Aedes aegypti aminopeptidase N3 is a functional binding receptor of Bacillus thuringiensis subsp. israelensis Cry4Ba toxin

Abstract

Bacillus thuringiensis is widely employed for biological control. It can effectively suppress populations of various mosquito species, including Aedes aegypti. However, the precise mechanism underlying the action of cry toxin secreted by Bacillus thuringiensis on Ae. aegypti remains elusive. In this study, we investigated one of the binding receptors of cry toxin, aminopeptidase N. Through comprehensive bioinformatics analysis involving whole-genome screening, genetic mapping, structural characterization, phylogenetic analysis, and spatiotemporal expression profiling, we identified twenty-nine homologs of Ae. aegypti aminopeptidase N. Further, we successfully expressed GST-APN3 protein in E. coli and demonstrated through ligand blot and ELISA assays that APN3 exhibits high affinity binding to Cry4Ba toxin (Kd = 20.53 nM). To elucidate the functional role of APN3 as a receptor mediating Cry4Ba activity in Ae. aegypti midgut cells (some of which express this gene at high levels), CRISPR/Cas9 technology was employed to knock out APN3. Our bioassay results revealed that APN3 knockout mosquito larvae had 2.9 to 4.1-fold higher resistance against Cry4Ba, indicating its crucial involvement as an active receptor mediating Cry4Ba activity. Overall, this study provides a foundation for elucidating the specific larvicidal mechanisms of Bt against mosquito populations.