title: Toxicity and mode of action of insecticidal Cry1A proteins from Bacillus thuringiensis in an insect cell line, CF-1.
abstract: Bacillus thuringiensis Cry toxins are insecticidal proteins used to control insect pests. The interaction of Cry toxins with the midgut of susceptible insects is a dynamic process involving activation of the toxin, binding to midgut receptors in the apical epithelium and conformational changes in the toxin molecule, leading to pore formation and cell lysis. An understanding of the molecular events underlying toxin mode of action is essential for the continued use of Cry toxins. In this work, we examined the mechanism of action of Cry1A toxins in the lepidopteran cell line CF-1, using native Cry1Ab and mutant forms of this protein that interfer with different steps in the mechanism of action, specifically, receptor binding, oligomerization or pore formation. These mutants lost activity against both Manduca sexta larvae and CF-1 cells. We also analyzed a mutation created in domain I of Cry1Ab, in which helix α-1 and part of helix α-2 were deleted (Cry1AbMod). Cry1AbMod is able to oligomerize in the absence of toxin receptors, and although it shows reduced activity against some susceptible insects, it kills insect pests that have developed resistance to native Cry1Ab. Cry1AbMod showed enhanced toxicity to CF-1, suggesting that oligomerization of native Cry1Ab may be a limiting step in its activity against CF-1 cells. The toxicity of Cry1Ac and Cry1AcMod were also analyzed. Our results suggest that some of the steps in the mode of action of Cry1A toxins are conserved in vivo in insect midgut cells and in vitro in an established cell line, CF-1.
authors.affiliation: Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apdo. Postal 510-3, Cuernavaca 620, Morelos, Mexico. Electronic address: leivi@ibt.unam.mx.
authors.forename: Leivi
authors.initials: L
authors.lastname: Portugal
authors.affiliation: Canadian Forest Service, Great Lakes Forestry Centre, 1219 Queen St. E., Sault Ste. Marie, ON P6A 2E5, Canada. Electronic address: lgringo@nrcan.gc.ca.
authors.forename: J Lawrence
authors.initials: JL
authors.lastname: Gringorten
authors.affiliation: Canadian Forest Service, Great Lakes Forestry Centre, 1219 Queen St. E., Sault Ste. Marie, ON P6A 2E5, Canada.
authors.forename: Guido F
authors.initials: GF
authors.lastname: Caputo
authors.affiliation: Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apdo. Postal 510-3, Cuernavaca 620, Morelos, Mexico. Electronic address: mario@ibt.unam.mx.
authors.forename: Mario
authors.initials: M
authors.lastname: Soberón
authors.affiliation: Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apdo. Postal 510-3, Cuernavaca 620, Morelos, Mexico. Electronic address: cgaray@ibt.unam.mx.
authors.forename: Carlos
authors.initials: C
authors.lastname: Muñoz-Garay
authors.affiliation: Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apdo. Postal 510-3, Cuernavaca 620, Morelos, Mexico. Electronic address: bravo@ibt.unam.mx.
authors.forename: Alejandra
authors.initials: A
authors.lastname: Bravo
mesh_terms: D000818:Animals; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000494:pharmacology*; D015153:Blotting, Western; D002460:Cell Line; D004731:Endotoxins / Q000494:pharmacology*; D006460:Hemolysin Proteins / Q000494:pharmacology*; D007313:Insecta / Q000187:drug effects*; D007306:Insecticides / Q000494:pharmacology*; D007814:Larva / Q000187:drug effects; D018402:Manduca / Q000187:drug effects
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; D007306:Insecticides; C030956:insecticidal crystal protein, Bacillus Thuringiensis
keywords: Bacillus thuringiensis; CF-1 cells; Cry proteins; Manduca sexta; Mode of action; Oligomerization; Toxicity