) [52], and considerably higher than previously reported values for other insecticidal G-subfamily ponericins, such as ponericin G1 (LD50 25.7 nmol g -1 in Acheta domestica, [19]). Overall, the current study aligns with previous work indicating that both W and G-subfamily peptides have insecticidal activity [19], and the moderate paralytic activity of Nc2a indicates that insecticidal activity extends to L-subfamily ponericins as well.…”