Neurotoxin Merging: A Strategy Deployed by the Venom of the Spider Cupiennius salei to Potentiate Toxicity on Insects

Abstract

The venom of Cupiennius salei is composed of dozens of neurotoxins, with most of them supposed to act on ion channels. Some insecticidal monomeric neurotoxins contain an α-helical part besides their inhibitor cystine knot (ICK) motif (type 1). Other neurotoxins have, besides the ICK motif, an α-helical part of an open loop, resulting in a heterodimeric structure (type 2). Due to their low toxicity, it is difficult to understand the existence of type 2 peptides. Here, we show with the voltage clamp technique in oocytes of Xenopus laevis that a combined application of structural type 1 and type 2 neurotoxins has a much more pronounced cytolytic effect than each of the toxins alone. In biotests with Drosophila melanogaster, the combined effect of both neurotoxins was enhanced by 2 to 3 log units when compared to the components alone. Electrophysiological measurements of a type 2 peptide at 18 ion channel types, expressed in Xenopus laevis oocytes, showed no effect. Microscale thermophoresis data indicate a monomeric/heterodimeric peptide complex formation, thus a direct interaction between type 1 and type 2 peptides, leading to cell death. In conclusion, peptide mergers between both neurotoxins are the main cause for the high cytolytic activity of Cupiennius salei venom.

Complex precursor structures of cytolytic cupiennins identified in spider venom gland transcriptomes

“…6.07. * Data are from 14 , ** from 25 , *** from 18 , using flesh flies. Peptide LD 50 95% Confidence Intervals (pmol/mg fly) Cu 1a* 5.9 4.2–8.3 Cu 1b* 4.7 3.9–5.7 Cu 1d* 6.4 5.4–7.6 Cu 3a 18.7 17.11–20.4 Cu 3e 38.5 34.2–43.3 Cu 3d 12.7 11.3–14.1 Cu 4a 12.4 11.0–14.1 Cu 6a 78.0 70.3–86.6 Cu 6b 39.5 35.6–43.9 Cu 6d 23.2 21.4–25.2 Cu 7b 438.1 423.6–453.0 Cu 8 159.7 154.7–164.8 CsTx-16a1b1 63.5 58.1–69.3 CsTx-16c1d1 53.6 49.1–58.5 CsTx-1** 0.53 0.515–0.555 CsTx-9** 45.54 43.30–47.78 CsTx-13** 111.2 105.5–116.9 CIT1a*** ~ 0.6 …”

“…Hence, C. salei always endeavors to optimize the venom activity by synergistic activities 20 , 22 , 24 , 33 in which the toxicity on insects is potentiated, e.g. by neurotoxin merging 25 .…”

“…Cupiennins 3a, 3b, 4a, 6a, 6b, 6d, 7b, 8, and the two-chain CsTx-16a1b1, and CsTx-16c1d1 were synthesized (all C-terminally amidated) by GeneCust, (Dulange, Luxembourg). Bioassays using Drosophila flies were performed as described elsewhere 25 . Briefly, all peptides were dissolved in 0.1 M ammonium acetate, pH 6.1 and 0.05 µl injected intrathoracically into the flight muscles of female flies (1- to 7-days old).…”

“…Optimization is the key function for venom usage 22 , venom composition 20 and synergistic interactions of its components 23 – 25 , as described for C. salei . Which optimization strategy is behind the production of numerous small, cationic peptides?…”

Caterpillar Venom: A Health Hazard of the 21st Century

“…All these observations may explain the disturbances of cell adhesion and migration events that are seen after envenomation [36]. Moreover, the authors speculate that there is a synergy with other toxins in the venom, a phenomena also seen in other venomous animals [68]. This could explain the formation of local hemorrhaging, characterized by a disruption of the subendothelial extracellular matrix causing blood vessel wall instability [36].…”