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

Préfontaine

et al. 2008

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Helix ␣4 of Bacillus thuringiensis Cry toxins is thought to line the lumen of the pores they form in the midgut epithelial cells of susceptible insect larvae. To define its functional role in pore formation, most of the ␣4 amino acid residues were replaced individually by a cysteine in the Cry1Aa toxin. The toxicities and pore-forming abilities of the mutated toxins were examined, respectively, by bioassays using neonate Manduca sexta larvae and by a light-scattering assay using midgut brush border membrane vesicles isolated from M. sexta. A majority of these mutants had considerably reduced toxicities and pore-forming abilities. Most mutations causing substantial or complete loss of activity map on the hydrophilic face of the helix, while most of those having little or only relatively minor effects map on its hydrophobic face. The properties of the pores formed by mutants that retain significant activity appear similar to those of the pores formed by the wild-type toxin, suggesting that mutations resulting in a loss of activity interfere mainly with pore formation.Bacillus thuringiensis is a gram-positive spore-forming bacterium that produces a variety of insecticidal toxins which accumulate as protoxins in the form of parasporal crystals. Once ingested by susceptible insect larvae, the crystals become soluble in the midgut, and the protoxins are converted to active toxins by intestinal proteases. The activated toxins act by forming pores in the midgut luminal membrane after binding to specific receptors located at the membrane surface of the intestinal epithelium columnar cells (25). These pores are large enough to disrupt the ionic gradients established across the membrane and to cause the osmotic lysis of the cells (27).The structures of several insecticidal Cry toxins have been elucidated by X-ray crystallography (4,5,13,16,19,20,24). With the exception of a recently described crystal protein of unknown toxicity (1), they all share a remarkably similar threedomain structure. Domain I is composed of a bundle of seven amphipathic ␣-helices, with helix ␣5 surrounded by the other helices. Domain II is composed of three ␤-sheets with a "Greek key" topology forming a ␤-prism, and domain III is composed of two antiparallel sheets forming a ␤-sandwich with a "jelly roll" topology. While domain I is thought to be responsible for membrane insertion and pore formation, domains II and III are thought to be involved in the binding of the toxin to its receptors (9,16,19,25,27).Receptor binding presumably triggers a conformational change in the toxin molecule that leads to its oligomerization and insertion into the membrane. According to the umbrella model of insertion, a hairpin composed of helices ␣4 and ␣5 inserts into the membrane, while the rest of the helices are deployed over the membrane surface (2,14,15,28). Results from chemical modification of preformed Cry1Aa pores in artificial membranes, using a mutant toxin possessing an aspartic acid-to-cysteine substitution at residue 136 in helix ␣4, indicated that at le...

Section: Resultssupporting

confidence: 64%

Cysteine Scanning Mutagenesis of α4, a Putative Pore-Lining Helix of the Bacillus thuringiensis Insecticidal Toxin Cry1Aa

Girard

Préfontaine

et al. 2008

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“…However, Cry1Ac, which was slightly more toxic to M. sexta than Cry1Aa and Cry1Ab in previous studies (7,40), was slightly less toxic than Cry1Aa (Table 1). On the other hand, although Coux et al (7) 2 ) than those found in the present study, the activity of the Cry1Aa mutant that they observed was closer to that measured in the present study.…”

Section: Discussionmentioning

confidence: 70%

“…The Cry1Aa mutant R233A was created by oligonucleotide-directed in vitro mutagenesis (7). The wild-type toxins Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ba, Cry1Ca, and Cry1Ea and the R233A mutant were prepared from B. thuringiensis strains producing the appropriate single recombinant toxins as described elsewhere (23).…”

Section: Chemicalsmentioning

confidence: 99%

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Effect of Insect Larval Midgut Proteases on the Activity of Bacillus thuringiensis Cry Toxins

Fortier

Frutos

et al. 2007

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To test the possibility that proteolytic cleavage by midgut juice enzymes could enhance or inhibit the activity of Bacillus thuringiensis insecticidal toxins, once activated, the effects of different toxins on the membrane potential of the epithelial cells of isolated Manduca sexta midguts in the presence and absence of midgut juice were measured. While midgut juice had little effect on the activity of Cry1Aa, Cry1Ac, Cry1Ca, Cry1Ea, and R233A, a mutant of Cry1Aa from which one of the four salt bridges linking domains I and II of the toxin was eliminated, it greatly increased the activity of Cry1Ab. In addition, when tested in the presence of a cocktail of protease inhibitors or when boiled, midgut juice retained almost completely its capacity to enhance Cry1Ab activity, suggesting that proteases were not responsible for the stimulation. On the other hand, in the absence of midgut juice, the cocktail of protease inhibitors also enhanced the activity of Cry1Ab, suggesting that proteolytic cleavage by membrane proteases could render the toxin less effective. The lower toxicity of R233A, despite a similar in vitro pore-forming ability, compared with Cry1Aa, cannot be accounted for by an increased susceptibility to midgut proteases. Although these assays were performed under conditions approaching those found in the larval midgut, the depolarizing activities of the toxins correlated only partially with their toxicities.

“…In the case of Cry1A toxins, the first 28 amino acid residues are removed from the N terminus and approximately half of the protoxin residues are removed from the C terminus (6). However, susceptibility of the activated toxins to further proteolysis in the midgut environment could possibly affect toxicity and explain apparent discrepancies that are occasionally observed between their in vitro and in vivo activities (15,41,49,60). For example, Cry1Ca can be completely degraded when incubated with midgut juice from advanced larval instars of Spodoptera littoralis (29).…”

mentioning

confidence: 99%

Protease Inhibitors Fail To Prevent Pore Formation by the Activated Bacillus thuringiensis Toxin Cry1Aa in Insect Brush Border Membrane Vesicles

Kirouac

Quievy

et al. 2006

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To investigate whether membrane proteases are involved in the activity of Bacillus thuringiensis insecticidal toxins, the rate of pore formation by trypsin-activated Cry1Aa was monitored in the presence of a variety of protease inhibitors with Manduca sexta midgut brush border membrane vesicles and by a light-scattering assay. Most of the inhibitors tested had no effect on the pore-forming ability of the toxin. However, phenylmethylsulfonyl fluoride, a serine protease inhibitor, promoted pore formation, although this stimulation only occurred at higher inhibitor concentrations than those commonly used to inhibit proteases. Among the metalloprotease inhibitors, o-phenanthroline had no significant effect; EDTA and EGTA reduced the rate of pore formation at pH 10.5, but only EDTA was inhibitory at pH 7.5. Neither chelator affected the properties of the pores already formed after incubation of the vesicles with the toxin. Taken together, these results indicate that, once activated, Cry1Aa is completely functional and does not require further proteolysis. The effect of EDTA and EGTA is probably better explained by their ability to chelate divalent cations that could be necessary for the stability of the toxin's receptors or involved elsewhere in the mechanism of pore formation.Bacillus thuringiensis is by far the most widely used alternative to chemical insecticides for the control of insect pests in forestry, agriculture, and public health. During sporulation, this bacterium produces insecticidal proteins that accumulate in the form of parasporal crystals. Following their ingestion by susceptible insect larvae, these protoxins are solubilized and converted to active toxins by midgut proteases. Activated toxins act by forming pores after binding to specific receptors at the surface of the insect midgut luminal membrane, leading to cell lysis, destruction of the epithelium, and death of the insect (52). In their activated form, all B. thuringiensis Cry toxins for which the crystal structure has been solved share a similar three-domain structure (7,21,24,33,34,44). Domain I is composed of a bundle of seven anti-parallel ␣-helices and is generally thought to be responsible for membrane insertion and pore formation, while domains II and III are mainly composed of ␤-sheets and involved in the binding, specificity, and stability of the toxin (24, 33, 52).Midgut proteases play an essential role in the activation of B. thuringiensis toxins (1, 6, 13). In the case of Cry1A toxins, the first 28 amino acid residues are removed from the N terminus and approximately half of the protoxin residues are removed from the C terminus (6). However, susceptibility of the activated toxins to further proteolysis in the midgut environment could possibly affect toxicity and explain apparent discrepancies that are occasionally observed between their in vitro and in vivo activities (15,41,49,60). For example, Cry1Ca can be completely degraded when incubated with midgut juice from advanced larval instars of Spodoptera littoralis (29). Synergisti...