Charged residue screening in helix 4 of the Bacillus thuringiensis Cry4B toxin reveals one critical residue for larvicidal activity

Abstract

The different Cry δ-endotoxins produced by Bacillus thuringiensis have been shown to kill susceptible insect larvae by forming a lytic pore in the target midgut epithelial cell membrane. We have previously employed single proline substitutions via PCR-based mutagenesis and demonstrated that helices 4 and 5 in the pore-forming domain of the 130-kDa Cry4B toxin are essential for mosquito-larvicidal activity against Aedes aegypti. To further identify critical residues for toxicity, substitutions with alanine of each of the charged amino acids (Arg-143, Lys-156, Arg-158 and Glu-159) and one polar residue (Asn-151) in the transmembrane helix 4 were performed. Similar to the wild-type Cry4B protoxin, all five mutant toxins were over-expressed as cytoplasmic inclusions in Escherichia coli and were structurally stable upon solubilisation and trypsin activation in carbonate buffer, pH 9.0. Interestingly, a complete loss of activity against A. aegypti larvae was observed for the alanine substitution at Arg-158, while replacements at the four other positions did not affect the toxicity. The results reveal a crucial role in toxin function for the positively charged side chain of Arg-158 in helix 4 of the Cry4B toxin.