Proteomic and biochemical characterization of Bacillus thuringiensis toxin Cry4Ba interaction with midgut proteins from Aedes (Stegomyia) aegypti larvae
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Cry4Ba toxin derived from Bacillus thuringiensis israelensis (Bti) is highly toxic to larval stages of the yellow fever mosquito Aedes aegypti, a vector for disease causing pathogens, including dengue, chikungunya and yellow fever. Cry4Ba toxin kills larvae first by binding to gut membrane surface receptors, but these receptor identities in the larval gut are mostly uncharacterized. The overall objective of this research was to investigate Cry4Ba toxin interaction with midgut brush border membrane vesicles (BBMV) and lipid rafts of Aedes larvae. BBMV were prepared from whole larvae, and the proteins were separated by two-dimensional electrophoresis followed by ligand blot analysis with the Cry4Ba toxin. Mass spectrometry analysis of Cry4Ba toxin bound proteins resulted in the identification of three alkaline phosphatase (ALP) isoforms, an aminopeptidase, lipid raft proteins (flotillin and prohibitin), VATPase B subunit, and actin. The identified ALPs were further validated by immunoblotting with ALP antibodies from Anopheles larvae. Multiple sequence alignment revealed that A. aegypti flotillin-1 (Aeflot-1) has high sequence similarity to other insect flotillin-1s and Aeflot-1 antibody can detect other insect flotillin-1s. Immunolocalization of Aeflot-1 was detected on the apical microvilli of larval posterior midgut and gastric caeca. Furthermore, lipid rafts from Aedes BBMV were enriched in the Aeflot-1 and cholesterol. These results indicate that specialized plasma membrane domains are present in A. aegypti, and Aeflot-1 antibody can be used as an important tool to characterize membrane lipid rafts. Previous research suggests that lipid rafts prepared from insect gut BBMV have an important role in Bt toxicity. We isolated and analyzed the protein components of Aedes larval BBMV lipid rafts that interact with Cry4Ba toxin. Lipid raft marker proteins, such as flotillin-1 and APN-1, were selectively associated with lipid rafts in the low density fraction. Also, upon Cry4Ba toxin incubation with BBMV, the toxin was enriched in the lipid raft fraction. Using LCMS/ MS we identified a total of 312 proteins including both known lipid raft markers and novel proteins implicated as Cry toxin putative receptors. We also identified a large number of other proteins that have substantial interest across numerous areas of research in mosquito biology and control.