Bacillus thuringiensis Cyt2Aa2 toxin disrupts cell membranes by forming large protein aggregates.
Bacillus thuringiensis (Bt) Cyt2Aa2 showed toxicity against Dipteran insect larvae and in vitro 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.
Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Putthamonthon 4 Rd., Salaya, Nakhonpathom 73170, Thailand.
Sudarat
S
Tharad
Department of Nanobiotechnology, Institute for Biophysics, University of Natural Resources and Life Sciences Vienna (BOKU), Muthgasse 11, Vienna 1190, Austria.
José L
JL
Toca-Herrera
National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), 113 Phahonyothin Rd., Pathumthani 12120, Thailand chartchai.kri@mahidol.ac.th boonhiang@biotec.or.th.
Boonhiang
B
Promdonkoy
Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Putthamonthon 4 Rd., Salaya, Nakhonpathom 73170, Thailand chartchai.kri@mahidol.ac.th boonhiang@biotec.or.th.
Chartchai
C
Krittanai
D000330:Aedes / Q000737:chemistry / Q000382:microbiology; D000818:Animals; D001413:Bacillus thuringiensis / Q000737:chemistry* / Q000378:metabolism / Q000472:pathogenicity / Q000648:ultrastructure; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D002463:Cell Membrane Permeability / Q000235:genetics; D004731:Endotoxins / Q000737:chemistry / Q000378:metabolism*; D006460:Hemolysin Proteins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D007814:Larva / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D050356:Lipid Metabolism / Q000235:genetics; D018625:Microscopy, Atomic Force; D066329:Protein Aggregates; D011485:Protein Binding
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; D008051:Lipid Bilayers; D066329:Protein Aggregates; C030956:insecticidal crystal protein, Bacillus Thuringiensis
Bacillus thuringiensis; complex formation; cytolytic activity; protein aggregation