A Detergent-like Mechanism of Action of the Cytolytic Toxin Cyt1A from Bacillus thuringiensis var. israelensis

Abstract

The cytolytic delta-endotoxin Cyt1A from Bacillus thuringiensis var. israelensis is used in commercial preparations of environmentally safe insecticides. The current hypothesis on its mode of action is that the toxin self-assembles into well-defined cation-selective channels or pores, which results in colloid-osmotic lysis of the cell. Recently, a new hypothesis has been put forward suggesting that Cyt1A rather nonspecifically aggregates on the membrane surface and acts in a detergent-like manner. To distinguish between these two hypotheses, we investigated whether in the presence of lipid Cyt1A self-assembles into stoichiometric oligomers, which are characteristic of pores or channels, or aggregates into nonstoichiometric complexes, which would support the detergent-like model. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that in the presence of lipid Cyt1A forms protein aggregates with a broad range of molecular weights, some being too large to enter the gel. Cyt1A tryptophan (Trp) fluorescence in the presence of lipid exhibited a decrease in anisotropy and quantum yield, but an unchanged lifetime, which is consistent with the presence of toxin aggregates in the membrane. Electrostatic interactions between the charged amino acid residues and the lipid headgroups are responsible for bringing the protein to the membrane surface, while hydrophobic and/or van der Waals interactions make the membrane binding irreversible. Fluorescence photobleaching recovery, a technique that measures the diffusion coefficient of fluorescently labeled particles, and epifluorescence microscopy revealed that upon addition of Cyt1A lipid vesicles were broken into smaller, faster diffusing objects. Since no change in size or morphology of the vesicles is expected when pores are formed in the osmotically equilibrated membranes, our results support the detergent-like mode of action of Cyt1A.

Partial Restoration of Antibacterial Activity of the Protein Encoded by a Cryptic Open Reading Frame ( cyt1Ca ) from Bacillus thuringiensis subsp. israelensis by Site-Directed Mutagenesis

“…The amino acids important for Cyt1Aa activity (37) are shown in large type; the amino acids replaced in this study are shown in large grey type and numbered. Underlined amino acids are predicted to be on the Cyt1Aa surface (22,26). Arrows point to Cyt1Ca truncations, according to the full-length Cyt1Aa (grey) and proteinase K activation (black).…”

Amino acids at N- and C-termini are required for the efficient production and folding of a cytolytic γ-endotoxin from Bacillus thuringiensis

“…Two possible hypotheses have been currently proposed to explain its mode of action, and these hypotheses are referred to as the detergent-liked and pore-forming models. The detergent-like model proposes that the activated toxin molecules aggregate on the membrane surface, thereby inducing membrane fragmentation and releasing intracellular molecules (10,11), whereas the pore-forming model suggests that parts of the toxin molecule are inserted into the lipid bilayers in order to form transmembrane pores, thus resulting in osmotic imbalances and cell lysis (12,13).…”

A 54-Kilodalton Protein Encoded by pBtoxis Is Required for Parasporal Body Structural Integrity in Bacillus thuringiensis subsp. israelensis

“…Thus, Cry4A, Cry4B, and Cry11A, cooperatively produce the lethal effect observed in susceptible larvae (39,40). Moreover, studies over the past decade have clearly established that Cyt1Aa, a lipophilic toxin with a different mode of action than the Cry proteins (4,19), synergizes the toxicity of Cry4A, Cry4B, and Cry11A and delays the evolution of resistance to these in Culex quinquefasciatus (25,39,40). As a result of the complex interactions among these toxins, resistance to B. thuringiensis subsp.…”

“…Activated Cry proteins bind to midgut microvillar proteins, oligomerize, and insert into the membrane, where they kill epithelial cells through formation of cation channels or pores and induction of cell death pathways (3,5,30). Alternatively, Cyt proteins are lipophilic, found primarily in subspecies toxic to dipterans, and do not require midgut microvillar proteins for binding; instead, they insert directly to the lipid bilayer, in which they form ion channels or lipid faults that lead to cell lysis (4,19).…”