Cry4Aa and Cry4Ba Mosquito-Active Toxins Utilize Different Domains in Binding to a Particular Culex ALP Isoform: A Functional Toxin Receptor Implicating Differential Actions on Target Larvae.

The three-domain Cry4Aa toxin produced from Bacillus thuringiensis subsp. israelensis was previously shown to be much more toxic to Culex mosquito larvae than its closely related toxin-Cry4Ba. The interaction of these two individual toxins with target receptors on susceptible larval midgut cells is likely to be the critical determinant in their differential toxicity. Here, two full-length membrane-bound alkaline phosphatase (mALP) isoforms from Culex quinquefasciatus larvae, Cq- mALP1263and Cq- mALP1264, predicted to be GPI-linked was cloned and functionally expressed in Spodoptera frugiperda ( Sf 9) cells as 57- and 61-kDa membrane-bound proteins, respectively. Bioinformatics analysis disclosed that both Cq- mALP isoforms share significant sequence similarity to Aedes aegypti -mALP-a Cry4Ba toxin receptor. In cytotoxicity assays, Sf 9 cells expressing Cq- mALP1264, but not Cq- mALP1263, showed remarkably greater susceptibility to Cry4Aa than Cry4Ba, while immunolocalization studies revealed that both toxins were capable of binding to each Cq- mALP expressed on the cell membrane surface. Molecular docking of the Cq -mALP1264-modeled structure with individual Cry4 toxins revealed that Cry4Aa could bind to Cq -mALP1264 primarily through particular residues on three surface-exposed loops in the receptor-binding domain-DII, including Thr512 , Tyr513 and Lys514 in the β10-β11loop. Dissimilarly, Cry4Ba appeared to utilize only certain residues in its C-terminal domain-DIII to interact with such a Culex counterpart receptor. Ala-substitutions of selected β10-β11loop residues (T512A, Y513A and K514A) revealed that only the K514A mutant displayed a drastic decrease in biotoxicity against C. quinquefasciatus larvae. Further substitution of Lys514 with Asp (K514D) revealed a further decrease in larval toxicity. Furthermore, in silico calculation of the binding affinity change (ΔΔGbind ) in Cry4Aa- Cq -mALP1264 interactions upon these single-substitutions revealed that the K514D mutation displayed the largest ΔΔGbind value as compared to three other mutations, signifying an adverse impact of a negative charge at this critical receptor-binding position. Altogether, our present study has disclosed that these two related-Cry4 mosquito-active toxins conceivably exploited different domains in functional binding to the same Culex membrane-bound ALP isoform- Cq- mALP1264 for mediating differential toxicity against Culex target larvae.