Bacillus thuringiensis subsp. israelensis 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 Aedes aegypti 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.
Keywords: Bacillus thuringiensis; Bti bacterium; Cyt1Aa toxin; detergent effect; fluorescence quenching; insecticide; larvicidal agent; membrane insertion; mosquito control; pore formation.
© 2020 Onofre et al.