Knockout of aminopeptidase N3 confers resistance to low concentrations of Bacillus thuringiensis Cry4Ba protoxin in Aedes aegypti

Abstract Background 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 protein produced by Bacillus thuringiensis on Ae. aegypti remains elusive. On the basis of our previous research findings, five Aedes aminopeptidase N proteins (AeAPNs) were identified from the brush border membrane vesicles (BBMV) of Ae. aegypti that could bind to Cry4Ba or Cry11Aa. Further analysis confirmed that AeAPN1 and AeAPN2 are not functional receptors for these proteins. In this study, we investigated an additional aminopeptidase N (AeAPN3, AAEL012774) as a potential binding receptor for Cry proteins. Methods Comprehensive bioinformatics analysis involving whole-genome screening, genetic mapping, structural characterization, phylogenetic analysis, and spatiotemporal expression profiling were used to identify Ae. aegypti aminopeptidase N homologs. Ligand blotting and enzyme-linked immunosorbent assay (ELISA) were used to measure binding affinity to Cry4Ba. To elucidate its functional role as a potential receptor mediating Cry4Ba activity in Ae. aegypti midgut cells, Ae APN3 was knocked out with CRISPR/Cas9 technology. Results A total of 29 homologs of Ae. aegypti aminopeptidase N were identified in this study. Then, we expressed GST–APN3 fusion protein in E. coli and found that it had high-affinity binding to Cry4Ba protein (K d = 20.53 nM). Mosquito larvae had approximately threefold higher resistance against Cry4Ba after AeAPN3 knockout, indicating its significant involvement as an active receptor mediating Cry4Ba activity. Conclusions Overall, this study provides a foundation for elucidating the specific larvicidal mechanisms of Bacillus thuringiensis (Bt) against mosquito populations. Graphical Abstract