Enhancing Bacillus thuringiensis Cry8Ea1 toxicity: Insights into protease sensitivity for the evolutionary adaptation...

Toxicity of Bacillus thuringiensis Cry protoxins relies on activation by larval midgut proteases, but overprocessing can reduce toxicity in various insects. Cry8Ea1 is effective against Coleopteran pests but shows limited toxicity towards Holotrichia parallela due to instability in its midgut proteases. Treatment of Cry8Ea1 protoxin with trypsin or midgut juice-proteases, produces a 55.6 kDa fragment by cleaving between residues V163 and Y655, removing the first three α-helices from domain I. To prevent protease degradation, the trypsin cleavage site was identified and mutated (Cry8Ea1R163H). Cry8Ea1R163H mutant resulted in a 67.2 kDa activated toxin after treatment with trypsin or midgut juice-proteases, and showed a correlative 8.5-fold increase insecticidal activity against H. parallela larvae. We analyzed the activation of other Cry8 proteins with trypsin or midgut juice-proteases from H. parallela, our data showed that Cry8Fa1 was activated into a 67.2 kDa protein, in contrast to Cry8Ha1, Cry8Ca1 and Cry8Ga2 that were activated into 55.6 kDa protein fragments. Finally, the structure of the trypsin cleavage site in all Cry8 protein members was predicted, revealing that in Cry8Fa, Cry8Q and Cry8I, the trypsin cleavage site is buried. Phylogenetic analyses suggest an adaptive evolution of certain Cry8 proteins against the host digesting enzymes.