Kinetics of pore formation by the Bacillus thuringiensis toxin Cry1Ac

After binding to specific receptors, Cry toxins form pores in the midgut apical membrane of susceptible insects. The receptors could form part of the pore structure or simply catalyze pore formation and consequently be recycled. To discriminate between these possibilities, the kinetics of pore formation in brush border membrane vesicles isolated from Manduca sexta was studied with an osmotic swelling assay. Pore formation, as deduced from changes in membrane permeability induced by Cry1Ac during a 60-min incubation period, was strongly dose-dependent, but rapidly reached a maximum as toxin concentration was increased. Following exposure of the vesicles to the toxin, the osmotic swelling rate reached a maximum shortly after a delay period. Under these conditions, at relatively high toxin concentrations, the maximal osmotic swelling rate increased linearly with toxin concentration. When vesicles were incubated for a short time with the toxin and then rapidly cooled to prevent the formation of new pores before and during the osmotic swelling experiment, a plateau in the rate of pore formation was observed as toxin concentration was increased. Taken together, these results suggest that the receptors do not act as simple catalysts of pore formation, but remain associated with the pores once they are formed.     

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.     

Antagonism between Cry1Ac1 and Cyt1A1 toxins of bacillus thuringiensis

Most strains of the insecticidal bacterium Bacillus thuringiensis have a combination of different protoxins in their parasporal crystals. Some of the combinations clearly interact synergistically, like the toxins present in B. thuringiensis subsp. israelensis. In this paper we describe a novel joint activity of toxins from different strains of B. thuringiensis. In vitro bioassays in which we used pure, trypsin-activated Cry1Ac1 proteins from B. thuringiensis subsp. kurstaki, Cyt1A1 from B. thuringiensis subsp. israelensis, and Trichoplusia ni BTI-Tn5B1-4 cells revealed contrasting susceptibility characteristics. The 50% lethal concentrations (LC50s) were estimated to be 4,967 of Cry1Ac1 per ml of medium and 11.69 ng of Cyt1A1 per ml of medium. When mixtures of these toxins in different proportions were assayed, eight different LC50s were obtained. All of these LC50s were significantly higher than the expected LC50s of the mixtures. In addition, a series of bioassays were performed with late first-instar larvae of the cabbage looper and pure Cry1Ac1 and Cyt1A1 crystals, as well as two different combinations of the two toxins. The estimated mean LC50 of Cry1Ac1 was 2.46 ng/cm2 of diet, while Cyt1A1 crystals exhibited no toxicity, even at very high concentrations. The estimated mean LC50s of Cry1Ac1 crystals were 15.69 and 19.05 ng per cm2 of diet when these crystals were mixed with 100 and 1,000 ng of Cyt1A1 crystals per cm2 of diet, respectively. These results indicate that there is clear antagonism between the two toxins both in vitro and in vivo. Other joint-action analyses corroborated these results. Although this is the second report of antagonism between B. thuringiensis toxins, our evidence is the first evidence of antagonism between toxins from different subspecies of B. thuringiensis (B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. israelensis) detected both in vivo and in vitro. Some possible explanations for this relationship are discussed.     

Genetics of pink bollworm resistance to Bacillus thuringiensis toxin Cry1Ac

Laboratory selection increased resistance of pink bollworm (Pectinophora gossypiella) to the Bacillus thuringiensis toxin Cry1Ac. Three selections with Cry1Ac in artificial diet increased resistance from a low level to >100-fold relative to a susceptible strain. We used artificial diet bioassays to test F1 hybrid progeny from reciprocal crosses between resistant and susceptible strains. The similarity between F1 progeny from the two reciprocal crosses indicates autosomal inheritance of resistance. The dominance of resistance to Cry1Ac depended on the concentration. Resistance was codominant at a low concentration of Cry1Ac, partially recessive at an intermediate concentration, and completely recessive at a high concentration. Comparison of the artificial diet results with previously reported results from greenhouse bioassays shows that the high concentration of Cry1Ac in bolls of transgenic cotton is essential for achieving functionally recessive inheritance of resistance.     

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

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 alpha-3 from Cry toxins could form coiled-coils. Our data shows that different mutations in helix alpha-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.     

Cadherin-like receptor binding facilitates proteolytic cleavage of helix alpha-1 in domain I and oligomer pre-pore formation of Bacillus thuringiensis Cry1Ab toxin

Cry toxins form lytic pores in the insect midgut cells. The role of receptor interaction in the process of protoxin activation was analyzed. Incubation of Cry1Ab protoxin with a single chain antibody that mimics the cadherin-like receptor and treatment with Manduca sexta midgut juice or trypsin, resulted in toxin preparations with high pore-forming activity in vitro. This activity correlates with the formation of a 250 kDa oligomer that lacks the helix alpha-1 of domain I. The oligomer, in contrast with the 60 kDa monomer, was capable of membrane insertion as judged by 8-anilino-1-naphthalenesulfonate binding. Cry1Ab protoxin was also activated to a 250 kDa oligomer by incubation with brush border membrane vesicles, presumably by the action of a membrane-associated protease. Finally, a model where receptor binding allows the efficient cleavage of alpha-1 and formation of a pre-pore oligomeric structure that is efficient in pore formation, is presented.     

Structural and functional analysis of the pre-pore and membrane-inserted pore of Cry1Ab toxin

Bacillus thuringiensis produces insecticidal Cry proteins that are active against different insect species. The primary action of Cry toxins is to lyse midgut epithelial cells in the target insect by forming lytic pores on the apical membrane. After interaction with cadherin receptor, Cry proteins undergo conformational changes from a monomeric structure to a pre-pore-oligomeric form that is able to interact with a second GPI-anchored aminopeptidase-N receptor and then insert into lipid membranes. Here, we review the recent advances in the understanding of the structural changes presented by Cry1Ab toxin upon membrane insertion. Based on analysis of the Trp fluorescence of pure monomeric and oligomeric Cry1Ab structures in solution and in membrane-bound state we reported that oligomerization caused 27% reduction of Trp exposed to the solvent. After membrane insertion there is another conformational change that allows an additional rearrangement of the Trp residues resulting in a total protection of these residues from exposure to the solvent. The oligomeric structure is membrane insertion competent since more than 96% of the Cry1Ab oligomer inserts into the membrane as a function of lipid:protein ratio, in contrast to the monomer of which only 5-10%, inserts into the membrane. Finally, analysis of the stability of monomeric, pre-pore and pore structures of Cry1Ab toxin after urea and thermal denaturation suggested that a more flexible conformation could be necessary for membrane insertion and this flexible structure is obtained by toxin oligomerization and by alkaline pH. Domain I is involved in the intermolecular interaction within the oligomeric Cry1Ab and this domain is inserted into the membrane in the membrane-inserted state.     

Functional display of Bacillus thuringiensis Cry1Ac toxin on T7 phage

The Cry1Ac toxin from Bacillus thuringiensis was displayed on the surface of T7 phage. The cry1Ac gene was fused to the C-terminal end of T7-10B capsid protein and displayed on the surface of T7 phage as revealed by Western blot analysis of the purified phage particles. The T7-Cry1Ac phages retained toxicity against Manduca sexta larvae. We demonstrated that the T7-Cry1Ac phage interacts with Cry1Ac receptors present in M. sexta BBMV either in solution or in overlay binding assays.     

Unfolding events in the water-soluble monomeric Cry1Ab toxin during transition to oligomeric pre-pore and membrane-inserted pore channel

The insecticidal crystal (Cry) proteins produced by Bacillus thuringiensis undergo several conformational changes from crystal inclusion protoxins to membrane-inserted channels in the midgut epithelial cells of the target insect. Here we analyzed the stability of the different forms of Cry1Ab toxin, monomeric toxin, pre-pore complex, and membrane-inserted channel, after urea and thermal denaturation by monitoring intrinsic tryptophan fluorescence of the protein and 1-anilinonaphthalene-8-sulfonic acid binding to partially unfolded proteins. Our results showed that flexibility of the monomeric toxin was dramatically enhanced upon oligomerization and was even further increased by insertion of the pre-pore into the membrane as shown by the lower concentration of chaotropic agents needed to achieve unfolding of the oligomeric species. The flexibility of the toxin structures is further increased by alkaline pH. We found that the monomer-monomer interaction in the pre-pore is highly stable because urea promotes oligomer denaturation without disassembly. Partial unfolding and limited proteolysis studies demonstrated that domains II and III were less stable and unfold first, followed by unfolding of the most stable domain I, and also that domain I is involved in monomer-monomer interaction. The thermal-induced unfolding and analysis of energy transfer from Trp residues to bound 1-anilinonaphthalene-8-sulfonic acid dye showed that in the membrane-inserted pore domains II and III are particularly sensitive to heat denaturation, in contrast to domain I, suggesting that only domain I may be inserted into the membrane. Finally, the insertion into the membrane of the oligomeric pre-pore structure was not affected by pH. However, a looser conformation of the membrane-inserted domain I induced by neutral or alkaline pH correlates with active channel formation. Our studies suggest for the first time that a more flexible conformation of Cry toxin could be necessary for membrane insertion, and this flexible structure is induced by toxin oligomerization. Finally the alkaline pH found in the midgut lumen of lepidopteran insects could increase the flexibility of membrane-inserted domain I necessary for pore formation.     

Bacillus thuringiensis Cry1Ac and Cry1Fa delta-endotoxin binding to a novel 110 kDa aminopeptidase in Heliothis virescens is not N-acetylgalactosamine mediated.

We determined that Bacillus thuringiensis Cry1Ac and Cry1Fa delta-endotoxins recognize the same 110, 120 and 170 kDa aminopeptidase N (APN) molecules in brush border membrane vesicles (BBMV) from Heliothis virescens. The 110 kDa protein, not previously identified as an APN, contained a variant APN consensus sequence identical to that found in Helicoverpa punctigera APN 2. PCR amplification of H. virescens cDNA based on this sequence and a conserved APN motif yielded a 0.9 kb product that has 89% sequence homology with H. punctigera APN 2. Western blots revealed that the 110 kDa molecule was not recognized by soybean agglutinin, indicating the absence of GalNAc. A 125I labeled-Cry1Ac domain III mutant (509QNR(511)-AAA) that has an altered GalNAc binding pocket (Lee et al., Appl. Environ. Microbiol. 65 (1999) 4513) showed abolished binding to the 120 APN, reduced binding to the 170 kDa APN, and enhanced binding to the 110 kDa APN. Periodate treated H. virescens BBMV blots were also probed with 125I labeled-Cry1Ac and 509QNR(511)-AAA toxins. Both toxins still recognized the 110 kDa APN and a >210 kDa molecule which may be a cadherin-like protein. Additionally, 125I-(509)QNR(511)-AAA recognized periodate treated 170 kDa APN. Results indicate that the 110 kDa APN is distinct from other Cry1 toxin binding APNs and may be the first described Cry1Ac-binding APN that does not contain GalNAc.     

Binding of Bacillus thuringiensis toxin Cry1Ac to multiple sites of cadherin in pink bollworm

Toxins from Bacillus thuringiensis (Bt) are widely used for pest control. In particular, Bt toxin Cry1Ac produced by transgenic cotton kills some key lepidopteran pests. We found that Cry1Ac binds to recombinant peptides corresponding to extracellular regions of a cadherin protein (BtR) in a major cotton pest, pink bollworm (Pectinophora gossypiella) (PBW). In conjunction with previous results showing that PBW resistance to Cry1Ac is linked with mutations in the BtR gene, the results reported here support the hypothesis that BtR is a receptor for Cry1Ac in PBW. Similar to other lepidopteran cadherins that bind Bt toxins, BtR has at least two Cry1Ac-binding domains in cadherin-repeat regions 10 and 11, which are immediately adjacent to the membrane proximal region. However, unlike cadherins from Manduca sexta and Bombyx mori, toxin binding was not seen in regions more distal from the membrane proximal region. We also found that both the protoxin and activated toxin forms of Cry1Ac bound to recombinant BtR fragments, suggesting that Cry1Ac activation may occur either before or after receptor binding.     

抗苏云金杆菌毒素Cry1Ac粉纹夜蛾细胞系的特性研究

为了从离体细胞水平探讨昆虫对苏云金芽孢杆菌杀虫晶体蛋白的部分抗性机制,本文采用活化的Cry1AC毒素对粉纹夜蛾BTI-TN-581-4细胞连续筛选86代,获得了高水平抗性细胞,研究了其某些特性。它对Cry1C产生了低水平的交互抗性,对低渗溶液的耐受性显著增强,双向电泳图谱表明抗性细胞膜蛋白组分发生了明显的变化。膜蛋白组分的变化可能导致了筛选细胞的耐低渗透压和抗Cry1C。     

Mutated cadherin alleles from a field population of Helicoverpa armigera confer resistance to Bacillus thuringiensis toxin Cry1Ac

The cotton bollworm Helicoverpa armigera is the major insect pest targeted by cotton genetically engineered to produce the Bacillus thuringiensis toxin (transgenic Bt cotton) in the Old World. The evolution of this pest's resistance to B. thuringiensis toxins is the main threat to the long-term effectiveness of transgenic Bt cotton. A deletion mutation allele (r(1)) of a cadherin gene (Ha_BtR) was previously identified as genetically linked with Cry1Ac resistance in a laboratory-selected strain of H. armigera. Using a biphasic screen strategy, we successfully trapped two new cadherin alleles (r(2) and r(3)) associated with Cry1Ac resistance from a field population of H. armigera collected from the Yellow River cotton area of China in 2005. The r(2) and r(3) alleles, respectively, were created by inserting the long terminal repeat of a retrotransposon (designated HaRT1) and the intact HaRT1 retrotransposon at the same position in exon 8 of Ha_BtR, which results in a truncated cadherin containing only two ectodomain repeats in the N terminus of Ha_BtR. This is the first time that the B. thuringiensis resistance alleles of a target insect of Bt crops have been successfully detected in the open field. This study also demonstrated that bollworm larvae carrying two resistance alleles can complete development on Bt cotton. The cadherin locus should be an important target for intensive DNA-based screening of field populations of H. armigera.     

家蚕氨肽酶和类钙粘蛋白与苏云金芽孢杆菌Cry1Ac毒素相互作用

苏云金芽孢杆菌Bacillus thuringiensis生产的晶体毒素被广泛用作农林害虫的杀虫剂。鳞翅目昆虫受体蛋白是阐明其与晶体毒素相互作用的重要模式。文中纯化了苏云金芽孢杆菌的晶体毒素蛋白,质谱鉴定为Cry1Ac毒素,然后重组表达家蚕氨肽酶N (BmAPN6) 和类钙粘蛋白 (CaLP) 结合结构域。利用免疫共沉淀、Far-Western印迹和酶联免疫吸附试验,证明Cry1Ac毒素蛋白和BmAPN6之间的相互作用。在Sf9细胞中,对Cry1Ac毒素的细胞毒活性分析,表明BmAPN6参与Cry1Ac毒素诱导的细胞形态异常和裂解死亡。文中也利用相同的方法,对钙粘蛋白的3个结合位点CR7、CR11和CR12进行相互作用分析,结果表明3个重复结构域是CaLP的Cry1Ac结合位点。上述结果表明,BmAPN6和CaLP可作为Cry1Ac毒素致病的功能性受体,为进一步揭示晶体毒素的致病机制和基因编辑增强家蚕抗病性提供了研究靶标。     

An ABC transporter mutation is correlated with insect resistance to Bacillus thuringiensis Cry1Ac toxin

Transgenic crops producing insecticidal toxins from Bacillus thuringiensis (Bt) are commercially successful in reducing pest damage, yet knowledge of resistance mechanisms that threaten their sustainability is incomplete. Insect resistance to the pore-forming Cry1Ac toxin is correlated with the loss of high-affinity, irreversible binding to the mid-gut membrane, but the genetic factors responsible for this change have been elusive. Mutations in a 12-cadherin-domain protein confer some Cry1Ac resistance but do not block this toxin binding in in vitro assays. We sought to identify mutations in other genes that might be responsible for the loss of binding. We employed a map-based cloning approach using a series of backcrosses with 1,060 progeny to identify a resistance gene in the cotton pest Heliothis virescens that segregated independently from the cadherin mutation. We found an inactivating mutation of the ABC transporter ABCC2 that is genetically linked to Cry1Ac resistance and is correlated with loss of Cry1Ac binding to membrane vesicles. ABC proteins are integral membrane proteins with many functions, including export of toxic molecules from the cell, but have not been implicated in the mode of action of Bt toxins before. The reduction in toxin binding due to the inactivating mutation suggests that ABCC2 is involved in membrane integration of the toxin pore. Our findings suggest that ABC proteins may play a key role in the mode of action of Bt toxins and that ABC protein mutations can confer high levels of resistance that could threaten the continued utilization of Bt-expressing crops. However, such mutations may impose a physiological cost on resistant insects, by reducing export of other toxins such as plant secondary compounds from the cell. This weakness could be exploited to manage this mechanism of Bt resistance in the field.     

New resistance mechanism in Helicoverpa armigera threatens transgenic crops expressing Bacillus thuringiensis Cry1Ac toxin

In Australia, the cotton bollworm, Helicoverpa armigera, has a long history of resistance to conventional insecticides. Transgenic cotton (expressing the Bacillus thuringiensis toxin Cry1Ac) has been grown for H. armigera control since 1996. It is demonstrated here that a population of Australian H. armigera has developed resistance to Cry1Ac toxin (275-fold). Some 70% of resistant H. armigera larvae were able to survive on Cry1Ac transgenic cotton (Ingard) The resistance phenotype is inherited as an autosomal semidominant trait. Resistance was associated with elevated esterase levels, which cosegregated with resistance. In vitro studies employing surface plasmon resonance technology and other biochemical techniques demonstrated that resistant strain esterase could bind to Cry1Ac protoxin and activated toxin. In vivo studies showed that Cry1Ac-resistant larvae fed Cy1Ac transgenic cotton or Cry1Ac-treated artificial diet had lower esterase activity than non-Cry1Ac-fed larvae. A resistance mechanism in which esterase sequesters Cry1Ac is proposed.     

一些化学物质对苏云金芽孢杆菌Cry1Ac毒素与昆虫离体细胞相互作用的影响

为探讨苏云金芽孢杆菌Bacillus thuringiensis（Bt）杀虫晶体蛋白与昆虫细胞的相互作用,以Bt Cry1Ac毒素和对该毒素敏感的粉纹夜蛾Trichoplusia ni离体细胞BTI-TN-5B1-4为材料,研究了一些化学物质对Cry1Ac毒素与昆虫离体细胞相互作用的影响.结果表明：N-糖基化抑制剂衣霉素、蛋白质合成抑制剂放线菌酮、胞吞作用抑制剂莫能菌素和胰蛋白酶预处理,都能不同程度地提高BTI-TN-5B1-4细胞对Cry1Ac毒素的敏感性,其中胰蛋白酶预处理的作用最明显;而N-乙酰半乳糖胺不能抑制Cry1Ac毒素对这种离体细胞的毒力.     

Binding of Bacillus thuringiensis Cry1A toxins to brush border membrane vesicles of midgut from Cry1Ac susceptible and resistant Plutella xylostella

Plutella xylostella strain resistant (PXR) to Bacillus thuringiensis Cry1Ac toxin was not killed at even more than 1000 μg Cry1Ac/g diet but killed by Cry1Ab at 0.5 μg/g diet. In contrast, susceptible strain (PXS) was killed by Cry1Ac at 1 μg/g diet. Cy3-labeld Cry1A(s) binding to brush border membrane vesicles (BBMV) prepared from both strains were analyzed with direct binding assay. The Kd value of Cry1Aa to both BBMV was almost identical: 213.2 and 205.8 nM, and 263.5 and 265.0 nM for Cry1Ac. The highest Kd values were in Cry1Ab which showed most effective insecticidal activity in PXS and PXR, 2126 and 2463 nM, respectively. These results clearly showed that the BBMV from PXR and PXS could equally bind to Cry1Ac. The binding between BBMV and Cy3-labeled Cry1Ac was inhibited only by anti-175 kDa cadherin-like protein (CadLP) and -252 kDa protein antisera, but not by anti-120 kDa aminopeptidase. This supports that resistance in PXR resulted from the abortion of pore formation after the binding of Cry1Ac to the BBMV. And furthermore, the importance of 175K CadLP and P252 proteins in those bindings was suggested. We briefly discuss possible mechanisms of the resistance.     

Effects of entomopathogenic nematodes on evolution of pink bollworm resistance to Bacillus thuringiensis toxin Cry1Ac

The evolution of resistance by pests can reduce the efficacy of transgenic crops that produce insecticidal toxins from Bacillus thuringiensis (Bt). However, fitness costs may act to delay pest resistance to Bt toxins. Meta-analysis of results from four previous studies revealed that the entomopathogenic nematode Steinernema riobrave (Rhabditida: Steinernematidae) imposed a 20% fitness cost for larvae of pink bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae), that were homozygous for resistance to Bt toxin Cry1Ac, but no significant fitness cost was detected for heterozygotes. We conducted greenhouse and laboratory selection experiments to determine whether S. riobrave would delay the evolution of pink bollworm resistance to Cry1Ac. We mimicked the high dose/refuge scenario in the greenhouse with Bt cotton (Gossypium hirsutum L.) plants and refuges of non-Bt cotton plants, and in the laboratory with diet containing Cry1Ac and refuges of untreated diet. In both experiments, half of the replicates were exposed to S. riobrave and half were not. In the greenhouse, S. riobrave did not delay resistance. In the laboratory, S. riobrave delayed resistance after two generations but not after four generations. Simulation modeling showed that an initial resistance allele frequency > 0.015 and population bottlenecks can diminish or eliminate the resistance-delaying effects of fitness costs. We hypothesize that these factors may have reduced the resistance-delaying effects of S. riobrave in the selection experiments. The experimental and modeling results suggest that entomopathogenic nematodes could slow the evolution of pest resistance to Bt crops, but only under some conditions.