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 alpha8 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.

Figure 1. The Cry11Aa toxin binding is inhibited by an anti-AaeCad antibody and the cadherin protein is detected in Aedes BBMV

(A) Cry11Aa (5 nM) binding to BBMVs (10 μg) is competed with varying dilutions of anti-AaeCad antibody but not by an anti-NHE3 antibody that also detects a membrane-bound protein in Aedes midgut [55]. (B) Western blot of BBMV proteins (10 μg) analysed with the anti-AaeCad antibody diluted 1:7500.

Figure 2. Immunolocalization of cadherin and Cry11A toxin binding sites in larval gut of 4^th instar Ae. aegypti larvae

Whole larvae (C–H) and gut sections (A and B) were incubated with affinity purified anti-AaeCad antibody diluted 1:100. Whole larvae sections (I–L) were incubated with Cry11Aa (100 nM) and then with anti-Cry11A antibody diluted 1:1000. Cy3-linked secondary antibody (1:1000, red) was used to determine cadherin localization or Cry11A toxin-binding sites. The cell and tissue structures were visualized by phalloidin (Alexa Fluor^® 488, 1:100, green). Red immunofluorescence shows cadherin localization on the apical side of the posterior midgut (A, C and D) but not in the apical membranes of anterior midgut (B and E) and hindgut (A) epithelial cells. Cadherin was also observed on the apical side of distal (F) and proximal (G) gastric caecae. No specific signal was observed in posterior midgut cells when tissues were probed with preimmune serum as a negative control (H). Cry11A toxin bound the apical membrane of epithelial cells in gastric caeca (J) and posterior midgut (K and L). No immunofluorescence was found in the apical membrane of anterior midgut (I). Scale bars, 50 μm (A, B, I–L); 100 μm (C–H). AMG, anterior midgut; DGC, distal gastric caecae; GC, gastric caecae; HG, hindgut; PGC, proximal gastric caecae; PMG, posterior midgut.

Figure 3. The amino acid sequence and structure features of the full-length Aedes cadherin protein

(A) Five overlapping cadherin cDNA fragments, G31, G7, C13, G10 and C3 were amplified from an Aedes midgut cDNA library or whole Aedes cDNA samples and these fragments were assembled by five sequential steps of subcloning. This Aedes cDNA contains a complete ORF, which encodes a full-length Aedes cadherin protein. The protein contains a signal peptide (SIG), 11 cadherin repeats (CR1–11), a membrane-proximal region (MPR), a transmembrane domain (TM) and a cytoplasmic domain (CD). Toxin-binding regions were localized in CR8–11. (B) The putative N-terminal signal peptide is underlined with dots and the transmembrane domain is double underlined. The 11 cadherin repeat sequences are in bold. Putative calcium-binding sites are underlined and the integrin recognition sequence RGD (aa 1098–1101) is in italics and underlined. The ATP/GTP-binding site is dash-underlined and the predicted N-glycosylated residues (Asn) are labelled with an asterisk above the letter N.

Figure 4. In toxin overlay assays and ELISA, the Cry11Aa toxin binds cadherin fragments and cadherin repeats

(A) Partial cDNA fragments, G7, C13 and G10 (90 pmol each) were separated by SDS/PAGE (8 % gel) and stained using Coomassie Blue (lanes 1–3). These fragments were electotransferred to a PVDF membrane, which was incubated with 20 nM Cry11Aa toxin. Unbound toxin was removed by washing, the membrane was incubated with anti-Cry11Aa antiserum (1:2000) and then visualized by luminol (lanes 5–7). (B) The purified cadherin repeats CR7–11 (30 pmol each) were resolved by SDS/PAGE (15 % gel) and stained using Coomassie Blue (lanes 1–5). (C) The cadherin repeats, CR8 (○), CR9 (■), CR10 (□) and CR11 (▲) show dose-dependent binding to immobilized Cry11Aa (0.4 μg), but CR7 (●) does not bind Cry11Aa. OD₄₀₅, A ₄₀₅.

Figure 5. Cry11Aa toxin binds the G10 cadherin fragment with high affinity

(A) The cadherin fragment G10 shows dose-dependent binding (insert, ●) to immobilized Cry11Aa (0.4 μg), but a cadherin fragment from the Heliothis virescens cadherin [24] (insert, ■) does not bind Cry11Aa. Bound G10 was determined with an anti-AaeCad antiserum followed by incubation with secondary antibody and colour detection. The binding affinity, 16.7 nM, of Cry11Aa toxin to the cadherin fragment was determined in a competition assay using 80 nM G10 with increasing concentrations of Cry11Aa toxin (0–1000 nM). OD₄₀₅, A ₄₀₅. (B) The Cry11Aa loop α8 mutant V262A (○) retains its ability to bind the G10 fragment and competes with Cry11Aa binding, whereas the mutant E266A (▲) does not bind the Aedes cadherin fragment. Wild-type Cry11Aa binding is shown as ■ in (A, main graph) and (B). Maximal binding was normalized to the maximal absorbance obtained in the absence of G10. Error bars indicate standard deviation obtained using three separate experiments.

Figure 6. Mosquitocidal Cry11Ba and Cry4Aa toxins, but not Cry4Ba, compete with the Cry11Aa-binding site on the Aedes cadherin fragment

Cry11Ba (●) and Cry4Aa (△) show displacement of Cry11Aa (■) binding to G10, whereas Cry4Ba (□) does not compete. Binding assays were performed as described in the legend to Figure 5 and in the Materials and methods section. Error bars indicate standard deviation obtained using three separate experiments.