title: Investigation of the pore-forming mechanism of a cytolytic delta-endotoxin from Bacillus thuringiensis.
abstract: Cyt2Aa1 is a cytolytic protein produced by Bacillus thuringiensis subsp. kyushuensis. Penetration of the toxin into membranes has been studied to learn more about membrane-insertion mechanisms and transmembrane-pore formation. The haemolysis assay of Cyt2Aa1 showed a steep and sigmoidal dose-response curve, indicating that toxin aggregation or oligomerization is required for pore formation. Studies of the effect of temperature on pore formation and fluorimetric studies of acrylodan-labelled toxin suggest that toxin inserts into the membrane before oligomerizing to form a pore. Low temperature neither inhibited membrane binding nor closed pores that have been formed, but markedly inhibited oligomerization of the toxin molecules. When toxin-treated red blood cells at 4 degrees C were transferred to a toxin-free solution at 37 degrees C, no significant increase in haemolysis was observed. This result suggests that membrane-bound toxin could not diffuse laterally and interact with other molecules to form a pore. From these results, we propose that Cyt2Aa1 binds and inserts into the membrane as a monomer. Oligomerization occurs when toxin molecules have bound in close proximity to each other and pores are formed from large oligomers.
authors.affiliation: National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, 113 Phaholyothin Road, Klong 1, Klong Luang, Pathumthani 12120, Thailand. boonhiang@biotech.or.th
authors.forename: Boonhiang
authors.initials: B
authors.lastname: Promdonkoy
authors.forename: David J
authors.initials: DJ
authors.lastname: Ellar
mesh_terms: D001413:Bacillus thuringiensis / Q000502:physiology*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000633:toxicity; D001427:Bacterial Toxins / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity; D004731:Endotoxins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000633:toxicity; D004910:Erythrocyte Membrane / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D004912:Erythrocytes / Q000187:drug effects / Q000473:pathology; D006460:Hemolysin Proteins; D006461:Hemolysis / Q000187:drug effects*; D006801:Humans; D018272:Porins / Q000097:blood / Q000187:drug effects; D013053:Spectrophotometry
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D018272:Porins; C030956:insecticidal crystal protein, Bacillus Thuringiensis