Bacillus thuringiensis Cry1Ab Mutants Affecting Oligomer Formation Are Non-toxic to Manduca sexta Larvae

Abstract

Pore-forming toxins are biological weapons produced by a variety of living organisms, particularly bacteria but also by insects, reptiles, and invertebrates. These proteins affect the cell membrane of their target, disrupting permeability and leading eventually to cell death. The pore-forming toxins typically transform from soluble, monomeric proteins to oligomers that form transmembrane channels. The Cry toxins produced by Bacillus thuringiensis are widely used as insecticides. These proteins have been recognized as pore-forming toxins, and their primary action is to lyse midgut epithelial cells in their target insect. To exert their toxic effect, a prepore oligomeric intermediate is formed leading finally to membrane-inserted oligomeric pores. To understand the role of Cry oligomeric pre-pore formation in the insecticidal activity we isolated point mutations that affected toxin oligomerization but not their binding with the cadherin-like, Bt-R 1 receptor. We show the helix ␣-3 in domain I contains sequences that could form coiled-coil structures important for oligomerization. Some single point mutants in this helix bound Bt-R 1 receptors with similar affinity as the wild-type toxin, but were affected in oligomerization and were severally impaired in pore formation and toxicity against Manduca sexta larvae. These data indicate the pre-pore oligomer and the toxin pore formation play a major role in the intoxication process of Cry1Ab toxin in insect larvae. Bacillus thuringiensis (Bt)2 is a Gram-positive bacterium that produces insecticidal Cry toxins. Cry proteins are widely used for insect control in agriculture and forestry and against mosquitoes, due to their high specificity and safety for humans and for the environment (1, 2).Although Bt Cry toxins are widely used as insecticides their mode of action is still not completely understood. These Cry toxins are pore-forming toxins that induce cell death by forming ionic pores following insertion into the membrane, causing osmotic lysis of larvae midgut cells (1-3). However, recently an alternative model proposed that these toxins activate a signal pathway through Bt-R 1 receptor interaction, which results in insect cell death without the participation of lytic pores into the membrane (4). It is important to note that this alternative model was proposed based on the effect of Cry1Ab toxin to cultured Trichoplusia ni H5 insect cells expressing the Manduca sexta toxin receptor, Bt-R 1 .Nevertheless, in both models, receptor interaction with exposed regions in domains II and III of Cry1A toxins (1-4) is a key step that determines insect toxicity. In the case of Cry1A toxins, two receptors have been characterized in several lepidopteran species: cadherin-like proteins, known as Bt-R receptors (5) (Bt-R 1 in the case of M. sexta), and glycosylphosphatidylinositol-anchored proteins, as aminopeptidase-N or alkaline phosphatase (6, 7).In the pore-forming model, it is proposed that both receptors are important and participate in a sequential manner (3,8,9). After proteoly...

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

“…Also, it was reported that Cry1Ab mutants in helix ␣-4 were affected in pore formation activity and were inactive. The mutants in helix ␣-4 are dominant-negative inhibitors of Cry1Ab toxin (5,40).…”

“…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)(6)(7)(8). Domain II and III are involved in the recognition and binding interaction with receptors in midgut cells (9 -11).…”

Domain II Loop 3 of Bacillus thuringiensis Cry1Ab Toxin Is Involved in a “Ping Pong” Binding Mechanism with Manduca sexta Aminopeptidase-N and Cadherin Receptors

“…For monomer production, Cry1Ab crystals were solubilized in alkaline buffer and activated by trypsin as reported previously (24). In order to obtain oligomeric structure, the crystals were activated with 5% M. sexta midgut juice in the presence of scFv73 antibody as described (16 (7,25,26). These constructions were transformed into Escherichia coli ER2566 cells; protein expression was induced by the addition of 1 mM isopropyl-thio-␤-galactopyranoside.…”

“…Also, it was shown that binding with APN was in the range of 100 nM (20). We previously cloned and produced in E. coli two cadherin fragments corresponding to CR7-CR12 (residues Met 810 -Ala 1485 ) and to CR12 (residues Gly 1370 -Ala 1485 ) of M. sexta Bt-R1 (7,25,26). It was reported that Cry1Ab bound to a cadherin peptide (CR12-MPED) similar to CR12 with a binding affinity of 9.5 nM (34).…”

“…Overall, these proteins show a similar organization in three different domains, suggesting a conserved mode of action as follows. Domain I contains seven ␣-helixes and is involved in oligomer formation, insertion into the membrane, and pore formation (1,7); domain II consists of three antiparallel ␤-sheets, and its structure is the most variable of three domains and is implicated in receptor recognition; and domain III is composed of two antiparallel ␤-sheets and is also involved in receptor binding (1).…”