A detergent-like mechanism of action of the cytolytic toxin Cyt1A from Bacillus thuringiensis var. israelensis.
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.
Manceva
Slobodanka D
SD
Department of Chemistry and Biochemistry, University of Southern Mississippi, Hattiesburg, Mississippi 39406-5043, USA.
Pusztai-Carey
Marianne
M
Russo
Paul S
PS
Butko
Peter
P
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism / Q000633:toxicity*; D001427:Bacterial Toxins / Q000737:chemistry* / Q000378:metabolism / Q000633:toxicity*; D003902:Detergents / Q000633:toxicity*; D004591:Electrophoresis, Polyacrylamide Gel; D004731:Endotoxins / Q000737:chemistry* / Q000378:metabolism / Q000633:toxicity*; D005454:Fluorescence Polarization; D031541:Fluorescence Resonance Energy Transfer; D006460:Hemolysin Proteins; D008081:Liposomes; D008563:Membrane Lipids / Q000737:chemistry* / Q000378:metabolism*; D008564:Membrane Potentials / Q000187:drug effects; D008856:Microscopy, Fluorescence; D008970:Molecular Weight; D009997:Osmotic Pressure / Q000187:drug effects; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D055672:Static Electricity; D014364:Tryptophan / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D003902:Detergents; D004731:Endotoxins; D006460:Hemolysin Proteins; D008081:Liposomes; D008563:Membrane Lipids; D010713:Phosphatidylcholines; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D014443:Tyrosine; D014364:Tryptophan