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

Abstract

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...

Cadherin, Alkaline Phosphatase, and Aminopeptidase N as Receptors of Cry11Ba Toxin from Bacillus thuringiensis subsp. jegathesan in Aedes aegypti

“…A number of proteins have been identified as Cry toxin receptors, such as cadherin, alkaline phosphatases (ALPs), and aminopeptidase (APN), and these were first identified in different lepidopteran insects as Cry1A toxin binding proteins (4,22,23,26,29,32,36,37,42,44). In mosquito larvae, similar proteins have been described.…”

Role of Pore-Forming Toxins in Bacterial Infectious Diseases

“…This shedding apparently did not result from proteolysis but was dependent upon cAMP and the ERK1/2 pathway, via cleavage of the GPI anchor by an endogenous GPI-specific phospholipase C (453), similar to what was found for B. anthracis (34). Since aminopeptidase and alkaline phosphatase are receptors for Cry toxins (454), the observed shedding may be a protective function that is under the control of the ERK1/2 (and p38) MAPK pathway.…”

Specificity determinants for Cry insecticidal proteins: Insights from their mode of action

“…Loop 2 seems critical for recognition of Cry receptors (Arenas et al, 2010;Jiménez et al, 2012;Pigott et al, 2008), through interactions proposed to involve hydropathic complementarity (Gómez et al, 2002).…”