The acyclotide ribe 31 from Rinorea bengalensis has selective cytotoxicity and potent insecticidal properties in Drosophila

Abstract

Cyclotides and acyclic versions of cyclotides (acyclotides) are peptides involved in plant defense. These peptides contain a cystine knot motif formed by three interlocked disulfide bonds, with the main difference between the two classes being the presence or absence of a cyclic backbone, respectively. The insecticidal activity of cyclotides is well documented, but no study to date explores the insecticidal activity of acyclotides. Here, we present the first in vivo evaluation of the insecticidal activity of acyclotides from Rinorea bengalensis on the vinegar fly Drosophila melanogaster. Of a group of structurally comparable acyclotides, ribe 31 showed the most potent toxicity when fed to D. melanogaster. We screened a range of acyclotides and cyclotides and found their toxicity toward human red blood cells was substantially lower than toward insect cells, highlighting their selectivity and potential for use as bioinsecticides. Our confocal microscopy experiments indicated their cytotoxicity is likely mediated via membrane disruption. Furthermore, our surface plasmon resonance studies suggested ribe 31 preferentially binds to membranes containing phospholipids with phosphatidyl-ethanolamine headgroups. Despite having an acyclic backbone, we determined the three-dimensional NMR solution structure of ribe 31 is similar to that of cyclotides. In summary, our results suggest that, with further optimization, ribe 31 could have applications as an insecticide due to its potent in vivo activity against D. melanogaster. More broadly, this work advances the field by demonstrating that acyclotides are more common than previously thought, have potent insecticidal activity, and have the advantage of potentially being more easily manufactured than cyclotides.

Keywords: Drosophila melanogaster; Rinorea bengalensis; acyclotides; bioinsecticides; cyclotides; cytotoxic.

Figure 1

Insecticidal activity of kalata B1, Cter M, and ribe 31 against different insects.A, sequence and insecticidal activity of kalata B1 against Helicoverpa punctigera, major pests of corn and cotton. B, sequence and insecticidal activity of Cter M against Helicoverpa armigera larvae (H. armigera), a cotton pest. C, sequence and insecticidal activity of ribe 31 against Drosophila melanogaster (D. melanogaster). Cysteine residues are marked red in yellow circles. Yellow lines connecting two cysteine residues represent disulfide bonds.

Figure 2

Cystine knot peptides derived from R. bengalensis.A, HPLC traces of stem and leaf extracts of R. bengalensis and the relative elution time of cystine knot peptides discovered from it are shown in top panel. B, the amino acid sequences of acyclotides ribe 10, 20, 24, and 31 and cyclotide ribe 33 are listed in the bottom panel. Yellow lines connecting two cysteine residues represent disulfide bonds.

Figure 3

The results of a CAFE assay of ribe 31 (A), cyO2 (B), and kalata B1 (C) on D. melanogaster at a range of concentrations (μM). The lifespan of flies fed a sucrose diet (20% sucrose) or a sucrose plus ribe 31 diet at varying concentrations is shown at the top. Differences in average lifespan of each experimental group compared to the sucrose-only control group (‘sucrose’) are shown at the bottom. Each dot in the scatter plot represents the survival of one fly. Error bars show the 95% CI. A, effect size statistics: ribe 31, 1.6 μM (n = 98) versus sucrose (n = 104) = −200 [95% CI −220, −180] p < 1∗10 to 4; ribe 31, 16 μM (n = 207) versus sucrose (n = 104) = −252 [95% CI −269, −236] p < 1∗10 to 4; ribe 31, 160 μM (n = 209) versus sucrose (n = 104) = −352 [95% CI −365, −337] p < 1∗10 to 4. B, effect size statistics: cyO2, 1.6 μM (n = 105) versus sucrose (n = 104) = −137 [95% CI −171, −103] p < 1∗10 to 4; cyO2, 16 μM (n = 105) versus sucrose (n = 104) = −174 [95% CI −202, −146] p < 1∗10 to 4; cyO2, 160 μM (n = 105) versus sucrose (n = 104) = −310 [95% CI −327, −291] p < 1∗10 to 4. C, effect size statistics: kalata B1, 1.7 μM (n = 99) versus sucrose (n = 102) = −39 [95% CI −76, 0] p= 0.0327; kalata B1, 17 μM (n = 105) versus sucrose (n = 102) = −99 [95% CI −136, −62] p < 1∗10 to 4; kalata B1: 170 μM (n = 207) versus sucrose (n = 102) = −140 [95% CI −170, −106] p < 1∗10 to 4. CI, confidence interval.

Figure 4

Secondary Hα chemical shift comparison between cyO2 and ribe 31 and 33 and the three-dimensional NMR structure of ribe 31.A, the comparison of secondary Hα chemical shifts of ribe 31 (orange), ribe 33 (blue) and cyO2 (lime green). The chemical shifts of cyO2 were obtained from the Biological Magnetic Resonance Data Bank (BMRB, ID: 16073 (62)). All cysteines are highlighted in red text with yellow boxes. The cyclic backbones of ribe 33 and cyO2 are indicated with a thick black line, and disulfide bond connectivities (Cys^I-IV, Cys^II-V and Cys^III-VI) are shown as thin black lines. B, superposition of 20 conformers representing the 3D NMR structure of acyclotide ribe 31. C, the superimposition of 3D NMR structure of cyclotide cyO2 (lime green) and acyclotide ribe 31 (orange).

Figure 5

Homology model and binding affinity of ribe peptides toward the model membrane.A, homology models of kalata B1 (pink), ribe 10 (purple), ribe 20 (green), ribe 24 (brown), ribe 31 (orange), and ribe 33 (blue) generated using the SWISS-MODEL web server (https://swissmodel.expasy.org) using the structure of cyO2 (PDB ID: 2KCG) as a template. All structures are represented in surface diagrams using PyMol, with the hydrophobic residues highlighted in corresponding colors. B, binding affinity of ribe peptides toward the model membrane composed of POPC/POPE (80:20) evaluated using surface plasmon resonance. C, sensorgrams of ribe 31 binding to the model membrane at a range of concentrations from 4 μM to 32 μM. PDB, Protein Data Bank; POPC, 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine; POPE, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine.

Figure 6

The interactions of Alexa Fluor 488–labeled cystine knot peptides (green) with live Sf9 cells were imaged using confocal microscopy. Sf9 insect cells were treated with (A) Alexa Fluor 488–labeled cyO2 (A488-cyO2) at 0.5 μM, (B) Alexa Fluor 488–labeled ribe 31 (A488-ribe 31) at 0.5 μM, and (C) Alexa Fluor 488–labeled kalata B1 (A488-kB1) at 0.5 μM. Images were captured after 30 min of treatment. The cell membrane was labeled with wheat germ agglutinin conjugated with Alexa Fluor 647 (blue).