Analysis of the Large Aqueous Pores Produced by a <i>Bacillus Turingiensis</i> Protein Insecticide in <i>Manduca Sexta</i> Midgut‐Brush‐Border‐Membrane Vesicles

Carroll, Joe; Ellar, David J.

doi:10.1111/j.1432-1033.1997.00797.x

Cited by 38 publications

(17 citation statements)

References 38 publications

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“…Among the intestinal factors that could affect toxin activity, the strongly alkaline pH characteristic of the larval midgut of lepidopteran insects (40,41) had only minor effects on the in vitro activity of Cry1Aa and Cry1Ac. A slightly higher permeability to sucrose and raffinose was nevertheless observed at pH 10.5 than at pH 7.5, consistent with a slight increase in pore size with increasing pH as was previously reported for Cry1Ac (57). A similar effect of pH was observed for all three Cry1Aa mutants, but it was not detected with either of the Cry1Ac mutants.…”

Section: Figsupporting

confidence: 73%

Role of Interdomain Salt Bridges in the Pore-forming Ability of the Bacillus thuringiensis Toxins Cry1Aa and Cry1Ac

Coux¹,

Vachon²,

Rang³

et al. 2001

Journal of Biological Chemistry

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linking domains I and II in Cry1Aa were abolished individually in ␣-helix 7 mutants D222A, R233A, R234A, and D242A. Two additional mutants targeting the fourth salt bridge (R265A) and the double mutant (D242A/R265A) were rapidly degraded during trypsin activation. Mutations were also introduced in the corresponding Cry1Ac salt bridge (D242E, D242K, D242N, and D242P), but only D242N and D242P could be produced. All toxins tested, except D242A, were shown by light-scattering experiments to permeabilize Manduca sexta larval midgut brush border membrane vesicles. The three active Cry1Aa mutants at pH 10.5, as well as D222A at pH 7.5, demonstrated a faster rate of pore formation than Cry1Aa, suggesting that increases in molecular flexibility due to the removal of a salt bridge facilitated toxin insertion into the membrane. However, all mutants were considerably less toxic to M. sexta larvae than to the respective parental toxins, suggesting that increased flexibility made the toxins more susceptible to proteolysis in the insect midgut. Interdomain salt bridges, especially the Asp 242 -Arg 265 bridge, therefore contribute greatly to the stability of the protein in the larval midgut, whereas their role in intrinsic pore-forming ability is relatively less important.During sporulation, Bacillus thuringiensis produces a parasporal crystal body composed of one or more proteins that are toxic to a number of insect larvae (1) or to other invertebrates (2). After solubilization in the insect midgut and activation by intestinal proteases, these proteins bind to specific receptors at the surface of the apical brush border membrane of epithelial columnar cells, insert into the membrane, and form pores that disrupt midgut cellular functions (3-5).Elucidation of the crystal structure of the coleopteranspecific Cry3A toxin (6) and the lepidopteran-specific Cry1Aa toxin (7) revealed a similar three-domain structure for both proteins. Domain I, composed of eight amphipathic ␣-helices, is thought to be involved in membrane insertion and pore formation (8 -15). Domain II, composed of three ␤-sheets and two short ␣-helices, is involved in the binding of the toxin to its receptor on the epithelial cell surface (16 -23). Domain III, composed of two ␤-sheets forming a face-to-face ␤-sandwich, appears to be involved in the stability (6), specificity (24 -26), and binding (27-34) of the toxin.These domains are closely packed together with the largest number of interdomain contacts found between domains I and II (6, 7). In Cry1Aa, domains I and II are linked by four salt bridges: Asp

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Section: Figsupporting

confidence: 73%

Role of Interdomain Salt Bridges in the Pore-forming Ability of the Bacillus thuringiensis Toxins Cry1Aa and Cry1Ac

Coux¹,

Vachon²,

Rang³

et al. 2001

Journal of Biological Chemistry

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“…Cry toxin channels are poorly selective, transporting different ions and solutes of higher size including sugars and amino acids [2,[23][24][25][26]. However, all these studies were done only with Cry toxins in their monomeric state [2,[23][24][25][26].…”

Section: Cry1camentioning

confidence: 99%

Permeability Changes of Manduca sexta Midgut Brush Border Membranes Induced by Oligomeric Structures of Different Cry Toxins

et al. 2006

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Abstract. The pore-formation activity of monomeric and oligomeric forms of different Cry1 toxins (from Cry1A to Cry1G) was analyzed by monitoring ionic permeability across Manduca sexta brush border membrane vesicles. The membrane vesicles were isolated from microvilli structures, showing a high enrichment of apical membrane markers and low intrinsic K + permeability. A fluorometric assay performed with 3,3¢-dipropylthiodicarbocyanine fluorescent probe, sensitive to changes in membrane potential, was used. Previously, it was suggested that fluorescence determinations with this dye could be strongly influenced by the pH, osmolarity and ionic strength of the medium. Therefore, we evaluated these parameters in control experiments using the K + -selective ionophore valinomycin. We show here that under specific ionic conditions changes in fluorescence can be correlated with ionic permeability without effects on osmolarity or ionic strength of the medium. It is extremely important to attenuate the background response due to surface membrane potential and the participation of the endogenous permeability of the membrane vesicles. Under these conditions, we analyzed the pore-formation activity induced by monomeric and oligomeric structures of different Cry1 toxins. The Cry1 toxin samples containing oligomeric structures correlated with high pore activity, in contrast to monomeric samples that showed marginal pore-formation activity, supporting the hypothesis that oligomer formation is a necessary step in the mechanism of action of Cry toxins.

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“…Those formed by R131H were also significantly less permeable to sucrose at pH 7.5 and to raffinose at pH 10.5. The pores formed by Cry1Aa and Cry1Ac have previously been shown to be more permeable to sucrose at pH 10.5 than at pH 7.5, suggesting a slightly larger pore diameter at the higher pH (7,9,40,41). Although higher permeabilities to sucrose were observed at pH 10.5, with most mutants capable of forming pores at both pH values, none of these increases was statistically significant (P Ͼ 0.05).…”

Section: Toxicitymentioning

confidence: 54%

Helix 4 Mutants of the Bacillus thuringiensis Insecticidal Toxin Cry1Aa Display Altered Pore-Forming Abilities

Vachon

Préfontaine

Rang

et al. 2004

Appl Environ Microbiol

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The role played by ␣-helix 4 of the Bacillus thuringiensis toxin Cry1Aa in pore formation was investigated by individually replacing each of its charged residues with either a neutral or an oppositely charged amino acid by using site-directed mutagenesis. The majority of the resulting mutant proteins were considerably less toxic to Manduca sexta larvae than Cry1Aa. Most mutants also had a considerably reduced ability to form pores in midgut brush border membrane vesicles isolated from this insect, with the notable exception of those with alterations at amino acid position 127 (R127N and R127E), located near the N-terminal end of the helix. Introducing a negatively charged amino acid near the C-terminal end of the helix (T142D and T143D), a region normally devoid of charged residues, completely abolished pore formation. For each mutant that retained detectable pore-forming activity, reduced membrane permeability to KCl was accompanied by an approximately equivalent reduction in permeability to N-methyl-D-glucamine hydrochloride, potassium gluconate, sucrose, and raffinose and by a reduced rate of pore formation. These results indicate that the main effect of the mutations was to decrease the toxin's ability to form pores. They provide further evidence that ␣-helix 4 plays a crucial role in the mechanism of pore formation.Bacillus thuringiensis is the most extensively used commercial biopesticide worldwide and is presently the sole source of toxin genes for the development of insect-resistant transgenic plants (13,15,42). The insecticidal activity of B. thuringiensis is primarily associated with its ability to synthesize a crystalline parasporal inclusion body containing highly specific insecticidal proteins (22,36). The mode of action of these insecticides involves solubilization of the crystal in the highly alkaline lepidopteran midgut lumen, activation of the toxins by intestinal proteases, recognition of one or more binding sites on the midgut brush border membrane surface followed by pore formation, and cell lysis leading ultimately to insect death (36).The elucidation by X-ray diffraction analysis of the threedimensional structures of the activated Cry1Aa (21), Cry2Aa (31), Cry3Aa (28), and Cry3Bb (16) toxins has revealed a common three-domain folding pattern. Domain I is made of seven ␣-helices, and domains II and III are composed mostly of ␤-sheets. While domain I is considered to be responsible for pore formation (37, 43, 44), domains II and III are involved in receptor binding and host specificity (11,12,24,47). Domain III is also thought to play a role in protein stability (28). The domains of the activated toxins were shown to interact with each other to yield their overall toxic effect (33, 34). Exchanging domain I from different toxins can affect crystal formation, stability, pore formation, and membrane permeability as well as the size of the pores and toxicity.The toxin is thought to form pores in the cell membrane by first inserting a hairpin composed of the hydrophobic ␣5 and the amphipathic ␣4 h...

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Analysis of the Large Aqueous Pores Produced by a Bacillus Turingiensis Protein Insecticide in Manduca Sexta Midgut‐Brush‐Border‐Membrane Vesicles

Cited by 38 publications

References 38 publications

Role of Interdomain Salt Bridges in the Pore-forming Ability of the Bacillus thuringiensis Toxins Cry1Aa and Cry1Ac

Role of Interdomain Salt Bridges in the Pore-forming Ability of the Bacillus thuringiensis Toxins Cry1Aa and Cry1Ac

Permeability Changes of Manduca sexta Midgut Brush Border Membranes Induced by Oligomeric Structures of Different Cry Toxins

Helix 4 Mutants of the Bacillus thuringiensis Insecticidal Toxin Cry1Aa Display Altered Pore-Forming Abilities

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