Amino Acid Substitutions in the C-Terminal Domains of Vip3Aa Enhance Insecticidal Toxicity

Abstract

Vip3 proteins secreted by the entomopathogenic bacterium Bacillus thuringiensis (Bt) have an important role in biological control against economically important lepidopteran pests. The elucidation of Vip3 protein structures has helped to address the roles of domains and amino acid positions involved in toxicity, especially in the N-terminal domains I and II, thereby supporting their more efficient utilization. In this study, we evaluated the impact of combinations of critical amino acid substitutions, selected from previous studies, in domains IV and V of the Vip3Aa90 protein on its insecticidal activity against three lepidopteran pests. The double mutant S543N/I544L, triple mutants S543N/I544L/E627A and S543N/I544L/S686R, and quadruple mutant S543N/I544L/E627A/S686R were constructed in Escherichia coli by site-directed mutagenesis. Among these, only the Vip3Aa mutant proteins S543N/I544L/E627A and S543N/I544L/E627A/S686R could be expressed and purified for bioassays. Both mutant proteins had similar toxicity against Spodoptera littoralis, showing higher insecticidal activity than the wild-type (WT) Vip3Aa90 at the LC90 level. However, at the LC50 level, only a slight improvement in toxicity was observed for the quadruple mutant. In the case of S. exigua, no significant difference in toxicity was observed for either of the two mutant proteins with respect to the WT at either LC level. Interestingly, for G. molesta, though the toxicity of the triple mutant did not differ significantly compared to that of the WT protein, that of the quadruple mutant showed a marked decrease in toxicity of over 10-fold. This study revealed that combining selected amino acid substitutions in domains IV and V can enhance Vip3Aa90 toxicity against some lepidopteran species but can be either neutral or even deleterious in others.