Understanding the basis of specificity of Bacillus thuringiensis Cry2A toxins towards Aedes aegypti

Bacillus thuringiensis is a gram-positive spore forming soil bacterium and one of the most successful, environmentally friendly, intensively used and studied microbial insecticides. The major characteristic of Bt is the production of proteinaceous crystals containing toxins with specific activity against many insects including diptera, lepidoptera and coleoptera. Understanding the basis of specificity of Cry2A toxins of Bacillus thuringiensis is important for the risk assessment of novel insecticidal toxins from this bacterium to ensure that they are not detrimental to non-target organisms within the environment. Cry2A toxins are a group of three-domain proteins with highly similar sequences, and this project sought to understand the basis of the specificity of Cry2A toxins against the mosquito Aedes aegypti. This was investigated through finding out which domain(s) and /or amino acid motif(s) were crucial for activity. Cry2A toxins in our lab were characterised and expressed, after which bioassays were conducted against Aedes aegypti, and several hybrid toxins and mutants were created based on the bioassay results and were used to determine the relationship between amino acid sequence and toxicity through bioassay and bioinformatic analyses. Domain I was found to be responsible for the specificity of Cry2A toxins against Aedes aegypti, specifically the 49-amino acids comprising the N-terminal region, which folds back onto domain II. The specificity-determining region was further found to consist of four amino acids (E/RTD) within this N-terminal region. Finally, the mechanism of proteolytic activation of Cry2A by Aedes aegypti was studied in vitro, leading to a proposed model of proteolytic activation, which was contrary to previously published reports.