The Cyt1Aa toxin from Bacillus thuringiensis inserts into target membranes via different mechanisms in insects, red blood cells, and lipid liposomes

Abstract

<jats:p><jats:italic>Bacillus thuringiensis</jats:italic> subsp. <jats:italic>israelensis</jats:italic> produces crystal inclusions composed of three-domain Cry proteins and cytolytic Cyt toxins, which are toxic to different mosquito larvae. A key component is the Cyt toxin, which synergizes the activity of the other Cry toxins, thereby resulting in high toxicity. The precise mechanism of action of Cyt toxins is still debated, and two models have been proposed: the pore formation model and the detergent effect. Here, we performed a systematic structural characterization of the Cyt toxin interaction with different membranes, including in <jats:italic>Aedes aegypti</jats:italic> larval brush border membrane vesicles, small unilamellar vesicle liposomes, and rabbit erythrocytes. We examined Cyt1Aa insertion into these membranes by analyzing fluorescence quenching in solution and in the membrane-bound state. For this purpose, we constructed several Cyt1Aa variants having substitutions with a single cysteine residue in different secondary structures, enabling Cys labeling with Alexa Fluor 488 for quenching analysis using I-soluble quencher in solution and in the membrane-bound state. We identified the Cyt1Aa residues exposed to the solvent upon membrane insertion, predicting a possible topology of the membrane-inserted toxin in the different membranes. Moreover, toxicity assays with these variants revealed that Cyt1Aa exerts its insecticidal activity and hemolysis through different mechanisms. We found that Cyt1Aa exhibits variable interactions with each membrane system, with deeper insertion into mosquito larva membranes, supporting the pore formation model, whereas in the case of erythrocytes and small unilamellar vesicles, Cyt1Aa's insertion was more superficial, supporting the notion that a detergent effect underlies its hemolytic activity.</jats:p>

Bacterial Toxins Active against Mosquitoes: Mode of Action and Resistance

“…It has been shown that the N-terminal region is responsible for the toxin oligomerization, and the C-terminal region is involved in the binding of the toxin to the membrane [ 84 ]. Specific amino acid residues and protein regions that affect Cyt1Aa binding, oligomerization, and membrane insertion have been investigated [ 75 , 85 , 86 , 87 ]. The second model is the detergent-like model, where it is proposed that the Cyt toxin aggregates nonspecifically on the cell membrane, leading to lipid bilayer disassembly and cell death [ 77 , 88 ].…”

“…aegypti larvae but not for red blood cells [ 85 ]. Therefore, Cyt toxin insertion by the pore formation model could occur in microvilli membranes, while a detergent membrane interaction seems to be related to its hemolytic activity [ 87 ].…”

Recombinant Beauveria bassiana expressing Bacillus thuringiensis toxin Cyt1Aa: a promising approach for enhancing Aedes mosquito control

“…Cyt1Aa acts by binding to specific receptors on the membrane of midgut epithelial cells ( 55 ). This binding triggers a conformational change in Cyt1Aa, allowing it to insert into the cell membrane and form pores or channels ( 56 , 57 ). These pores disrupt the integrity of the midgut membrane, leading to the leakage of cellular contents and loss of osmotic balance.…”

The use of antigens derived from Bacillus thuringiensis bacteria for further differentiation

“…Thuringiensis has been found to have unique biological activities. Among them are cytotoxicity specific to certain human cancer cells (Mizuki et al, 2000;Aboul-Soud et al, 2019;Mendoza-Almanza et al, 2020), lectin activity against mammalian red blood cells (Torres-Quintero et al, 2018;Onofre et al, 2020), and activity against trichomonads (Lee et al, 2017). Future research will include tests of parasporal proteins, e.g., animal feces, which may affect indicators of biological activity unrelated to pathogenicity.…”