Characterization of the mechanism of action of the genetically modified Cry1AbMod toxin that is active against Cry1Ab-resistant insects.
Bacillus thuringiensis Cry toxins are used in the control of insect pests. They are pore-forming toxins with a complex mechanism that involves the sequential interaction with receptors. They are produced as protoxins, which are activated by midgut proteases. Activated toxin binds to cadherin receptor, inducing an extra cleavage including helix alpha-1, facilitating the formation of a pre-pore oligomer. The toxin oligomer binds to secondary receptors such as aminopeptidase and inserts into lipid rafts forming pores and causing larval death. The primary threat to efficacy of Bt-toxins is the evolution of insect resistance. Engineered Cry1AMod toxins, devoid of helix alpha-1, could be used for the control of resistance in lepidopterans by bypassing the altered cadherin receptor, killing resistant insects affected in this receptor. Here we analyzed the mechanism of action of Cry1AbMod. We found that alkaline pH and the presence of membrane lipids facilitates the oligomerization of Cry1AbMod. In addition, tryptophan fluorescence emission spectra, ELISA binding to pure aminopeptidase receptor, calcein release assay and analysis of ionic-conductance in planar lipid bilayers, indicated that the secondary steps in mode of action that take place after interaction with cadherin receptor such as oligomerization, receptor binding and pore formation are similar in the Cry1AbMod and in the wild type Cry1Ab. Finally, the membrane-associated structure of Cry1AbMod oligomer was analyzed by electron crystallography showing that it forms a complex with a trimeric organization.
Muñóz-Garay
Carlos
C
Instituto de Biotecnología, Universidad Nacional Autónoma de México. Apdo. Morelos, Mexico.
Portugal
Leivi
L
Pardo-López
Liliana
L
Jiménez-Juárez
Nuria
N
Arenas
Ivan
I
Gómez
Isabel
I
Sánchez-López
Rosana
R
Arroyo
Raquel
R
Holzenburg
Andreas
A
Savva
Christos G
CG
Soberón
Mario
M
Bravo
Alejandra
A
D000330:Aedes / Q000187:drug effects; D000818:Animals; D000852:Anopheles / Q000187:drug effects; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D001427:Bacterial Toxins / Q000494:pharmacology*; D001681:Biological Assay; D015153:Blotting, Western; D018826:CD13 Antigens / Q000378:metabolism; D018360:Crystallography, X-Ray; D004352:Drug Resistance, Microbial / Q000187:drug effects*; D004731:Endotoxins / Q000235:genetics* / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005818:Genetic Engineering; D006460:Hemolysin Proteins / Q000235:genetics* / Q000378:metabolism*; D007313:Insecta / Q000187:drug effects* / Q000378:metabolism; D007306:Insecticides / Q000494:pharmacology; D007814:Larva / Q000187:drug effects* / Q000378:metabolism / Q000382:microbiology; D008051:Lipid Bilayers; D018402:Manduca / Q000187:drug effects; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010572:Pest Control, Biological; D055503:Protein Multimerization; D014364:Tryptophan
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D007306:Insecticides; D008051:Lipid Bilayers; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D014364:Tryptophan; D018826:CD13 Antigens