title: Functional significance of membrane associated proteolysis in the toxicity of Bacillus thuringiensis Cry3Aa toxin against Colorado potato beetle.
abstract: Bacillus thuringiensis Cry toxins are widely used as biocontrol agents in bioinsecticides and transgenic plants. In the three domain-Cry toxins, domain II has been identified as an important determinant of their highly specific activity against insects. In this work, we assessed the role in membrane associated proteolysis and toxicity in Colorado potato beetle (CPB) of a previously reported ADAM recognition motif present in Cry3Aa toxin domain II. We used site-directed mutagenesis to modify the Bacillus thuringiensis cry3A gene in amino acid residues 344, 346, 347, 351 and 353 of the ADAM recognition motif in Cry3Aa toxin. Cry3Aa toxin mutants displayed decreased toxicity when compared to the wild type toxin and impaired ability to compete CPB brush border membrane associated cleavage of an ADAM fluorogenic substrate. Although the proteolytic profile of Cry3Aa toxin mutants generated by brush border membrane associated proteases was similar to that of Cry3Aa toxin, the metalloprotease inhibitor 1,10-phenanthroline was less efficient on the proteolysis of mutants than on that of the wild type toxin. The relevance of the Cry3Aa-ADAM interaction through the predicted recognition sequence was further confirmed by analyzing the effect of membrane integrity disturbance on Cry3Aa toxin membrane associated proteolysis and CPB larvae toxicity. Data support that Cry3Aa proteolysis, as a result of the interaction with ADAM through the Cry3Aa recognition motif, is essential for Cry3Aa toxic action in CPB. Detailed knowledge of Cry3Aa interaction with CPB midgut membrane should facilitate the development of more effective Bt based products against this devastating pest and other Coleoptera.
authors.affiliation: Departamento de Genética, Facultad de Ciencias Biológicas, Universidad de Valencia, Dr. Moliner 50, Burjassot 46100, Valencia, Spain.
authors.forename: Inmaculada
authors.initials: I
authors.lastname: García-Robles
authors.forename: Camila
authors.initials: C
authors.lastname: Ochoa-Campuzano
authors.forename: Jorge
authors.initials: J
authors.lastname: Sánchez
authors.forename: Estefanía
authors.initials: E
authors.lastname: Contreras
authors.forename: M Dolores
authors.initials: MD
authors.lastname: Real
authors.forename: Carolina
authors.initials: C
authors.lastname: Rausell
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics / Q000494:pharmacology*; D002478:Cells, Cultured; D001517:Coleoptera / Q000187:drug effects*; D003120:Colorado; D004731:Endotoxins / Q000235:genetics / Q000494:pharmacology*; D006460:Hemolysin Proteins / Q000235:genetics / Q000494:pharmacology*; D008871:Microvilli / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010572:Pest Control, Biological; D059748:Proteolysis / Q000187:drug effects*; D017422:Sequence Analysis, DNA
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis