Aedes aegypti cadherin serves as a putative receptor of the Cry11Aa toxin from Bacillus thuringiensis subsp. israelensis

Abstract

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.

Aedes cadherin mediates the in vivo toxicity of the Cry11Aa toxin to Aedes aegypti

“…A full-length Aedes cadherin cDNA (AaeCad) cloned into pCR2.1 vector was previously reported [8]. To remove the 5′ and 3′ UTRs, partial AaeCad fragments (5EM and 3EM) were prepared with a set of primers (Supplementary Table S1).…”

“…The 100 kDa protein was identified as an APN and two of these have been characterized [9, 10]. In a previous study, we showed that the Aedes cadherin (AAEL007478 and AAEL007488), which is homologous to the lepidopteran Bt-R 1 and mediates Cry1A toxicity in Lepidoptera, bound Cry11Aa with high affinity [8]. This finding suggests that the cadherin is associated with the insecticidal activity of the Cry11Aa toxin.…”

Alkaline phosphatases and aminopeptidases are altered in a Cry11Aa resistant strain of Aedes aegypti

“…Previously identified receptor proteins of Cry11Aa in Aedes are cadherin (AAEL018140), APN1 (AAEL012778), APN2 (AAEL08155), and ALP1 (AAEL009077) (Chen et al, 2009a; Chen et al, 2009b; Chen et al, 2013; Fernandez et al, 2009). These receptors were further analyzed to determine if there were any mutations or if receptor expression was altered.…”

“…Antibodies to the cadherin peptide ( Aedes cadherin-specific antibody), and ALP and APN proteins were previously reported (Chen et al, 2009a; Chen et al, 2009b; Fernandez et al, 2009). The cadherin peptide antibody is specific for Aedes cadherin, but the ALP (AAEL009077) antibody cross reacts with a number of other ALPs (Jiménez et al, 2012).…”

“…Cry11Aa binds a number of BBMV proteins (Fernandez et al, 2006). Among them are the Aedes cadherin (AAEL018140) and two aminopeptidases N (APNs) (AAEL012778 and AAEL008155), all binding Cry11Aa with high affinity (Chen et al, 2009a; Chen et al, 2009b; Chen et al, 2013). These proteins are also present in the posterior midgut epithelial cells and/or the gastric caeca (Chen et al, 2009b; Chen et al, 2013).…”