Bacillus thuringiensis (Bt) bacteria are insect pathogens that rely on insecticidal pore forming proteins known as Cry and Cyt toxins to kill their insect larval hosts. At least four different non-structurally related families of proteins form the Cry toxin group of toxins. The expression of certain Cry toxins in transgenic crops has contributed to an efficient control of insect pests resulting in a significant reduction in chemical insecticide use. The mode of action of the three domain Cry toxin family involves sequential interaction of these toxins with several insect midgut proteins facilitating the formation of a pre-pore oligomer structure and subsequent membrane insertion that leads to the killing of midgut insect cells by osmotic shock. In this manuscript we review recent progress in understanding the mode of action of this family of proteins in lepidopteran, dipteran and coleopteran insects. Interestingly, similar Cry-binding proteins have been identified in the three insect orders, as cadherin, aminopeptidase-N and alkaline phosphatase suggesting a conserved mode of action. Also, recent data on insect responses to Cry toxin attack is discussed. Finally, we review the different Bt based products, including transgenic crops, that are currently used in agriculture.

Bacillus thuringiensis (Bt) produce inclusions that are composed of proteins known as crystal proteins or Cry toxins. Due to their high specificity and their safety to humans and the environment these Cry toxins are considered valuable alternatives to chemical pesticides in insect control programs. It is believed that Cry toxin-induced membrane pore formation is responsible for insect toxicity. The molecular mechanism of pore formation involves recognition and subsequent binding of the toxin to membrane receptors. This binding is accompanied by toxin oligomerization and transfer of domain I helices of the toxin to the lipid-water interface. This toxin insertion creates pores that lyse the cells. Several receptors from lepidopteran, coleopteran, and dipteran insects have been well characterized. Here we provide an overview of our understanding of the interactions between Cry toxin and multiple receptors in mosquitoes, in particular Aedes aegypti. We review the manner by which the receptors were identified and characterized, with a focus on three proteins – cadherin, alkaline phosphatase and aminopeptidase-N.

The Cry11Aa protein produced in Bacillus thuringiensis subsp. israelensis, a bacterial strain used worldwide for the control of Aedes aegypti larvae, binds midgut brush border membrane vesicles (BBMV) with an apparent Kd of 29.8 nM. Previously an aminopeptidase N (APN), named AaeAPN2, was identified as a putative Cry11Aa toxin binding protein by pull-down assays using biotinylated Cry11Aa toxin (Chen et al., (2009) Insect Biochem Mol Biol., 39: 688–696). Here we show this protein localizes to the apical membrane of epithelial cells in proximal and distal regions of larval caeca. The AaeAPN2 protein binds Cry11Aa with high affinity, 8.6 nM. The full-length and fragments of AaeAPN2 were cloned and expressed in Escherichia coli. The toxin-binding region was identified and further competitive assays demonstrated that Cry11Aa binding to BBMV was efficiently competed by the full-length AaeAPN2 and the fragments of AaeAPN2b and AaeAPN2e. In bioassays against Ae. aegypti larvae, the presence of full-length and a partial fragment (AaeAPN2b) of AaeAPN2 enhanced Cry11Aa larval mortality. Taken together, we conclude that AaeAPN2 is a binding protein and plays a role in Cry11Aa toxicity.

No abstract

Cry11Ba is one of the most toxic proteins to mosquito larvae produced by Bacillus thuringiensis. It binds Aedes aegypti brush border membrane vesicles (BBMV) with high affinity, showing an apparent dissociation constant (K d ) of 8.2 nM. We previously reported that an anticadherin antibody competes with Cry11Ba binding to BBMV, suggesting a possible role of cadherin as a toxin receptor. Here we provide evidence of specific cadherin repeat regions involved in this interaction. Using cadherin fragments as competitors, a C-terminal fragment which contains cadherin repeat 7 (CR7) to CR11 competed with Cry11Ba binding to BBMV. This binding was also efficiently competed by the CR9, CR10, and CR11 peptide fragments. Moreover, we show CR11 to be an important region of interaction with Cry11Ba toxin. An alkaline phosphatase (AaeALP1) and an aminopeptidase-N (AaeAPN1) also competed with Cry11Ba binding to Ae. aegypti BBMV. Finally, we found that Cry11Ba and Cry4Ba share binding sites. Synthetic peptides corresponding to loops ␣8, ␤2-␤3 (loop 1), ␤8-␤9, and ␤10-␤11 (loop 3) of Cry4Ba compete with Cry11Ba binding to BBMV, suggesting Cry11Ba and Cry4Ba have common sites involved in binding Ae. aegypti BBMV. The data suggest that three different Ae. aegypti midgut proteins, i.e., cadherin, AaeALP1, and AaeAPN1, are involved in Cry11Ba binding to Ae. aegypti midgut brush border membranes.

Bacillus thuringiensis subsp. israelensis produces three Cry toxins (Cry4Aa, Cry4Ba and Cry11Aa) that are active against Aedes aegypti larvae. The identification of the rate-limiting binding steps of Cry toxins that are used for insect control in the field, such as those of B. thuringiensis subsp. israelensis, should provide targets for improving insecticides against important insect pests. Previous studies showed that Cry11Aa binds to cadherin receptor fragment CR7–11 (cadherin repeats 7–11) with high affinity. Binding to cadherin has been proposed to facilitate Cry toxin oligomer formation. In the present study, we show that Cry4Ba binds to CR7–11 with 9-fold lower binding affinity compared with Cry11Aa. Oligomerization assays showed that Cry4Ba is capable of forming oligomers when proteolytically activated in vitro in the absence of the CR7–11 fragment in contrast with Cry11Aa that formed oligomers only in the presence of CR7–11. Pore-formation assays in planar lipid bilayers showed that Cry4Ba oligomers were proficient in opening ion channels. Finally, silencing the cadherin gene by dsRNA (double-stranded RNA) showed that silenced larvae were more tolerant to Cry11Aa in contrast with Cry4Ba, which showed similar toxic levels to those of control larvae. These findings show that cadherin binding is not a limiting step for Cry4Ba toxicity to A. aegypti larvae.

No abstract

a b s t r a c tUsing a Cry11Ba toxin model, predicted loops in domain II were analyzed for their role in receptor binding and toxicity. Peptides corresponding to loops a8, 1 and 3, but not loop 2, competed with toxin binding to Aedes midgut membranes. Mutagenesis data reveal loops a8, 1 and 3 are involved in toxicity. Loops 1 and 3 are of greater significance in toxicity to Aedes and Culex larvae than to Anopheles. Cry11Ba binds the apical membrane of larval caecae and posterior midgut, and binding can be competed by loop 1 but not by loop 2 peptides. Cry11Ba binds the same regions to which anticadherin antibody binds, and this antibody competes with Cry11Ba binding suggesting a possible role of cadherin in toxication.