Mutations in Domain I of Bacillus thuringiensis δ-Endotoxin CryIAb Reduce the Irreversible Binding of Toxin to Manduca sexta Brush Border Membrane Vesicles

Chen, Xue Jun; Curtiss, April; Alcantara, Edwin P.; Dean, Donald H.

doi:10.1074/jbc.270.11.6412

Cited by 74 publications

(69 citation statements)

References 32 publications

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“…The authors of this study concluded that a positive charge in this position could participate in the formation of specific binding domain or that it could be important for the arrangement of α-helices in the amino terminal region of the toxin, and that the conformation of this domain could indirectly disrupt the specificity domain of the toxin [44]. The introduction of a negative charge in residue A92 supported this hypothesis since mutations A92E and A92D were severely affected in toxicity and correlated with loss of pore formation [8,44]. It was finally proposed that the irreversible insertion into the BBMV was affected in A92E and A92D mutants [8].…”

Section: Mutagenesis Of Helix α-3 Of Different Cry Toxinsmentioning

confidence: 71%

“…The introduction of a negative charge in residue A92 supported this hypothesis since mutations A92E and A92D were severely affected in toxicity and correlated with loss of pore formation [8,44]. It was finally proposed that the irreversible insertion into the BBMV was affected in A92E and A92D mutants [8]. Residue R93 is located in position a of the predicted coiled coil structure of helix α-3 (Fig 1), and as stated above this is a highly conserved amino acid within the Cry toxin family.…”

Section: Mutagenesis Of Helix α-3 Of Different Cry Toxinsmentioning

confidence: 79%

“…Other important mutations performed in helix α-3 of Cry1Ac toxin are those introduced in residues R93 and A92. These mutants were also affected toxicity, but only the conservative change R93K was fully active, while other changes of R93 (G, A, and S) showed 100-1000 fold reduced toxicity and many other changes resulted in complete loss of toxicity [8,44]. The authors of this study concluded that a positive charge in this position could participate in the formation of specific binding domain or that it could be important for the arrangement of α-helices in the amino terminal region of the toxin, and that the conformation of this domain could indirectly disrupt the specificity domain of the toxin [44].…”

Section: Mutagenesis Of Helix α-3 Of Different Cry Toxinsmentioning

confidence: 98%

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The pre-pore from Bacillus thuringiensis Cry1Ab toxin is necessary to induce insect death in Manduca sexta

et al. 2008

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The insecticidal Cry toxins from Bacillus thuringiensis bacteria are pore-forming toxins that lyse midgut epithelial cells in insects. We have previously proposed that they form pre-pore oligomeric intermediates before membrane insertion.For formation of these oligomers coiled-coil structures are important, and helix α-3 from Cry toxins could form coiled-coils. Our data shows that different mutations in helix α-3 are affected in pore formation and toxicity. Mutants affected in toxicity bind Bt-R 1 receptor with a similar K D as the wild type toxin but do not form oligomers nor induce pore formation in planar lipid bilayers, indicating that the pre-pore oligomer is an obligate intermediate in the intoxication of Cry1Ab toxin and that interaction of monomeric Cry1Ab with Bt-R 1 is not enough to kill susceptible larvae.

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Section: Mutagenesis Of Helix α-3 Of Different Cry Toxinsmentioning

confidence: 71%

Section: Mutagenesis Of Helix α-3 Of Different Cry Toxinsmentioning

confidence: 79%

Section: Mutagenesis Of Helix α-3 Of Different Cry Toxinsmentioning

confidence: 98%

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The pre-pore from Bacillus thuringiensis Cry1Ab toxin is necessary to induce insect death in Manduca sexta

et al. 2008

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“…The authors concluded that a positive charge in this position could participate in the formation of specific binding domain or that the conformation of this domain could indirectly disrupt the specificity domain of the toxin (52). The mutations A92E and A92D were also severely affected in toxicity and correlated with loss of pore formation (52,53). It was proposed that the irreversible insertion of these mutants into the BBMV was affected (53).…”

Section: Discussionmentioning

confidence: 99%

“…All these data could be explained if the isolated mutants in helix ␣-3 were unable to form stable oligomers that are important for making pore channels. In other reports the Arg-93 and Ala-92 of Cry1Ac toxin (located at the beginning of helix ␣-3) were changed to different amino acids, only the conservative change R93K was fully active, some other changes of Arg-93 (Gly, Ala, and Ser) showed 100-to 1000-fold reduced toxicity, whereas all other changes resulted in complete loss of toxicity (52,53). The authors concluded that a positive charge in this position could participate in the formation of specific binding domain or that the conformation of this domain could indirectly disrupt the specificity domain of the toxin (52).…”

Section: Discussionmentioning

confidence: 99%

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

Jiménez-Juárez

Muñóz-Garay

Gómez

et al. 2007

Journal of Biological Chemistry

103

108

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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...

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Mode of Action of Bacillus thuringiensis Toxins

Zhuang

Gill

2002

Chemistry of Crop Protection

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Mutations in Domain I of Bacillus thuringiensis δ-Endotoxin CryIAb Reduce the Irreversible Binding of Toxin to Manduca sexta Brush Border Membrane Vesicles

Cited by 74 publications

References 32 publications

The pre-pore from Bacillus thuringiensis Cry1Ab toxin is necessary to induce insect death in Manduca sexta

The pre-pore from Bacillus thuringiensis Cry1Ab toxin is necessary to induce insect death in Manduca sexta

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

Mode of Action of Bacillus thuringiensis Toxins

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