Permeabilization of Model Lipid Membranes by Bacillus sphaericus Mosquitocidal Binary Toxin and its Individual Components

Abstract

The high larvicidal effect of Bacillus sphaericus (Bs), a mosquito control agent, originates from the presence of a binary toxin (Bs Bin) composed of two proteins (BinA and BinB) that work together to lyse gut cells of susceptible larvae. We demonstrate for the first time that the binary toxin and its individual components permeabilize receptor-free large unilamellar phospholipid vesicles (LUVs) and planar lipid bilayers (PLBs) by a mechanism of pore formation. Calcein-release experiments showed that LUV permeabilization was optimally achieved at alkaline pH and in the presence of acidic lipids. BinA was more efficient than BinB, BinB facilitated the BinA effect, and their stoichiometric mixture was more effective than the full Bin toxin. In PLBs, BinA formed voltage-dependent channels of approximately 100-200 pS with long open times and a high open probability. Larger channels (> or =400 pS) were also observed. BinB, which inserted less easily, formed smaller channels (< or =100 pS) with shorter mean open times. Channels observed after sequential addition of the two components, or formed by their 1:1 mixture (w/w), displayed BinA-like activity. Bs Bin toxin was less efficient at forming channels than the BinA/BinB mixture, with channels displaying the BinA channel behavior. Our data support the concept of BinA being principally responsible for pore formation in lipid membranes with BinB, the binding component of the toxin, playing a role in promoting channel activity.

Essential role of tryptophan residues in toxicity of binary toxin from Bacillus sphaericus

“…Large unilamellar vesicles (LUVs) were prepared from 2 mg ml -1 of a lipid mixture of phosphatidylcholine (PC)/phosphatidic acid (PA) in ratio 1:1 (w/w) dissolved in chloroform as de-scribed previously (8). The lipid mixture was evaporated under a nitrogen stream and the lipid film was resuspended in 200 μl of 60 mM calcein (pre-dissolved in 500 mM Na2CO3 buffer, pH 10) in 50 mM Na2CO3 buffer, pH 10.…”

“…Finally, 0.1% (v/v) Triton X-100 was added at time 900 s to totally release entrapped calceins. The degree of LUVs perturbation was determined as the percentage of calcein release as described previously (8).…”

“…Recently, the cellular response by vacuolation of the binary toxin-treated mammalian epithelial cell line (MDCK) expressing the binary toxin Cpm1 (Culex pipiens maltase 1) receptor was reported to associate with the induction of autophagy process (7). Although direct evidence to support the pore-forming activity of the binary toxin on epithelial midgut cells is unavailable, membrane permeabilization using a receptor-free planar lipid bilayer and large unilamellar phospholipid vesicles has been shown as the in vitro effects of the binary toxin components (8). Also, the ability of the binary toxin to insert into the model neutral lipid monolayers has been demonstrated (9).…”

Binary Toxins from Bacillus thuringiensis Active against the Western Corn Rootworm, Diabrotica virgifera virgifera LeConte

“…Binding to the target site (i.e., receptor) on the midgut epithelium is mediated by BinB, which appears to direct the subsequent binding of BinA (7,10,12). Ion channel studies with planar lipid bilayers (PLBs) and permeabilization assays with phospholipid vesicles indicate that (i) BinA-BinB mixtures form ion channels in PLBs that resemble those of BinA alone, (ii) BinA is more efficient than BinB in promoting ion channel activity in PLBs, and (iii) BinB enhances the vesicle-permeabilizing activity of BinA (41). These results suggest that BinA is primarily responsible for the ion channel-and pore-forming activity of the binary toxin and is facilitated by BinB.…”

Role of cysteine at positions 67, 161 and 241 of a Bacillus sphaericus binary toxin BinB

“…However, amino acids essential for receptor binding have not been identified. Some evidences suggest that BinB is inserted into membrane and form pores (24)(25)(26), although the precise mechanism in which the toxin kills the target cells is unclear. Therefore, C67 and C161 could also play a part in conformational change, receptor binding, or membrane insertion.…”