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.
We used site-directed mutagenesis to probe the function of four alternating arginines located at amino acid positions 525, 527, 529, and 531 in a highly conserved region of domain III in the Cry1Ac toxin of Bacillus thuringiensis. We created 10 mutants: eight single mutants, with each arginine replaced by either glycine (G) or aspartic acid (D), and two double mutants (R525G/R527G and R529G/R531G). In lawn assays of the 10 mutants with a cultured Choristoneura fumiferana insect cell line (Cf1), replacement of a single arginine by either glycine or aspartic acid at position 525 or 529 decreased toxicity 4-to 12-fold relative to native Cry1Ac toxin, whereas replacement at position 527 or 531 decreased toxicity only 3-fold. The reduction in toxicity seen with double mutants was 8-fold for R525G/R527G and 25-fold for R529G/R531G. Five of the mutants (R525G, R525D, R527G, R529D, and R525G/R527G) were tested in bioassays with Plutella xylostella larvae and ion channel formation in planar lipid bilayers. In the bioassays, R525D, R529D, and R525G/R527G showed reduced toxicity. In planar lipid bilayers, the conductance and the selectivity of the mutants were similar to those of native Cry1Ac. Toxins with alteration at position 527 or 529 tended to remain in their subconducting states rather than the maximally conducting state. Our results suggest that the primary role of this conserved region is to maintain both the structural integrity of the native toxin and the full functionality of the formed membrane pore.Bacillus thuringiensis is a gram-positive bacterium that produces one or more insecticidal crystal (Cry) proteins deposited in the form of an intracellular parasporal crystal during sporulation (20). Shortly after ingestion by a susceptible insect, most Cry proteins dissolve in the insect midgut, are activated by midgut proteases, and bind to a specific midgut epithelial cell receptor. After binding to specific docking proteins on the microvillous surface of susceptible epithelial cells in the larval midgut (12, 27), the toxin undergoes a conformational change in which the helix-rich domain I (DI) separates from the other two domains (23). A hairpin, composed of helices ␣4 and ␣5, subsequently inserts into the membrane with the other five helices spreading, in an umbrella-like fashion, over the membrane surface (8), with ␣4 lining the lumen of the pore to create a functional ion channel (19). The toxin-exposed midgut epithelial cells eventually die by a colloid-osmotic lysis mechanism (14).The atomic structures of two activated Cry proteins, Cry1Aa and Cry3A, have been reported (11, 16). The two proteins have similar three-domain structures. DI is directly involved in ion channel formation (8,23,28). The second domain, DII, is involved in binding (9, 17, 21), whereas DIII is thought to play both a structural role and a toxin recognition role (2,5,15,17).A multiple sequence alignment of cry genes shows five conserved blocks (13), with the fourth conserved block (in DIII) consisting of an intriguing series of four al...
The role of the third domain of CryIAa, a Bacillus thuringiensis insecticidal toxin, in toxin-induced membrane permeabilization in a receptor-free environment was investigated. Planar lipid bilayer experiments were conducted with the parental toxin and five proteins obtained by site-directed mutagenesis in block 4, an argininerich, highly conserved region of the protein. Four mutants were constructed by replacing the first arginine in position 21 by a lysine (R521K), a glutamine (R521Q), a histidine (R521H), or a glutamic acid (R521E). A fifth mutant was obtained by replacing the fourth arginine by a lysine (R527K). Like CryIAa, the mutants formed cation-selective channels. A limited but significant reduction in channel conductance was observed for all mutants except R521H. The effect was more dramatic for the voltage dependence of the channels formed by R521K and R521Q, which was reversed compared to that of the parental toxin. This study provides the first direct evidence of a functional role for domain III in membrane permeabilization. Our results suggest that residues of the positive arginine face of block 4 interact with domain I, the putative pore-forming region of CryIAa.
The effect of high K+ concentration, insulin and the L-type Ca2+ channel blocker PN 200-110 on cytosolic intracellular free calcium ([Ca2+]i) was studied in single ventricular myocytes of 10-day-old embryonic chick heart, 20-week-old human fetus and rabbit aorta (VSM) single cells using the Ca(2+)-sensitive fluorescent dye, Fura-2 microfluorometry and digital imaging technique. Depolarization of the cell membrane of both heart and VSM cells with continuous superfusion of 30 mM [K+]o induced a rapid transient increase of [Ca2+]i that was followed by a sustained component. The early transient increase of [Ca2+]i by high [K+]o was blocked by the L-type calcium channel antagonist nifedipine. However, the sustained component was found to be insensitive to this drug. PN 200-110 another L-type Ca2+ blocker was found to decrease both the early transient and the sustained increase of [Ca2+]i induced by depolarization of the cell membrane with high [K+]o. Insulin at a concentration of 40 to 80 microU/ml only produced a sustained increase of [Ca2+]i that was blocked by PN 200-110 or by lowering the extracellular Ca2+ concentration with EGTA. The sustained increase of [Ca2+]i induced by high [K+]o or insulin was insensitive to metabolic inhibitors such as KCN and ouabain as well to the fast Na+ channel blocker, tetrodotoxin and to the increase of intracellular concentrations of cyclic nucleotides. Using the patch clamp technique, insulin did not affect the L-type Ca2+ current and the delayed outward K+ current. These results suggest that the early increase of [Ca2+]i during depolarization of the cell membrane of heart and VSM cells with high [K+]o is due to the opening and decay of an L-type Ca2+ channel. However, the sustained increase of [Ca2+]i during a sustained depolarization is due to the activation of a resting (R) Ca2+ channel that is insensitive to lowering [ATP]i and sensitive to insulin.
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