Abstract
Bacillus thuringiensis (Bt) Cyt2Aa2 showed toxicity against Dipteran insect larvae and invitro lysis activity on several cells. It has potential applications in the biological control of insect larvae. Although pore-forming and/or detergent-like mechanisms were proposed, the mechanism underlying cytolytic activity remains unclear. Analysis of the haemolytic activity of Cyt2Aa2 with osmotic stabilizers revealed partial toxin inhibition, suggesting a distinctive mechanism from the putative pore formation model. Membrane permeability was studied using fluorescent dye entrapped in large unilamellar vesicles (LUVs) at various protein/lipid molar ratios. Binding of Cyt2Aa2 monomer to the lipid membrane did not disturb membrane integrity until the critical protein/lipid molar ratio was reached, when Cyt2Aa2 complexes and cytolytic activity were detected. The complexes are large aggregates that appeared as a ladder when separated by agarose gel electrophoresis. Interaction of Cyt2Aa2 with Aedes albopictus cells was investigated by confocal microscopy and total internal reflection fluorescent microscopy (TIRF). The results showed that Cyt2Aa2 binds on the cell membrane at an early stage without cell membrane disruption. Protein aggregation on the cell membrane was detected later which coincided with cell swelling. Cyt2Aa2 aggregations on supported lipid bilayers (SLBs) were visualized by AFM. The AFM topographic images revealed Cyt2Aa2 aggregates on the lipid bilayer at low protein concentration and subsequently disrupts the lipid bilayer by forming a lesion as the protein concentration increased. These results supported the mechanism whereby Cyt2Aa2 binds and aggregates on the lipid membrane leading to the formation of non-specific hole and disruption of the cell membrane.
Highlights
Bacillus thuringiensis (Bt) is an aerobic gram-positive bacterium, which produces insecticidal proteins during the sporulation phase [1]
Our results suggested that the lipid membrane disruption by Cyt2Aa2 occurs after binding, with the formation of protein aggregations and a subsequence disruption rather than through pore formation followed by cell swelling and lysis
Total internal reflection fluorescent microscopy A. albopictus cells cultured on slide cover glass were treated with 10 μg/ml of active labelled Cyt2Aa2 in Leibovitz’s L-15 medium at room temperature
Summary
Bacillus thuringiensis (Bt) is an aerobic gram-positive bacterium, which produces insecticidal proteins during the sporulation phase [1]. These insecticidal proteins are parasporal crystals consisting of two delta-endotoxin families, crystal (Cry) and cytolytic (Cyt) toxins [2]. The 3D structure of inactivated Cyt2Aa shows a monomeric structure with high similarity to protease-activated Cyt2Ba [7], with their secondary and tertiary structures being very similar. These crystallographic structures suggest a respective conformational change of the active toxin when binding to lipid bilayers. It shows specific in vivo toxicity against Dipteran insect larvae, such as mosquitoes and black flies [12,13]
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