Cytotoxicity of the Bacillus thuringiensis Cry4B toxin is mediated by the cadherin receptor BT-R3 of Anopheles gambiae

We demonstrate for the first time the selective cytotoxicity of Bacillus thuringiensis subsp. israelensis Cry4B toxin mediated by BT-R-3 using a cell-based system, which employs High Five insect cells stably expressing BT-R-3. Discovery and validation of BT-R-3 as the Cry4B receptor was accomplished using a web-based computational pipeline platform that facilitates high-throughput insecticidal target identification utilizing the Anopheles gambiae genome. Once the Cry4B toxin binds to the BT-R-3 receptor, a cell death pathway is manifested by sequential cytological changes that include membrane blebbing, cell swelling and lysis. Cry4B toxin associates with cell membrane in both oligomeric and monomeric forms. Monomeric toxin binds specifically to BT-R-3 whereas oligomer interacts with cell membrane non-specifically. Cytotoxicity and cell death are the direct result of binding of toxin monomer to BT-R-3. The oligomeric form of Cry4B toxin is not involved in cell death. Both the location of the toxin-binding region within BT-R-3 and its structural motif are critical to the binding affinity and specificity of the toxin. The toxin-binding region of BT-R-3 appears to be located in EC11, the most membrane proximal EC module within the extracellular domain. It is characterized by the presence of two highly conserved amino acid sequences within their N- and C-termini that flank EC11. These sequences represent signature motifs that mark the toxin-binding function in BT-R-3. The two sequences form two adjacent beta-strands within the beta-barrel of EC11, the positioning of which is a hallmark of all Cry toxin receptors thus far reported.