The Bacillus thuringiensis Cry4Ba toxin weakens the protective function of the midgut peritrophic membrane in Aedes aegypti larvae

Background Bacillus thuringiensis (Bt) toxin Cry4Ba specifically targets the mosquito larval midgut epithelial cells for its insecticidal toxicity. Prior to reaching the midgut cells, the toxin must penetrate the midgut peritrophic membrane (PM), an acellular gut protective structure lining the midgut epithelium. However, the mechanism of toxin passage through the PM is currently unknown. To examine the correlation between the permeability of Ae. aegypti larval PM and larval toxicity of the Cry4Ba toxin, larvae were fed with Cry4Ba and an inactive mutant, respectively. The toxin was subsequently localized in the larvae to examine the passage of Cry4Ba through the PM. Results In Cry4Ba fed larvae, Cry4Ba was localized in the PM, within the gastric caeca, and in the midgut epithelial cell surface at 10 min after ingestion of the toxin. Furthermore, in vivo PM permeability assays using FITC-dextran (2000 KDa) demonstrated that the permeability of the PM in Cry4Ba fed larvae was significantly increased. However, in larvae fed with the inactive Cry4Ba mutant, the toxin was restricted within the endoperitrophic space of the larval gut, indicating that alanine substitution at arginine 158 (Arg 158 ) in domain I resulted in failure of the toxin to pass through the PM. Conclusion We therefore conclude that the PM became more permeable upon larval exposure to Cry4Ba, thus weakening its protective function. The amino acid residue Arg 158 in Cry4Ba may play an important role in its ability to penetrate the mosquito larval PM.