An analysis of Bacillus thuringiensisδ‐endotoxin action on insect‐midgut‐membrane permeability using a light‐scattering assay

Abstract

Changes in the membrane permeability of Manduca sexta midgut brush-border-membrane vesicles (BBMV) after addition of Bacillus thuringiensis S-endotoxins were studied using osmotic swelling experiments, volume changes being monitored as the change in 90" light scattering. Typically, control BBMV exhibited limited permeability for sucrose and salts (KCl), while being permeable for urea and glucose. The action of d-endotoxin was examined using proteolytically activated Cry-IA(c) and CryIB toxins. CryIA(c) produced a marked change in the solute permeability of M. sexta BBMV, significant effects being observed at 3.75 pmol/mg BBMV. The permeability change was relatively non-selective with cations, anions and neutral solutes all traversing the membrane to an increased extent in the presence of CryIA(c). In contrast, the CryIB toxin had no effect on BBMV permeability.

Permeability Changes of Manduca sexta Midgut Brush Border Membranes Induced by Oligomeric Structures of Different Cry Toxins

“…Cry toxin channels are poorly selective, transporting different ions and solutes of higher size including sugars and amino acids [2,[23][24][25][26]. However, all these studies were done only with Cry toxins in their monomeric state [2,[23][24][25][26].…”

δ‐Endotoxins induce cation channels in Spodoptera frugiperda brush border membranes in suspension and in planar lipid bilayers

“…Osmotic swelling experiments in BBMV from Manduca sexta have indicated that CryIA(c) toxin increases anion permeability [16]. Experiments where MeGluC1 was substituted by MeGlumethanesulfonate in the incubation buffer showed that the toxin does not induce anion permeability changes, but a hyperpolarization (8 + 2 mV, n = 3) due to K ÷ efflux (as in Fig.…”

“…Recent in vitro assays using brush border membrane vesicles (BBMV) have shown permeability changes induced by Cry toxins at physiological concentrations (nM) [15,16]. However, the toxin effects were recorded after 1 h of incubation with the vesicles, missing the initial response produced by the toxin once it interacts with its receptor.…”

Bacillus thuringiensis Cry1Ac toxin interaction with Manduca sexta aminopeptidase N in a model membrane environment

“…kurstaki HD73 were grown as described for Bacillus megaterium KM [28]. Purification of crystals, solubilization, activation with Pieris brassicae gut extract and SDS\PAGE analysis were performed as described previously [7]. Activation of soluble Cry1Ac with trypsin was performed with 1 : 10 (w\w) trypsin (Sigma)\toxin at 37 mC for 1 h followed by the addition of a further 1 : 10 (w\w) trypsin\toxin and 10 % (v\v) 2 M Tris\HCl, pH 7.5, and incubation overnight at 37 mC.…”

“…After ingestion by a susceptible insect the inclusion is solubilized and the protoxins are activated proteolytically within the insect midgut [2]. After activation, the Cry toxin binds to specific receptors located on the midgut epithelial cell membrane [3][4][5], subsequently inducing a membrane permeability change [6,7] that leads to cell death by colloid osmotic lysis [8].…”