Bacillus thuringiensis subsp. israelensis produces crystal proteins, Cry (4Aa, 4Ba, 10Aa, and 11Aa) and Cyt (1Aa and 2Ba) proteins, toxic to mosquito vectors of human diseases. Cyt1Aa overcomes insect resistance to Cry11Aa and Cry4 toxins and synergizes the toxicity of these toxins. However, the molecular mechanism of synergism remains unsolved. Here, we provide evidence that Cyt1Aa functions as a receptor of Cry11Aa. Sequential-binding analysis of Cyt1Aa and Cry11Aa revealed that Cyt1Aa binding to Aedes aegypti brush border membrane vesicles enhanced the binding of biotinylated-Cry11Aa. The Cyt1Aa-and Cry11Aa-binding epitopes were mapped by means of the yeast two-hybrid system, peptide arrays, and heterologous competition assays with synthetic peptides. Two exposed regions in Cyt1Aa, loop ␤6-␣E and part of ␤7, bind Cry11Aa. On the other side, Cry11Aa binds Cyt1Aa proteins by means of domain II-loop ␣8 and ␤-4, which are also involved in midgut receptor interaction. Characterization of single-point mutations in Cry11Aa and Cyt1Aa revealed key Cry11Aa (S259 and E266) and Cyt1Aa (K198, E204 and K225) residues involved in the interaction of both proteins and in synergism. Additionally, a Cyt1Aa loop ␤6-␣E mutant (K198A) with enhanced synergism to Cry11Aa was isolated. Data provided here strongly indicates that Cyt1Aa synergizes or suppresses resistance to Cry11Aa toxin by functioning as a membrane-bound receptor. Bacillus thuringiensis subsp. israelensis is a highly effective pathogenic bacterium because it produces a toxin and also its functional receptor, promoting toxin binding to the target membrane and causing toxicity.receptor interaction ͉ binding epitopes ͉ insect resistance ͉ mode of action

A 65 kDa GPI (glycosylphosphatidyl-inositol)-anchored ALP (alkaline phosphatase) was characterized as a functional receptor of the Bacillus thuringiensis subsp. israelensis Cry11Aa toxin in Aedes aegypti midgut cells. Two (a 100 kDa and a 65 kDa) GPI-anchored proteins that bound Cry11Aa toxin were preferentially extracted after treatment of BBMV (brush boder membrane vesicles) from Ae. aegypti midgut epithelia with phospholipase C. The 65 kDa protein was further purified by toxin affinity chromatography. The 65 kDa protein showed ALP activity. The peptide-displaying phages (P1.BBMV and P8.BBMV) that bound to the 65 kDa GPI-ALP (GPI-anchored ALP) and competed with the Cry11Aa toxin to bind to BBMV were isolated by selecting BBMV-binding peptide-phages by biopanning. GPI-ALP was shown to be preferentially distributed in Ae. aegypti in the posterior part of the midgut and in the caeca, by using P1.BBMV binding to fixed midgut tissue sections to determine the location of GPI-ALP. Cry11Aa binds to the same regions of the midgut and competed with P1.BBMV and P8.BBMV to bind to BBMV. The importance of this interaction was demonstrated by the in vivo attenuation of Cry11Aa toxicity in the presence of these phages. Our results shows that GPI-ALP is an important receptor molecule involved in Cry11Aa interaction with midgut cells and toxicity to Ae. aegypti larvae.

Cry11Aa of Bacillus thuringiensis subsp. israelensis is the most active toxin to Aedes aegypti in this strain. We previously reported that, in addition to a 65 kDa GPI (glycosylphosphatidylinositol)-anchored ALP (alkaline phosphatase), the toxin also binds a 250 kDa membrane protein. Since this protein is the same size as cadherin, which in lepidopteran insects is an important Cry toxin receptor, we developed an anti-AaeCad antibody. This antibody detects a 250 kDa protein in immunoblots of larval BBMVs (brush border membrane vesicles). The antibody inhibits Cry11Aa toxin binding to BBMVs and immunolocalizes the cadherin protein to apical membranes of distal and proximal caecae and posterior midgut epithelial cells. This localization is consistent with areas to which Cry11Aa toxin binds and causes pathogenicity. Therefore, the full-length Aedes cadherin cDNA was isolated from Aedes larvae and partial overlapping fragments that covered the entire protein were expressed in Escherichia coli. Using toxin overlay assays, we showed that one cadherin fragment, which contains CR7–11 (cadherin repeats 7–11), bound Cry11Aa and this binding was primarily through toxin domain II loops α8 and 2. Cadherin repeats CR8–11 but not CR7 bound Cry11Aa under non-denaturing conditions. Cry11Aa bound the cadherin fragment with high affinity with an apparent Kd of 16.7 nM. Finally we showed that this Cry11Aa-binding site could also be competed by Cry11Ba and Cry4Aa but not Cry4Ba. These results indicate that Aedes cadherin is possibly a receptor for Cry11A and, together with its ability to bind an ALP, suggest a similar mechanism of toxin action as previously proposed for lepidopteran insects.

Bacillus thuringiensis subs israelensis produces Cry toxins active against mosquitoes. Receptor binding is a key determinant for specificity of Cry toxins composed of three domains. We found that exposed loop a-8 of Cry11Aa toxin, located in domain II, is an important epitope involved in receptor interaction. Synthetic peptides corresponding to exposed regions in domain II (loop a-8, b-4 and loop 3) competed binding of Cry11Aa to membrane vesicles from Aedes aegypti midgut microvilli. The role of loop a-8 of Cry11A in receptor interaction was demonstrated by phage display and site-directed mutagenesis. We isolated a peptide-displaying phage (P5.tox), that recognizes loop a-8 in Cry11Aa, interferes interaction with the midgut receptor and attenuates toxicity in bioassay. Loop a-8 mutants affected in toxicity and receptor binding were characterized.

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Cry11Aa is the most active Bacillus thuringiensis israelensis toxin against Aedes aegypti larvae. Ae. aegypti alkaline phosphatase (ALP) was previously identified as a Cry11Aa receptor mediating toxicity. Here we report the cloning and functional characterization of this Ae. aegypti Cry11Aa-ALP receptor. Of three ALP’s cDNA clones, the recombinant produced ALP1 isoform was shown to bind Cry11Aa and P1.BBMV peptide phage that specifically binds the midgut ALP-Cry11Aa receptor. An anti-ALP1 antibody inhibited binding to brush border membrane vesicles and toxicity of Cry11Aa in isolated cultured guts. Two ALP1 Cry11Aa binding regions (R59–G102 and N257–I296) were mapped by characterizing binding of Cry11Aa to nine recombinant overlapping peptides covering the ALP1 sequence. Finally, by using a peptide spot array of Cry11Aa domain III and site-directed mutagenesis, we show that the ALP1 R59–G102 region binds Cry11Aa through domain II loop α-8 while ALP1 N257–I296 interacts with Cry11Aa through domain III 561RVQSQNSGNN570 located in β18-β19. Our results show that Cry11Aa domain II and domain III are involved in the binding with two distinct binding sites in the ALP1 receptor.

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Phage display is an in vitro method for selecting polypeptides with desired properties from a large collections of variants. The insecticidal Cry toxins produced by Bacillus thuringiensis are highly specific to different insects. Various proteins such as cadherin, aminopeptidase-N (APN) and alkaline phosphatase (ALP) have been characterized as potential Cry-receptors. We used phage display to characterize the Cry toxin-receptor interaction(s). By employing phage-libraries that display singlechain antibodies (scFv) from humans or from immunized rabbits with Cry1Ab toxin or random 12-residues peptides, we have identified the epitopes that mediate binding of lepidopteran Cry1Ab toxin with cadherin and APN receptors from Manduca sexta and the interaction of dipteran Cry11Aa toxin with the ALP receptor from Aedes aegypti. Finally we displayed in phages the Cry1Ac toxin and discuss the potential for selecting Cry variants with improved toxicity or different specificity.