The mode of action of the Bacillus thuringiensis vegetative insecticidal protein Vip3A differs from that of Cry1Ab delta-endotoxin

Abstract

The Vip3A protein, secreted by Bacillus spp. during the vegetative stage of growth, represents a new family of insecticidal proteins. In our investigation of the mode of action of Vip3A, the 88-kDa Vip3A full-length toxin (Vip3A-F) was proteolytically activated to an approximately 62-kDa core toxin either by trypsin (Vip3A-T) or lepidopteran gut juice extracts (Vip3A-G). Biotinylated Vip3A-G demonstrated competitive binding to lepidopteran midgut brush border membrane vesicles (BBMV). Furthermore, in ligand blotting experiments with BBMV from the tobacco hornworm, Manduca sexta (Linnaeus), activated Cry1Ab bound to 120-kDa aminopeptidase N (APN)-like and 250-kDa cadherin-like molecules, whereas Vip3A-G bound to 80-kDa and 100-kDa molecules which are distinct from the known Cry1Ab receptors. In addition, separate blotting experiments with Vip3A-G did not show binding to isolated Cry1A receptors, such as M. sexta APN protein, or a cadherin Cry1Ab ecto-binding domain. In voltage clamping assays with dissected midgut from the susceptible insect, M. sexta, Vip3A-G clearly formed pores, whereas Vip3A-F was incapable of pore formation. In the same assay, Vip3A-G was incapable of forming pores with larvae of the nonsusceptible insect, monarch butterfly, Danaus plexippus (Linnaeus). In planar lipid bilayers, both Vip3A-G and Vip3A-T formed stable ion channels in the absence of any receptors, supporting pore formation as an inherent property of Vip3A. Both Cry1Ab and Vip3A channels were voltage independent and highly cation selective; however, they differed considerably in their principal conductance state and cation specificity. The mode of action of Vip3A supports its use as a novel insecticidal agent.

FIG. 1.

Proteolytic activation of Vip3A-F toxin by trypsin (A) or lepidopteran gut juice extracts (B). (A) A 2.5-μg aliquot of Vip3A-F toxin (lane 1) was incubated with 1% trypsin for 1 h (lane 2) or overnight (lane 3) at 37°C. M = MW markers (10⁻³) as indicated. (B) A 2.5-μg aliquot of Vip3A-F toxin (lane 1) was treated with O. nubilalis (lanes 2 and 4) or M. sexta (lanes 3 and 5) gut juice extracts for 1 h (lanes 2 and 3) or overnight (lanes 4 and 5). After digestion, reactions were stopped by adding Complete protease inhibitor (Roche Molecular Biochemicals), and samples were separated on SDS-8 to 12% PAGE.

FIG. 2.

Binding of biotinylated Vip3A-G to M. sexta (lanes 1 and 2) and O. nubilalis BBMV (lanes 3 and 4). Biotinylated Vip3A-G toxin (5 nM) was incubated with 10 μg of BBMV in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of a 250-fold excess of unlabeled Vip3A-G toxin. BBMV-bound toxins were visualized as described in Materials and Methods.

FIG. 3.

Binding of biotinylated Cry1Ab but not Vip3A-G toxin to isolated Cry1A receptor proteins. (A) M. sexta APN (5 μg) was separated on SDS-PAGE and transferred to PVDF membrane. The blot was blocked with 3% BSA for 1 h and incubated with 10 nM biotinylated Cry1Ab (lane 1) or Vip3A-G (lane 2). Bound toxins were visualized as described in Materials and Methods. (B) The recombinant-expressed TBR of the cadherin ectodomain (6 μg) was separated on SDS-PAGE, transferred to nitrocellulose membrane, and then probed before (lanes 1 and 2) or after (lanes 3 and 4) incubation with toxin as described in Materials and Methods. M = MW markers (10⁻³) as indicated. Lanes 1 and 2, TBR alone; lanes 3 and 4, TBR plus biotinylated Cry1Ab or biotinylated Vip3A-G, respectively. Protein was detected by virtue of an S-tag (lanes M and 1) or with the streptavidin conjugate (lanes 2 to 4) as described in Materials and Methods.

FIG. 4.

M. sexta BBMV ligand blotting with biotinylated Cry1Ab and Vip3A-G toxins. Twenty micrograms of M. sexta BBMV was separated on an SDS-8 to 12% PAGE and transferred to PVDF membrane. The membrane was blocked with 3% BSA and probed with 10 nM biotinylated Cry1Ab (lane 1) or Vip3A-G (lane 2) for 5 h. Toxin-binding proteins were visualized as described in Materials and Methods. M = MW markers (10⁻³) as indicated.

FIG. 5.

Inhibition of I_sc across M. sexta (A) and D. plexippus (B) as measured by voltage clamping of dissected midguts. To M. sexta, a total of 15 nM Cry1Ab toxin (▪) or Vip3A-G (×) or 150 nM Vip3A-F (⧫) or Vip3A-G (▴) was injected (first arrow) into the lumen side of the chamber, and the drop in I_sc was measured over time. For Vip3A-F (⧫), 15 nM Cry1Ab was also added at 35 min to verify membrane viability (second arrow). To D. plexippus, a total of 15 nM Cry1Ab toxin (▪) or 150 nM Vip3A-G toxin (⧫) was injected. After 22.5 min with no response from the Vip3A-G injection, 15 nM Cry1Ab toxin (second arrow) was added. The I_sc measured before the addition of the toxin was considered 100%.

FIG. 6.

In vitro-activated Vip3A ion channels formed in planar lipid bilayers. (A) Channels seen after activation with M. sexta gut fluid. Channels open down; holding voltage (V_h) = −40 mV. (B and C) Channels seen after activation with trypsin. Panel B contains two consecutive traces (channels open down) with V_h = −80 mV, whereas panel C contains two consecutive traces (channels open up) with V_h = +80 mV. Dashed lines indicate closed current level. (D) Current-voltage relations for the principal conducting state of trypsin-activated Vip3A under symmetrical (•) or asymmetrical (○) KCl conditions as described in Materials and Methods. Linear regression equations were y = 0.306x + 0.244, r² = 0.998 (•); or y = 0.227x − 4.22, r² = 0.992 (○), respectively.

FIG. 7.

Cry1Ab channels formed in planar lipid bilayers. (A) Variation observed in the conductance levels of Cry1Ab channels. V_h = +40 mV in the two consecutive traces (channels open up). (B) In a separate experiment, Cry1Ab channels are displayed at +20 mV (upper trace, channels open up) or at −20 mV (lower trace, channels open down). Dashed lines indicate closed current level. (C) Current-voltage relations for the principal conducting state of trypsin-activated Cry1Ab under symmetrical (•) or asymmetrical (○) KCl conditions as described in Materials and Methods. Linear regression equations were y = 0.730x − 0.806, r² = 0.999 (•); or y = 0.330x − 5.48, r² = 0.991 (○), respectively.