title: Functional assembly of 260-kDa oligomers required for mosquito-larvicidal activity of the Bacillus thuringiensis Cry4Ba toxin.
abstract: Oligomerization has been shown to contribute to the toxicity of Bacillus thuringiensis Cry toxins. Mutations have been made in the Cry4Ba toxin and resulted in toxic to non-toxic mutants toward Aedes aegypti larvae. In this study, Cry4Ba wild type and mutants were analyzed for oligomer formation in vitro, biochemical properties and their relationships with larvicidal activity. In vitro, the Cry4Ba forms two-main types of the oligomers including (1) the 260-kDa and larger oligomers, which assembled in the carbonate buffer, pH 10.0 and completely dissociated by heating at 90°C and (2) 190-kDa oligomer, which was induced by heat, sodium-salt and detergent addition. Polar and charge residues in the toxin domain I and II may contribute to formation of the 260-kDa oligomers. A single Cys-525 in domain III was replaced with serine resulting in the C525S mutant, which exhibited a 50% reduction in larvicidal activity compared to the Cry4Ba wild-type. The mutant exhibited partial loss in larger oligomer of the 260kDa and total loss of 190-kDa oligomer. The results revealed an important role of the Cys-525 in intermolecular disulfide formation of larger oligomer as well as the 190-kDa oligomer. Despite of their formations in the receptor free condition, the 260-kDa and larger oligomers were found to strongly correspond to Cry4Ba toxicity suggesting their functional roles in the A. aegypti larvae. Also, possible roles of the 260-kDa and larger oligomers have been proposed in this report.
authors: Department of Protozoology, Faculty of Tropical Medicine, Mahidol University Ratchathewi, Bangkok 10400, Thailand.
authors: Narumol
authors: N
authors: Khomkhum
authors: Bacterial Protein Toxin Research Group, Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakornpathom 73170, Thailand.
authors: Somphob
authors: S
authors: Leetachewa
authors: Bacterial Protein Toxin Research Group, Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakornpathom 73170, Thailand.
authors: Chanan
authors: C
authors: Angsuthanasombat
authors: Department of Protozoology, Faculty of Tropical Medicine, Mahidol University Ratchathewi, Bangkok 10400, Thailand. Electronic address: saengduen.moo@mahidol.ac.th.
authors: Saengduen
authors: S
authors: Moonsom
mesh_terms: D000330:Aedes / Q000187:drug effects* / Q000502:physiology; D000818:Animals; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry* / Q000494:pharmacology; D004731:Endotoxins / Q000737:chemistry* / Q000494:pharmacology; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000494:pharmacology; D020128:Inhibitory Concentration 50; D007306:Insecticides / Q000737:chemistry* / Q000494:pharmacology; D007814:Larva / Q000187:drug effects / Q000502:physiology; D008970:Molecular Weight; D055503:Protein Multimerization
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; D007306:Insecticides; C030956:insecticidal crystal protein, Bacillus Thuringiensis
keywords: Aedes aegypti; Bacillus thuringiensis; Cry4Ba; Larvicidal activity; Oligomerization