Article

Mutations in domain I interhelical loops affect the rate of pore formation by the Bacillus thuringiensis Cry1Aa toxin in insect midgut brush border membrane vesicles.

Université de Montréal, Quebec, Canada.
Applied and environmental microbiology (impact factor: 3.69). 05/2009; 75(12):3842-50. DOI:10.1128/AEM.02924-08 pp.3842-50
Source: PubMed

ABSTRACT Pore formation in the apical membrane of the midgut epithelial cells of susceptible insects constitutes a key step in the mode of action of Bacillus thuringiensis insecticidal toxins. In order to study the mechanism of toxin insertion into the membrane, at least one residue in each of the pore-forming-domain (domain I) interhelical loops of Cry1Aa was replaced individually by cysteine, an amino acid which is normally absent from the activated Cry1Aa toxin, using site-directed mutagenesis. The toxicity of most mutants to Manduca sexta neonate larvae was comparable to that of Cry1Aa. The ability of each of the activated mutant toxins to permeabilize M. sexta midgut brush border membrane vesicles was examined with an osmotic swelling assay. Following a 1-h preincubation, all mutants except the V150C mutant were able to form pores at pH 7.5, although the W182C mutant had a weaker activity than the other toxins. Increasing the pH to 10.5, a procedure which introduces a negative charge on the thiol group of the cysteine residues, caused a significant reduction in the pore-forming abilities of most mutants without affecting those of Cry1Aa or the I88C, T122C, Y153C, or S252C mutant. The rate of pore formation was significantly lower for the F50C, Q151C, Y153C, W182C, and S252C mutants than for Cry1Aa at pH 7.5. At the higher pH, all mutants formed pores significantly more slowly than Cry1Aa, except the I88C mutant, which formed pores significantly faster, and the T122C mutant. These results indicate that domain I interhelical loop residues play an important role in the conformational changes leading to toxin insertion and pore formation.

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Keywords

activated Cry1Aa toxin
 
activated mutant toxins
 
amino acid
 
apical membrane
 
Bacillus thuringiensis insecticidal toxins
 
conformational changes
 
cysteine residues
 
form pores
 
formed pores
 
key step
 
Manduca sexta neonate larvae
 
midgut epithelial cells
 
permeabilize M. sexta midgut brush border membrane vesicles
 
Pore formation
 
pore-forming abilities
 
pore-forming-domain
 
S252C mutants
 
site-directed mutagenesis
 
toxin insertion
 
weaker activity