Blocking binding of Bacillus thuringiensis Cry1Aa to Bombyx mori cadherin receptor results in only a minor reduction of toxicity

2009

Cell. Mol. Life Sci.

234

174

Cry proteins, produced by Bacillus thuringiensis (Bt), are widely used for the control of insect pests in agriculture as spray products or expressed in transgenic crops, such as maize and cotton. Little was known regarding the mechanism of action of these toxins when the first commercial Bt product was introduced fifty years ago. However, research on the mechanism of action over the last two decades has enhanced our knowledge of toxin interaction with membrane receptors and their effects in insect midgut cells. All this information allowed for the rational design of improved toxins with higher toxicity or toxins that overcome insect resistance, which could compromise Bt use and effectiveness in the field. In this review we discuss and evaluate the different models of the mode of action of Cry toxins, including a discussion about the role of various receptors in toxin action.

Section: Expression Of Cadherin Receptors In Cell Linesmentioning

confidence: 92%

Signaling versus punching hole: How do Bacillus thuringiensis toxins kill insect midgut cells?

Soberón

Gill

2009

Cell. Mol. Life Sci.

234

174

“…Nevertheless, Cry1Ac mutants in domain III that are affected in binding to APN-1 had a marginal effect on toxicity against M. sexta larvae (23). Also, in the case of Bombyx mori, it was shown that anti-cadherin antibodies protected detached midgut cells from the toxic effects of Cry1Aa, in contrast to anti-APN antibodies that had no effect on the toxicity of Cry1Aa (24). These apparently contradictory data could be explained if an additional secondary receptor could play the same role as APN in M. sexta midgut cells.…”

mentioning

confidence: 99%

Role of Alkaline Phosphatase from Manduca sexta in the Mechanism of Action of Bacillus thuringiensis Cry1Ab Toxin

Arenas

Journal of Biological Chemistry

Soberón

et al. 2010

155

157

Cry toxins produced by Bacillus thuringiensis have been recognized as pore-forming toxins whose primary action is to lyse midgut epithelial cells in their target insect. In the case of the Cry1A toxins, a prepore oligomeric intermediate is formed after interaction with cadherin receptor. The Cry1A oligomer then interacts with glycosylphosphatidylinositol-anchored receptors. Two Manduca sexta glycosylphosphatidylinositol-anchored proteins, aminopeptidase (APN) and alkaline phosphatase (ALP), have been shown to bind Cry1Ab, although their role in toxicity remains to be determined. Detection of Cry1Ab binding proteins by ligand blot assay revealed that ALP is preferentially expressed earlier during insect development, because it was found in the first larval instars, whereas APN is induced later after the third larval instar. The binding of Cry1Ab oligomer to pure preparations of APN and ALP showed that this toxin structure interacts with both receptors with high affinity (apparent K d ‫؍‬ 0.6 nM), whereas the monomer showed weaker binding (apparent K d ‫؍‬ 101.6 and 267.3 nM for APN and ALP, respectively). Several Cry1Ab nontoxic mutants located in the exposed loop 2 of domain II or in ␤-16 of domain III were affected in binding to APN and ALP, depending on their oligomeric state. In particular monomers of the nontoxic domain III, the L511A mutant did not bind ALP but retained APN binding, suggesting that initial interaction with ALP is critical for toxicity. Our data suggest that APN and ALP fulfill two roles. First APN and ALP are initial receptors promoting the localization of toxin monomers in the midgut microvilli before interaction with cadherin. Then APN and ALP function as secondary receptors mediating oligomer insertion into the membrane. However, the expression pattern of these receptors and the phenotype of L511A mutant suggest that ALP may have a predominant role in toxin action because Cry toxins are highly effective against the neonate larvae that is the target for pest control programs.The Cry toxins produced by Bacillus thuringiensis are used worldwide as effective biological control agents for many species of insects including agricultural and forest pests and several vectors of human and animal diseases. Its insecticidal property results from crystalline inclusions produced during sporulation that are formed by ␦-endotoxins known as Cry toxins (1).The Cry protein family is composed of more than 54 groups, among which the three-domain Cry family forms the major group, having members that show toxicity to different insect orders and to nematodes. The crystal structure has been solved for six three-domain Cry toxins and one protoxin that display different insect specificities (2). The activated Cry toxins are globular molecules consisting of a bundle of seven ␣-helices (domain I), a three-␤-sheet prism (domain II), and a ␤-sandwich (domain III) (3, 4). The N-terminal domain I is involved in oligomer formation, membrane insertion, and pore formation (5-8). Domain II and III are involved in the recogn...

“…Nevertheless, some Cry1Ac mutants affected in binding to APN1 showed a marginal effect on toxicity against M. sexta larvae, suggesting that other Cry1Ac binding proteins may be involved in Cry1Ac toxicity (16). In the case of Bombyx mori, it was shown that anticadherin antibodies protected detached midgut cells from the toxic effects of Cry1Aa, in contrast to anti-APN antibodies, which had no effect on the toxicity of this toxin (17). These apparently contradictory data could be explained if an additional secondary receptor could play the same role as APN in midgut cells.…”

mentioning

confidence: 99%

Differential Role of Manduca sexta Aminopeptidase-N and Alkaline Phosphatase in the Mode of Action of Cry1Aa, Cry1Ab, and Cry1Ac Toxins from Bacillus thuringiensis

Flores‐Escobar

Rodríguez-Magadán

et al. 2013

Appl Environ Microbiol

Aminopeptidase-N (APN1) and alkaline phosphatase (ALP) proteins located in the midgut epithelium of Manduca sexta have been implicated as receptors for Cry1Aa, Cry1Ab, and Cry1Ac insecticidal proteins produced by Bacillus thuringiensis subsp. kurstaki. In this study, we analyzed the roles of ALP and APN1 in the toxicity of these three Cry1A proteins. Ligand blot analysis using brush border membrane vesicles of M. sexta showed that Cry1Aa and Cry1Ab bind preferentially to ALP during early instars while binding to APN was observed after the third instar of larval development. Cry1Ac binds to APN throughout all larval development, with no apparent binding to ALP. ALP was cloned from M. sexta midgut RNA and expressed in Escherichia coli. Surface plasmon resonance binding analysis showed that recombinant ALP binds to Cry1Ac with 16-fold lower affinity than to Cry1Aa or Cry1Ab. Downregulation of APN1 and ALP expression by RNA interference (RNAi) using specific double-stranded RNA correlated with a reduction of transcript and protein levels. Toxicity analysis of the three Cry1A proteins in ALP-or APN1-silenced larvae showed that Cry1Aa relies similarly on both receptor molecules for toxicity. In contrast, RNAi experiments showed that ALP is more important than APN for Cry1Ab toxicity, while Cry1Ac relied principally on APN1. These results indicated that ALP and APN1 have a differential role in the mode of action of Cry1A toxins, suggesting that B. thuringiensis subsp. kurstaki produces different Cry1A toxins that in conjunction target diverse midgut proteins to exert their insecticidal effect.