Heterodimeric Insecticidal Peptide Provides New Insights into the Molecular and Functional Diversity of Ant Venoms

Abstract

Ants use venom for predation, defense, 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 modeling, stability studies, and 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 cross-linked by two disulfide bonds in an antiparallel orientation. We chemically synthesized 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 blowfly assays (LD50 = 3 nmol/g). Molecular modeling and circular dichroism studies revealed strong α-helical features, indicating its cytotoxic effects could derive from cell membrane pore formation or disruption. The native heterodimer was substantially more stable against proteolytic degradation (t 1/2 = 13 h) than its homodimers or monomers (t 1/2 < 20 min), indicating an evolutionary advantage of the more complex structure. The proteomic analysis of Pseudomyrmex penetrator venom and in-depth characterization 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 highly relevant for the design and development of peptide therapeutics, molecular probes, and bioinsecticides.

Discovery of an Insect Neuroactive Helix Ring Peptide from Ant Venom

“…Since most of the reported ant venom peptides that exhibit insecticidal activities are cytotoxic [21,[26][27][28][29] (see Table 2), we tested U 11 cytotoxicity using in vitro assays on the dipteran S2 Drosophila embryonic cell line. Both CCK-8 and LDH assays showed that U 11 was not cytotoxic to S2 cells after 1 h and 24 h of incubation at 5 and 50 µM (Figure 2A,B).…”

Adaptive trade-offs between vertebrate defense and insect predation drive ant venom evolution

“…We found that P. gracilis shares few similarities with the other three Pseudomyrmex species, and that the different venom families are present in different proportions among the species. Among these families, family 39 corresponded to the myrmexins (renamed here the dimeric pseudomyrmeciitoxin (PSDTX)), a group of dimeric peptides first described in the venom of P. triplarinus (49) which are highly cytotoxic to insect cells (50). Dimeric PSDTXs largely dominate the venom of P. viduus (94% of relative venom expression), which was found to be the most paralytic and lethal venom on blowflies.…”

The mechanism underlying toxicity of a venom peptide against insects reveals how ants are master at disrupting membranes

“…As cytotoxic peptides, they participate in the offensive role of the venom by inducing paralysis of the prey insect, as has been reported for other membrane-active venom peptides. 26 , 27 So far, the effects of several peptides from various venoms (from Hymenoptera, 10 , 11 , 13 , 14 , 15 , 26 , 27 , 37 , 38 , 39 , 40 , 41 spiders, 42 , 43 scorpions 44 …) have been evaluated on insects using in vivo assays. However, few studies have focused on insect cells 16 , 38 , 45 , 46 , 47 despite the relevance of this cellular model to elucidate the mode of action of cytotoxic peptides, to exploit the diversity of peptide libraries to discover new insecticides, or to extend the knowledge of the functions of venom peptides.…”

“… 26 , 27 So far, the effects of several peptides from various venoms (from Hymenoptera, 10 , 11 , 13 , 14 , 15 , 26 , 27 , 37 , 38 , 39 , 40 , 41 spiders, 42 , 43 scorpions 44 …) have been evaluated on insects using in vivo assays. However, few studies have focused on insect cells 16 , 38 , 45 , 46 , 47 despite the relevance of this cellular model to elucidate the mode of action of cytotoxic peptides, to exploit the diversity of peptide libraries to discover new insecticides, or to extend the knowledge of the functions of venom peptides. U 9 and M-Tb1a have a cytotoxic effect on S2 cells as potent as that of the previously studied ant venom cytotoxic peptides.…”

“…U 9 and M-Tb1a have a cytotoxic effect on S2 cells as potent as that of the previously studied ant venom cytotoxic peptides. 38 , 46 …”