Cry11Aa Interacts with the ATP-Binding Protein from Culex quinquefasciatus To Improve the Toxicity

Abstract

Cry11Aa displays high toxicity to the larvae of several mosquito species, including Aedes, Culex, and Anopheles. To study its binding characterization against Culex quinquefasciatus, Cry11Aa was purified and western blot results showed that Cry11Aa could bind successfully to the brush border membrane vesicles. To identify Cry11Aa-binding proteins in C. quinquefasciatus, a biotin-based protein pull-down experiment was performed and seven Cry11Aa-binding proteins were isolated from the midgut of C. quinquefasciatus larvae. Analysis of liquid chromatography-tandem mass spectrometry showed that one of the Cry11Aa-binding proteins is the ATP-binding domain 1 family member B. To investigate its binding property and effect on the toxicity of Cry11Aa, western blot, far-western blot, enzyme-linked immunosorbent assay, and bioassays of Cry11Aa in the presence and absence of the recombinant ATP-binding protein were performed. Our results showed that the ATP-binding protein interacted with Cry11Aa and increased the toxicity of Cry11Aa against C. quinquefasciatus. Our study suggests that midgut proteins other than the toxin receptors may modulate the toxicity of Cry toxins against mosquitoes.

Toxic Activity, Molecular Modeling and Docking Simulations of Bacillus thuringiensis Cry11 Toxin Variants Obtained via DNA Shuffling

“…In A. aegypti , this toxin interacts with two midgut brush border membrane receptors; a GPI anchored and alkaline phosphatase (ALP1) ( Fernandez et al, 2006 ) and also binds to Cyt1Aa as a kind of membrane-bound receptor of Cry11Aa increasing the toxic activity ( Perez et al, 2005 ). Other midgut proteins different to the receptor alkaline phosphatase (ALP1) such as ATP binding protein, increases the toxicity of Cry11Aa against C. quinquefasciatus ( Zhang L. et al, 2017 ). Other two toxins, Cry11Bb (94 kDa) and Cry11Ba (81 kDa), share a similar insect specificity and are phylogenetically related to Cry11Aa.…”

C-Type Lectin-20 Interacts with ALP1 Receptor to Reduce Cry Toxicity in Aedes aegypti

“…The use of Cry toxins against insect pests depends upon the interaction between Cry toxins and putative receptors, such as alkaline phosphatase (ALP), aminopeptidase-N (APN), and ATP-binding cassette (ABC) transporters [ 12 , 16 , 17 , 18 , 19 ]. However, if some third-party proteins could interfere with the interaction between Cry toxins and receptors, toxicity of Cry would be altered [ 37 ]. In order to test whether CTL-20 binds to Cry and/or putative receptors, recombinant CTL-20 and ALP1, as well as Cry11Aa protoxin, were expressed and purified in this study ( Figure 2 (A-a),(B-a), and Figure 3 a).…”

“…Among these toxins, Cry11Aa was the most promising for mosquitocidal activity [ 39 ]. The interaction of Cry11Aa with important toxin receptors such as ALP in the midgut cells causes toxicity to A. aegypti larvae [ 17 ], as interaction of active toxins with their membrane receptors is the key step for the molecular mechanism of pore formation [ 37 ]. Toxicity of Bt will thus be greatly altered if the key interaction between toxins and their receptors is diminished or even eliminated.…”

“…The membrane was blocked with PBSM (PBS + 0.05% skim milk) for 2 h at 37 °C incubator or overnight at 4 °C, washed with PBST (PBS + 0.05% Tween-20) and incubated with primary rabbit polyclonal antibody (1:3000) to each protein and bind with goat anti-rabbit antibody (1:3000) as secondary antibody. Antibody binding was visualized by a color reaction catalyzed by conjugated alkaline phosphatase as described previously [ 37 ].…”

Toxic Potential ofBacillus thuringiensis: An Overview

“…Although the mechanism of action of Cry toxins against various insects has been widely investigated, there are still many controversies. Therefore, there are currently different models in the literature that seek to explain it [28].…”