Helix 4 Mutants of the Bacillus thuringiensis Insecticidal Toxin Cry1Aa Display Altered Pore-Forming Abilities

AUTOR(ES)

Vachon, Vincent

FONTE

American Society for Microbiology

RESUMO

The role played by α-helix 4 of the Bacillus thuringiensis toxin Cry1Aa in pore formation was investigated by individually replacing each of its charged residues with either a neutral or an oppositely charged amino acid by using site-directed mutagenesis. The majority of the resulting mutant proteins were considerably less toxic to Manduca sexta larvae than Cry1Aa. Most mutants also had a considerably reduced ability to form pores in midgut brush border membrane vesicles isolated from this insect, with the notable exception of those with alterations at amino acid position 127 (R127N and R127E), located near the N-terminal end of the helix. Introducing a negatively charged amino acid near the C-terminal end of the helix (T142D and T143D), a region normally devoid of charged residues, completely abolished pore formation. For each mutant that retained detectable pore-forming activity, reduced membrane permeability to KCl was accompanied by an approximately equivalent reduction in permeability to N-methyl-d-glucamine hydrochloride, potassium gluconate, sucrose, and raffinose and by a reduced rate of pore formation. These results indicate that the main effect of the mutations was to decrease the toxin's ability to form pores. They provide further evidence that α-helix 4 plays a crucial role in the mechanism of pore formation.

Documentos Relacionados

• Differential Effects of pH on the Pore-Forming Properties of Bacillus thuringiensis Insecticidal Crystal Toxins
• Analysis of Mutations in the Pore-Forming Region Essential for Insecticidal Activity of a Bacillus thuringiensis δ-Endotoxin
• Sensitivity of Polarized Epithelial Cells to the Pore-Forming Toxin Aerolysin
• Pore-forming peptide of pathogenic Entamoeba histolytica.
• Luminal loop of the ryanodine receptor: A pore-forming segment?