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

Abstract

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...

Cytotoxicity of Bacillus thuringiensis Cry1Ab toxin depends on specific binding of the toxin to the cadherin receptor BT-R1 expressed in insect cells

“…For example, (i) Cry toxin can insert into bilayer lipid rafts and BBMV irrespective of toxin receptors, 20 (ii) toxin oligomer complexes can form in BBMV prepared from insects that are not susceptible to Cry toxin 29,47 and (iii) Cry toxin mutants that are not insecticidal induce changes in membrane permeability similar to wild-type toxin. 27,28 In the present study, we used an H5 insect cell-based system that allowed us to simultaneously correlate toxinreceptor and toxin-membrane interactions with cellular responses associated with cytotoxicity. An important distinction of this system is the ability to examine incorporation of Cry1Ab toxin into cell membrane and to monitor the interaction of the toxin with BT-R 1 in live insect cells.…”

“…Studies of mutated Cry toxin proteins have shown that neither the toxin oligomer complex nor commensurate changes in membrane vesicle permeability correlate directly with toxicity. [26][27][28][29] Furthermore, Cry toxin proteins can interact with lipid bilayer rafts and BBMV in the absence of a cadherin receptor, which determines specific insecticidal activity of Cry toxins.…”

A mechanism of cell death involving an adenylyl cyclase/PKA signaling pathway is induced by the Cry1Ab toxin of Bacillus thuringiensis

“…Changes in membrane permeability were correlated with the incorporation of Cry toxin oligomers into lipid bilayer rafts and brush border membrane vesicles (8,16). However, studies of mutated Cry toxin proteins have shown that neither the toxin oligomer complex nor commensurate changes in membrane vesicle permeability correlate directly with toxicity (17)(18)(19). Interestingly, Cry toxin oligomers also are incorporated into the cell membrane of nonsusceptible cabbage looper cells and are carried by the cells for several generations with no adverse effect (14).…”

Two disulfide mutants in domain I of <i>Bacillus thuringiensis</i> Cry3Aa δ-endotoxin increase stability with no effect on toxicity

“…There is also evidence that other domains of the protein are able to insert into membrane [16], and that several mutations in the domain I (α-helices 3 and 4) affect oligomer formation [18][19][20]. Several disulfide-bridge mutations generated by protein engineering which are related to the membrane partitioning mechanisms of Cry toxins are reviewed in [21].…”