The toxicity test and hypothetical model ofBacillus thuringiensis Cry1Aa helix4

Development of targeted biological agents against agricultural insect pests is of prime importance for the elaboration and implementation of integrated pest management strategies that are environment-friendly, respectful of bio-diversity and safer to human health through reduced use of chemical pesticides. A major goal to understand how Bt toxins work is to elucidate the functions of their three domains. Domains II and III are involved in binding specificity and structural integrity, but the function of Domain I remains poorly understood. Using a Manduca sexta BBMV (brush border membrane vesicles) system, we analyzed its responses to Cry1Aa 15 single-point mutations with altered Domain I helix 4 residues. Light scattering assay showed that toxicity was almost lost in 3 mutants, and we observed significantly reduced toxicity in other 7 mutants. However, 5 mutants retained wild-type toxicity. Using computer software, we simulated the three-dimensional structures of helix 4. Both experimental and bioinformatic analysis showed that residues in Cry1Aa Domain I helix 4 were involved in the formation of ion channels that is critical for its insect toxicity.     

The toxicity test and hypothetical model of Bacillus thuringiensis Cry1Aa helix4

Development of targeted biological agents against agricultural insect pests is of prime importance for the elaboration and implementation of integrated pest management strategies that are environment-friendly, respectful of bio-diversity and safer to human health through reduced use of chemical pesticides. A major goal to understand how Bt toxins work is to elucidate the functions of their three domains. Domains II and III are involved in binding specificity and structural integrity, but the function of Domain I remains poorly understood. Using a Manduca sexta BBMV (brush border membrane vesicles) system, we analyzed its responses to Cry1Aa 15 single-point mutations with altered Domain I helix 4 residues. Light scattering assay showed that toxicity was almost lost in 3 mutants, and we observed significantly reduced toxicity in other 7 mutants. However, 5 mutants retained wild-type toxicity. Using computer software, we simulated the three-dimensional structures of helix 4. Both experimental and bioinform     

Helix 4 of the Bacillus thuringiensis Cry1Aa toxin lines the lumen of the ion channel.

The mode of action of Bacillus thuringiensis insecticidal proteins is not well understood. Based on analogies with other bacterial toxins and ion channels, we hypothesized that charged amino acids in helix 4 of the Cry1Aa toxin are critical for toxicity and ion channel function. Using Plutella xylostella as a model target, we analyzed responses to Cry1Aa and eight proteins with altered helix 4 residues. Toxicity was abolished in five charged residue mutants (E129K, R131Q, R131D, D136N, D136C), however, two charged (R127E and R127N) and one polar (N138C) residue mutant retained wild-type toxicity. Compared with Cry1Aa and toxic mutants, nontoxic mutants did not show greatly reduced binding to brush border membrane vesicles, but their ion channel conductance was greatly reduced in planar lipid bilayers. Substituted cysteine accessibility tests showed that in situ restoration of the negative charge of D136C restored conductance to wild-type levels. The results imply that charged amino acids on the Asp-136 side of helix 4 are essential for toxicity and passage of ions through the channel. These results also support a refined version of the umbrella model of membrane integration in which the side of helix 4 containing Asp-136 faces the aqueous lumen of the ion channel.     

Helix α4 of the Bacillus thuringiensis Cry1Aa Toxin Plays a Critical Role in the Postbinding Steps of Pore Formation

Helix α4 of Bacillus thuringiensis Cry toxins is thought to play a critical role in the toxins' mode of action. Accordingly, single-site substitutions of many Cry1Aa helix α4 amino acid residues have previously been shown to cause substantial reductions in the protein's pore-forming activity. Changes in protein structure and formation of intermolecular disulfide bonds were investigated as possible factors responsible for the inactivity of these mutants. Incubation of each mutant with trypsin and chymotrypsin for 12 h did not reveal overt structural differences with Cry1Aa, although circular dichroism was slightly decreased in the 190- to 210-nm region for the I132C, S139C, and V150C mutants. The addition of dithiothreitol stimulated pore formation by the E128C, I132C, S139C, T142C, I145C, P146C, and V150C mutants. However, in the presence of these mutants, the membrane permeability never reached that measured for Cry1Aa, indicating that the formation of disulfide bridges could only partially explain their loss of activity. The ability of a number of inactive mutants to compete with wild-type Cry1Aa for pore formation in brush border membrane vesicles isolated from Manduca sexta was also investigated with an osmotic swelling assay. With the exception of the L147C mutant, all mutants tested could inhibit the formation of pores by Cry1Aa, indicating that they retained receptor binding ability. These results strongly suggest that helix α4 is involved mainly in the postbinding steps of pore formation.     

Structural and functional studies of alpha-helix 5 region from Bacillus thuringiensis Cry1Ab delta-endotoxin

The crystal insecticidal proteins from Bacillus thuringiensis are modular proteins comprised of three domains connected by single linkers. Domain I is a seven alpha-helix bundle, which has been involved in membrane insertion and pore formation activity. Site-directed mutagenesis has contributed to identify regions that might play an important role in the structure of the pore-forming domain within the membrane. There are several evidences that support that the hairpin alpha4-alpha5 inserts into the membrane in an antiparallel manner, while other helices lie on the membrane surface. We hypothesized that highly conserved residues of alpha5 could play an important role in toxin insertion, oligomerization and/or pore formation. A total of 15 Cry1Ab mutants located in six conserved residues of Cry1Ab, Y153, Y161, H168, R173, W182 and G183, were isolated. Eleven mutants were located within helix alpha5, one mutant was located in the loop alpha4-alpha5 and three mutants, W182P, W182I and G183C, were located in the loop alpha5-alpha6. Their effect on binding, K(+) permeability and toxicity against Manduca sexta larvae was analyzed and compared. The results provide direct evidence that some residues located within alpha5 have an important role in stability of the toxin within the insect gut, while some others also have an important role in pore formation. The results also provide evidence that conserved residues within helix alpha5 are not involved in oligomer formation since mutations in these residues are able to make pores in vitro.     

Influence of Mutagenesis of Bacillus thuringiensis Cry1Aa Toxin on Larvicidal Activity

Domain III of Bacillus thuringiensis Cry δ-endotoxins are considered to be related to the stability of the structure and avoidance of overdigestion by proteases. In this study, some residues of potential chymotrypsin and trypsin sites in Domain III of B. thuringiensis Cry1Aa were replaced individually with alanine by site-directed mutagenesis, in order to investigate their functional roles. Except F574A, all mutants F536A, R543A, F550A, F565A, R566A, F570A, F576A, F583A, and F590A were highly expressed the 130 kD protoxins at levels comparable to the wild-type tested by SDS-PAGE. In bioassays, F536A, R566A, and F590A increased toxicity against Spodoptera exigua Hüner larve by 20, 40, and 40%, respectively, as compared to the wild-type. F536A and F565A showed an increase of 6 and 10% in toxicity against Heliothis armigera Hubner than the wild-type. Toxicities of some mutants were altered greatly, and the same mutants were shown to have different toxicities against those two insects. Structural analyses showed that mutants R543A, F574A, F576A-affecting insecticidal activity might be relational to structural stability of toxin or decreased affinity for receptor binding. These results indicated that those residues were involved in the larvicidal activity of the Cry1Aa toxin.     

Helix 4 mutants of the Bacillus thuringiensis insecticidal toxin Cry1Aa display altered pore-forming abilities

The role played by alpha-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 alpha-helix 4 plays a crucial role in the mechanism of pore formation.     

Aedes aegypti alkaline phosphatase ALP1 is a functional receptor of Bacillus thuringiensis Cry4Ba and Cry11Aa toxins

Bacillus thuringiensis subs. israelensis produces at least three Cry toxins (Cry4Aa, Cry4Ba, and Cry11Aa) that are active against Aedes aegypti larvae. Previous work characterized a GPI-anchored alkaline phosphatase (ALP1) as a Cry11Aa binding molecule from the gut of A. aegypti larvae. We show here that Cry4Ba binds ALP1, and that the binding and toxicity of Cry4Ba mutants located in loop 2 of domain II is correlated. Also, we analyzed the contribution of ALP1 toward the toxicity of Cry4Ba and Cry11Aa toxins by silencing the expression of this protein though RNAi. Efficient silencing of ALP1 was demonstrated by real-time quantitative PCR (qPCR) and Western blot. ALP1 silenced larvae showed tolerance to both Cry4Ba and Cry11Aa although the silenced larvae were more tolerant to Cry11Aa in comparison to Cry4Ba. Our results demonstrate that ALP1 is a functional receptor that plays an important role in the toxicity of the Cry4Ba and Cry11Aa proteins.     

Binding of Bacillus thuringiensis subsp. israelensis Cry4Ba to Cyt1Aa has an important role in synergism

Bacillus thuringiensis subsp. israelensis (Bti) produces at least four different crystal proteins that are specifically toxic to different mosquito species and that belong to two non-related family of toxins, Cry and Cyt named Cry4Aa, Cry4Ba, Cry11Aa and Cyt1Aa. Cyt1Aa enhances the activity of Cry4Aa, Cry4Ba or Cry11Aa and overcomes resistance of Culex quinquefasciatus populations resistant to Cry11Aa, Cry4Aa or Cry4Ba. Cyt1Aa synergized Cry11Aa by their specific interaction since single point mutants on both Cyt1Aa and Cry11Aa that affected their binding interaction affected their synergistic insecticidal activity. In this work we show that Cyt1Aa loop β6-αE K198A, E204A and β7 K225A mutants affected binding and synergism with Cry4Ba. In addition, site directed mutagenesis showed that Cry4Ba domain II loop α-8 is involved in binding and in synergism with Cyt1Aa since Cry4Ba SI303-304AA double mutant showed decreased binding and synergism with Cyt1Aa. These data suggest that similarly to the synergism between Cry11Aa and Cyt1Aa toxins, the Cyt1Aa also functions as a receptor for Cry4Ba explaining the mechanism of synergism between these two Bti toxins.     

Effects of disulfide bridges in domain I of Bacillus thuringiensis Cry1Aa delta-endotoxin on ion-channel formation in biological membranes

The delta-endotoxin family of toxic proteins represents the major component of the insecticidal capability of the bacterium Bacillus thuringiensis. Domain I of the toxins, which is largely alpha-helical, has been proposed to unfold at protein entry into the membrane of a target insect, following models known as the penknife and umbrella models. We extended the analysis of a previous work in which four disulfide bridges were constructed in domain I of the Cry1Aa delta-endotoxin that putatively prevented unfolding during membrane partitioning. Using bioassays and voltage clamping of whole insect midgut instead of artificial lipid bilayers, it was found that, while toxicity and inhibition of the short-circuit current were reduced, only one of the disulfide bridges eliminated the activity of the toxins in the insect midgut membrane, and in that case, the loss of toxicity was due to the single amino acid substitution, R99C. It is proposed that at least alpha helices 4, 5, 6, and 7 and domain II partition in the midgut membranes of target insects, in support of an insertion model in which the whole protein translocates into the midgut membrane.     

The Bacillus thuringiensis Cry1Aa toxin: effects of trypsin and chymotrypsin site mutations on toxicity and stability

The objective of the present work was to create an active Cry1Aa toxin showing enhanced resistance to degradation by spruce budworm (Choristoneura fumiferana) midgut proteases by mutating potential chymotrypsin and trypsin sites. Fourteen Cry1Aa mutants were created in an Escherichia coli-Bacillus shuttle vector and expressed in a crystal minus Bacillus thuringiensis host. Using spruce budworm gut juice, commercial bovine trypsin and chymotrypsin we performed protease resistance assays with Cry1Aa wild type and mutant toxins. Although many mutants showed little or no change, several mutants showed a > 2-fold increase (R543S, R566G, and F570S) up to a > 4-fold increase in toxicity (F576S), in bioassay studies against C. fumiferana. The in vitro protease resistance assay results indicated a possible involvement of other gut juice components in toxin overdigestion.     

Bacillus thuringiensis Cry4Aa insecticidal protein: Functional importance of the intrinsic stability of the unique α4–α5 loop comprising the Pro-rich sequence

The long loop connecting transmembrane α4 and α5 of the Bacillus thuringiensis Cry4Aa toxin possesses a unique feature with Pro-rich sequence (Pro¹⁹³Pro¹⁹⁴_Pro¹⁹⁶) which was shown to be crucial for toxicity. Here, the structural role in the intrinsic stability of the Pro-rich sequence toward toxin activity was investigated. Three Val-substituted mutants (P193V, P194V and P196V) and one Phe-substituted mutant (P193F) were generated and over-expressed in Escherichia coli as inclusions at levels equal to the wild-type. Bioassays demonstrated that all mutants, particularly P193V and P193F whose inclusions were hardly soluble in carbonate buffer (pH 9.0), exhibited reduced toxicity, suggesting an essential role in toxin function by the specific cyclic structure of individual Pro residues. Analysis of the 65-kDa Cry4Aa structure from 10-ns molecular dynamics (MD) simulations revealed that the α4–α5 loop is substantially stable as it showed low structural fluctuation with a 1.2-Å RMSF value. When the flexibility of the α4–α5 loop was increased through P193G, P194G and P196G substitutions, decreased toxicity was also observed for all mutants, mostly for the P193G mutant with low alkali-solubility, suggesting a functional importance of loop-rigidity attributed by individual Pro-cyclic side-chains, particularly Pro¹⁹³. Further MD simulations revealed that the most critical residue−Pro¹⁹³ for which mutations vastly affect toxin solubility and larval toxicity is in close contact with several surrounding residues, thus playing an additional role in the structural arrangement of the Cry4Aa toxin molecule. Altogether, our data signify that the intrinsic stability of the unique Cry4Aa α4–α5 loop structure comprising the Pro-rich sequence plays an important role in toxin activity.     

Bombyx mori ABC transporter C2 structures responsible for the receptor function of Bacillus thuringiensis Cry1Aa toxin

Because Bombyx mori ABC transporter C2 (BmABCC2) has 1000-fold higher potential than B. mori cadherin-like protein as a receptor for Bacillus thuringiensis Cry1Aa toxin (Tanaka et al., 2013), the gate-opening ability of the latent pore under six extracellular loops (ECLs) of BmABCC2 was expected to be the reason for its higher potential (Heckel, 2012). In this study, cell swelling assays in Sf9 cells showed that BmABCC2 mutants lacking substrate-excreting activity retained receptor activity, indicating that the gate-opening activity of BmABCC2 is not responsible for Cry1Aa toxicity. The analysis of 29 BmABCC2 mutants demonstrated that ⁷⁷⁰DYWL⁷⁷³ of ECL 4 comprise a putative binding site to Cry1Aa. This suggests that specific toxicity of Cry1Aa toxin to a restricted range of lepidopteran insects is dependent on conservation and variation in the amino acid residues around ⁷⁷⁰DYWL⁷⁷³ of ECL 4 in the ABCC2.     

Cadherin binding is not a limiting step for Bacillus thuringiensis subsp. israelensis Cry4Ba toxicity to Aedes aegypti larvae

Bacillus thuringiensis subsp. israelensis produces three Cry toxins (Cry4Aa, Cry4Ba and Cry11Aa) that are active against Aedes aegypti larvae. The identification of the rate-limiting binding steps of Cry toxins that are used for insect control in the field, such as those of B. thuringiensis subsp. israelensis, should provide targets for improving insecticides against important insect pests. Previous studies showed that Cry11Aa binds to cadherin receptor fragment CR7-11 (cadherin repeats 7-11) with high affinity. Binding to cadherin has been proposed to facilitate Cry toxin oligomer formation. In the present study, we show that Cry4Ba binds to CR7-11 with 9-fold lower binding affinity compared with Cry11Aa. Oligomerization assays showed that Cry4Ba is capable of forming oligomers when proteolytically activated in vitro in the absence of the CR7-11 fragment in contrast with Cry11Aa that formed oligomers only in the presence of CR7-11. Pore-formation assays in planar lipid bilayers showed that Cry4Ba oligomers were proficient in opening ion channels. Finally, silencing the cadherin gene by dsRNA (double-stranded RNA) showed that silenced larvae were more tolerant to Cry11Aa in contrast with Cry4Ba, which showed similar toxic levels to those of control larvae. These findings show that cadherin binding is not a limiting step for Cry4Ba toxicity to A. aegypti larvae.     

A system for the directed evolution of the insecticidal protein from Bacillus thuringiensis

Theoretically, the activity of AB-type toxin molecules such as the insecticidal toxin (Cry toxin) from B. thuringiensis, which have one active site and two binding site, is improved in parallel with the binding affinity to its receptor. In this experiment, we tried to devise a method for the directed evolution of Cry toxins to increase the binding affinity to the insect receptor. Using a commercial T7 phage-display system, we expressed Cry1Aa toxin on the phage surface as fusions with the capsid protein 10B. These recombinant phages bound to a cadherin-like protein that is one of the Cry1Aa toxin receptors in the model target insect Bombyx mori. The apparent affinity of Cry1Aa-expressing phage for the receptor was higher than that of Cry1Ab-expressing phage. Phages expressing Cry1Aa were isolated from a mixed suspension of phages expressing Cry1Ab and concentrated by up to 130,000-fold. Finally, random mutations were made in amino acid residues 369–375 in domain 2 of Cry1Aa toxin, the mutant toxins were expressed on phages, and the resulting phage library was screened with cadherin-like protein-coated beads. As a result, phages expressing abnormal or low-affinity mutant toxins were excluded, and phages with high-affinity mutant toxins were selected. These results indicate that a method combining T7 phage display with selection using cadherin-like protein-coated magnetic beads can be used to increase the activity of easily obtained, low-activity Cry toxins from bacteria.     

Bacillus thuringiensis ssp. israelensis Cyt1Aa enhances activity of Cry11Aa toxin by facilitating the formation of a pre-pore oligomeric structure

Bacillus thuringiensis ssp. israelensis (Bti) has been used worldwide for the control of dipteran insect pests. This bacterium produces several Cry and Cyt toxins that individually show activity against mosquitoes but together show synergistic effect. Previous work demonstrated that Cyt1Aa synergizes the toxic activity of Cry11Aa by functioning as a membrane-bound receptor. In the case of Cry toxins active against lepidopteran insects, receptor interaction triggers the formation of a pre-pore oligomer that is responsible for pore formation and toxicity. In this work we report that binding of Cry11Aa to Cyt1Aa facilitates the formation of a Cry11Aa pre-pore oligomeric structure that is capable of forming pores in membrane vesicles. Cry11Aa and Cyt1A point mutants affected in binding and in synergism had a correlative effect on the formation of Cry11Aa pre-pore oligomer and on pore-formation activity of Cry11Aa. These data further support that Cyt1Aa interacts with Cry11Aa and demonstrate the molecular mechanism by which Cyt1Aa synergizes or suppresses resistance to Cry11Aa, by providing a binding site for Cry11Aa that will result in an efficient formation of Cry11Aa pre-pore that inserts into membranes and forms ionic pores.     

The alpha-helix 4 residue, Asn135, is involved in the oligomerization of Cry1Ac1 and Cry1Ab5 Bacillus thuringiensis toxins.

The insecticidal Cry toxins produced by the bacterium Bacillus thuringiensis are comprised of three structural domains. Domain I, a seven-helix bundle, is thought to penetrate the insect epithelial cell plasma membrane through a hairpin composed of alpha-helices 4 and 5, followed by the oligomerization of four hairpin monomers. The alpha-helix 4 has been proposed to line the lumen of the pore, whereas some residues in alpha-helix 5 have been shown to be responsible for oligomerization. Mutation of the Cry1Ac1 alpha-helix 4 amino acid Asn135 to Gln resulted in the loss of toxicity to Manduca sexta, yet binding was still observed. In this study, the equivalent mutation was made in the Cry1Ab5 toxin, and the properties of both wild-type and mutant toxin counterparts were analyzed. Both mutants appeared to bind to M. sexta membrane vesicles, but they were not able to form pores. The ability of both N135Q mutants to oligomerize was also disrupted, providing the first evidence that a residue in alpha-helix 4 can contribute to toxin oligomerization.     

Cysteine scanning mutagenesis of alpha4, a putative pore-lining helix of the Bacillus thuringiensis insecticidal toxin Cry1Aa

Helix alpha4 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 alpha4 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.     

IDENTIFICATION OF A CALMODULIN-BINDING SITE WITHIN THE DOMAIN I OF BACILLUS THURINGIENSIS Cry3Aa TOXIN

Bacillus thuringiensis Cry3Aa toxin is a coleopteran specific toxin highly active against Colorado Potato Beetle (CPB).We have recently shown thatCry3Aa toxin is proteolytically cleaved by CPBmidgut membrane associated metalloproteases and that this cleavage is inhibited by ADAMmetalloprotease inhibitors. In the present study, we investigated whether the Cry3Aa toxin is a calmodulin (CaM) binding protein, as it is the case of several different ADAMshedding substrates. In pull-down assays using agarose beads conjugated with CaM, we demonstrated that Cry3Aa toxin specifically binds to CaMin a calcium-independent manner. Furthermore, we used gel shift assays and (1) H NMRspectra to demonstrate that CaMbinds to a 16-amino acid synthetic peptide corresponding to residues N256-V271 within the domain I of Cry3Aa toxin. Finally, to investigate whether CaM has any effect on Cry3Aa toxin CPBmidgut membrane associated proteolysis, cleavage assays were performed in the presence of the CaM-specific inhibitor trifluoperazine. We showed that trifluoperazine significantly increased Cry3Aa toxin proteolysis and also decreased Cry3Aa larval toxicity.