Removal of Adsorbed Toxin Fragments That Modify Bacillus thuringiensis CryIC δ-Endotoxin Iodination and Binding by Sodium Dodecyl Sulfate Treatment and Renaturation

We report that 10- and 25-kDa toxin fragments adhere to CryIC prepared from Bacillus thuringiensis insecticidal crystals, block iodination, and alter membrane binding. There is no apparent affect on CryIC toxicity against Spodoptera exigua. Associated peptides remained bound to CryIC in the presence of 50 mM dithiothreitol or 6 M urea. A novel detergent-renaturation procedure was developed for the purification of B. thuringiensis CryIC toxin. Sodium dodecyl sulfate (SDS) treatment followed by gel filtration chromatography yielded a homogeneous 62-kDa CryIC toxin. After removal of SDS and renaturation, the purified CryIC toxin was fully insecticidal to S. exigua larvae. ¹²⁵I-labeled CryIC bound with high affinity to brush border membrane vesicles from S. exigua larvae.     

Development of Bacillus thuringiensis CryIC Resistance by Spodoptera exigua (Hubner) (Lepidoptera: Noctuidae)

Selection of resistance in Spodoptera exigua (Hubner) to an HD-1 spore-crystal mixture, CryIC (HD-133) inclusion bodies, and trypsinized toxin from Bacillus thuringiensis subsp. aizawai and B. thuringiensis subsp. entomocidus was attempted by using laboratory bioassays. No resistance to the HD-1 spore-crystal mixture could be achieved after 20 generations of selection. Significant levels of resistance (11-fold) to CryIC inclusion bodies expressed in Escherichia coli were observed after seven generations. Subsequent selection of the CryIC-resistant population with trypsinized CryIC toxin resulted, after 21 generations of CryIC selection, in a population of S. exigua that exhibited only 8% mortality at the highest toxin concentration tested (320 (mu)g/g), whereas the 50% lethal concentration was 4.30 (mu)g/g for the susceptible colony. Insects resistant to CryIC toxin from HD-133 also were resistant to trypsinized CryIA(b), CryIC from B. thuringiensis subsp. entomocidus, CryIE-CryIC fusion protein (G27), CryIH, and CryIIA. In vitro binding experiments with brush border membrane vesicles showed a twofold decrease in maximum CryIC binding, a fivefold difference in K(infd), and no difference in the concentration of binding sites for the CryIC-resistant insects compared with those for the susceptible insects. Resistance to CryIC was significantly reduced by the addition of HD-1 spores. Resistance to the CryIC toxin was still observed 12 generations after CryIC selection was removed. These results suggest that, in S. exigua, resistance to a single protein is more likely to occur than resistance to spore-crystal mixtures and that once resistance occurs, insects will be resistant to many other Cry proteins. These results have important implications for devising S. exigua resistance management strategies in the field.     

Domain III substitution in Bacillus thuringiensis delta-endotoxin CryIA(b) results in superior toxicity for Spodoptera exigua and altered membrane protein recognition.

To test our hypothesis that substitution of domain III of Bacillus thuringiensis delta-endotoxin (Cry) proteins might improve toxicity to pest insects, e.g., Spodoptera exigua, in vivo recombination was used to produce a number of cryIA(b)-cryIC hybrid genes. A rapid screening assay was subsequently exploited to select hybrid genes encoding soluble protoxins. Screening of 120 recombinants yielded two different hybrid genes encoding soluble proteins with domains I and II of CryIA(b) and domain III of CryIC. These proteins differed by only one amino acid residue. Both hybrid protoxins gave a protease-resistant toxin upon in vitro activation by trypsin. Bioassays showed that one of these CryIA(b)-CryIC hybrid proteins (H04) was highly toxic to S. exigua compared with the parental CryIA(b) protein and significantly more toxic than CryIC. In semiquantitative binding studies with biotin-labelled toxins and intact brush border membrane vesicles of S. exigua, this domain III substitution appeared not to affect binding-site specificity. However, binding to a 200-kDa protein by CryIA(b) in preparations of solubilized and blotted brush border membrane vesicle proteins was completely abolished by the domain III substitution. A reciprocal hybrid containing domains I and II of CryIC and domain III of CryIA(b) did bind to the 200-kDa protein, confirming that domain III of CryIA(b) was essential for this reaction. These results show that domain III of CryIC protein plays an important role in the level of toxicity to S. exigua, that substitution of domain III may be a powerful tool to increase the repertoire of available active toxins for pest insects, and that domain III is involved in binding to gut epithelium membrane proteins of S. exigua.     

Insecticidal properties of a crystal protein gene product isolated from Bacillus thuringiensis subsp. kenyae.

A protoxin gene, localized to a high-molecular-weight plasmid from Bacillus thuringiensis subsp. kenyae, was cloned on a 19-kb BamHI DNA fragment into Escherichia coli. Characterization of the gene revealed it to be a member of the CryIE toxin subclass which has been reported to be as toxic as the CryIC subclass to larvae from Spodoptera exigua in assays with crude E. coli extracts. To directly test the purified recombinant gene product, the gene was subcloned as a 4.8-kb fragment into an expression vector resulting in the overexpression of a 134-kDa protein in the form of phase-bright inclusions in E. coli. Treatment of solubilized inclusion bodies with either trypsin or gut juice from the silkworm Bombyx mori resulted in the appearance of a protease-resistant 65-kDa protein. In force-feeding bioassays, the purified activated protein was highly toxic to larvae of B. mori but not to larvae of Choristoneura fumiferana. In diet bioassays with larvae from S. exigua, the purified protoxin was nontoxic. However, prior activation of the protoxin by tryptic digestion resulted in the appearance of some toxic activity. These results demonstrate that this new subclass of protein toxin may not be useful for the control of Spodoptera species as previously reported. Hierarchical clustering of the nine known lepidopteran-specific CryI toxin subclasses through multiple sequence alignment suggests that the toxins fall into four possible subgroups or clusters.     

Gene knockout demonstrates that vip3A contributes to the pathogenesis of Bacillus thuringiensis toward Agrotis ipsilon and Spodoptera exigua

Vip3A is an 89-kDa protein secreted by Bacillus thuringiensis during vegetative growth. To determine the importance of Vip3A for the insect pathogenicity of B. thuringiensis the vip3A gene was deleted from strain HD1, yielding strain HD1Deltavip3A. Compared with HD1, strain HD1Deltavip3A was one-fourth as toxic to Agrotis ipsilon larvae and less than one-tenth as toxic to Spodoptera exigua larvae. When streptomycin was included in the S. exigua diet the toxicity of HD1Deltavip3A was approximately half that of HD1. Addition of HD1 spores increased the toxicity of purified Cry1 protein more than 600-fold against S. exigua, whereas addition of HD1Deltavip3A spores increased toxicity of Cry1 protein approximately 10-fold. These results demonstrate that an important component of B. thuringiensis insecticidal activity against S. exigua is the synthesis of Vip3A protein by B. thuringiensis cells after ingestion of spores and crystal proteins by insect larvae.     

Mosquitocidal activity of the CryIC delta-endotoxin from Bacillus thuringiensis subsp. aizawai.

The cloned 135-kDa CryIC delta-endotoxin from Bacillus thuringiensis is a lepidopteran-active toxin, displaying high activity in vivo against Spodoptera litoralis and Spodoptera frugiperda larvae and in vitro against the S. frugiperda Sf9 cell line. Here, we report that the CryIC delta-endotoxin cloned from B. thuringienesis subsp. aizawai HD-229 and expressed in an acrystalliferous B. thuringiensis strain is also toxic to Aedes aegypti, Anophles gambiae, and Culex quinquefasciatus mosquito larvae. Furthermore, when solubilized and proteolytically activated by insect gut extracts, CryIC is cytotoxic to cell lines derived from the first two of these dipteran insects. This activity was not observed for two other lepidopteran-active delta-endotoxins, CryIA(a) and CryIA(c). However, in contrast to the case with a lepidopteran and dipteran delta-endotoxin cloned from B. thuringiensis subsp. aizawai IC1 (M.Z. Haider, B. H. Knowles, and D. J. Ellar, Eur. J. Biochem. 156:531-540, 1986), no differences in the in vitro specificity or processing of CryIC were found when it was activated by lepidopteran or dipteran gut extract. The recombinant CryIC delta-endotoxin expressed in Escherichia coli was also toxic to A. aegypti larvae. By contrast, a second cryIC gene cloned from B. thuringiensis subsp. aizawai 7.29 (V. Sanchis, D. Lereclus, G. Menou, J. Chaufaux, S. Guo, and M. M. Lecadet, Mol. Microbiol. 3:229-238, 1989) was nontoxic. DNA sequencing showed that the two genes were identical. However, CryIC from B. thuringiensis subsp. aizawai 7.29 had been cloned with a truncated C terminus, and when it was compared with the full-length CryIC delta-endotoxin, it was found to be insoluble under alkaline reducing conditions. These results show that CryIC from B. thuringiensis subsp. aizawai is a dually active delta-endotoxin.     

Identification of putative insect brush border membrane-binding molecules specific to Bacillus thuringiensis delta-endotoxin by protein blot analysis.

Binding sites for insecticidal toxins of Bacillus thuringiensis are located in the brush border membranes of insect midguts. Two approaches were used to investigate the interactions of B. thuringiensis subsp. kurstaki HD-73 CryIA(c) toxin with brush border membrane vesicles from sensitive and naturally resistant insects: 125I-toxin-vesicle binding assays and protein blots probed with 125I-CryIA(c) toxin. In bioassays, Manduca sexta and Heliothis virescens larvae were highly sensitive, Helicoverpa zea larvae were moderately sensitive, and Spodoptera frugiperda larvae were resistant to CryIA(c) toxin. Studies of binding of 125I-CryIA(c) toxin to brush border membrane vesicles from the larval midguts revealed that all insects tested had high-affinity, saturable binding sites. Significantly, S. frugiperda larvae bind but are not killed by CryIA(c) toxin. Labeled CryIA(c) toxin incubated with protein blots identifies a major binding molecule of 120 kDa for M. sexta and 148 kDa for S. frugiperda. H. virescens and H. zea are more complex, containing 155-, 120-, 103-, 90-, and 63-kDa proteins as putative toxin-binding molecules. H. virescens also contains a minor toxin-binding protein of 81 kDa. These experiments provide information that can be applied toward a more detailed characterization of B. thuringiensis toxin-binding proteins.     

Identification of putative insect brush border membrane-binding molecules specific to Bacillus thuringiensis delta-endotoxin by protein blot analysis.

Binding sites for insecticidal toxins of Bacillus thuringiensis are located in the brush border membranes of insect midguts. Two approaches were used to investigate the interactions of B. thuringiensis subsp. kurstaki HD-73 CryIA(c) toxin with brush border membrane vesicles from sensitive and naturally resistant insects: 125I-toxin-vesicle binding assays and protein blots probed with 125I-CryIA(c) toxin. In bioassays, Manduca sexta and Heliothis virescens larvae were highly sensitive, Helicoverpa zea larvae were moderately sensitive, and Spodoptera frugiperda larvae were resistant to CryIA(c) toxin. Studies of binding of 125I-CryIA(c) toxin to brush border membrane vesicles from the larval midguts revealed that all insects tested had high-affinity, saturable binding sites. Significantly, S. frugiperda larvae bind but are not killed by CryIA(c) toxin. Labeled CryIA(c) toxin incubated with protein blots identifies a major binding molecule of 120 kDa for M. sexta and 148 kDa for S. frugiperda. H. virescens and H. zea are more complex, containing 155-, 120-, 103-, 90-, and 63-kDa proteins as putative toxin-binding molecules. H. virescens also contains a minor toxin-binding protein of 81 kDa. These experiments provide information that can be applied toward a more detailed characterization of B. thuringiensis toxin-binding proteins.     

Synergistic activity of a Bacillus thuringiensis delta-endotoxin and a bacterial endochitinase against Spodoptera littoralis larvae.

In an attempt to increase the insecticidal effect of the delta-endotoxin crystal protein CryIC on the relatively Cry-insensitive larvae of Spodoptera littoralis, a combination of CryIC and endochitinase was used. CryIC comprising the first 756 amino acids from Bacillus thuringiensis K26-21 and endochitinase ChiAII encoded by Serratia marcescens were separately produced in Escherichia coli carrying the genes in overexpression vectors. The endochitinase on its own, even at very low concentrations (0.1 microgram/ml), perforated the larval midgut peritrophic membrane. When applied together with low concentrations of CryIC, a synergistic toxic effect was obtained. In the absence of chitinase, about 20 micrograms of CryIC per ml was required to obtain maximal reduction in larval weight, while only 3.0 micrograms of CryIC per ml caused a similar toxic effect in the presence of endochitinase. Thus, a combination of the Cry protein and an endochitinase could result in effective insect control in transgenic systems in which the Cry protein is not expressed in a crystalline form.     

Early response of cultured lepidopteran cells to exposure to delta-endotoxin from Bacillus thuringiensis: involvement of calcium and anionic channels.

The role of ion channels in the initial steps following exposure of SF-9 lepidopteran insect cells in culture to the delta-endotoxin CryIC from the insecticidal bacterium Bacillus thuringiensis was investigated using single ionic channel measurements and microspectrofluorescence of the calcium-sensitive probe fura-2. It was found that: (1) the toxin triggers an immediate rise in intracellular calcium; (2) the surge is due to calcium entering the cells via calcium channels; (3) the toxin recruits or introduces anionic channels in the cell's plasma membrane in a time-dependent manner. These channels, not seen in the absence of the toxin, are induced by toxin exposure to either side of the cell membrane. They have a conductance of 26 picosiemens (pS) and are mainly permeable to chloride. This study provides the first evidence of the primary role of calcium and chloride ions in the action of delta-endotoxin on cultured insect cells.     

Cytolytic activity of Bacillus thuringiensis CryIC and CryIAc toxins to Spodoptera sp. midgut epithelial cells in vitro

Summary A sensitive lactate dehydrogenase (LDH) assay was modified to determine the cytolytic activity of Bacillus thuringiensis CryIC and CryIAc delta endotoxins to viable collagenase-dissociated midgut epithelial cells (MEC) from larvae of Spodoptera frugiperda and Spodoptera exigua. The MEC preparations from these Spodoptera sp. consisted predominantly of columnar cells (65–75%) and goblet cells (25–35%). Time course microscopy experiments indicated that only the columnar cells became swollen during CryIC toxin incubation. Also, comparative cytotoxicity studies were run with cell lines of nonmidgut origin established from S. frugiperda (SF21AE) and S. exigua (SEUCR1A). Optimum conditions for the cytotoxicity assay were similar for MEC and cell lines of both species, and were met in an assay in which 0.1-ml cell concentrations (8.5±0.5×10⁴ cells) were incubated with toxin dilutions (0.01–20 μg) for 1 h at 24° C at a final pH of 7.8. The Spodoptera sp. MEC were twofold more sensitive to CryIC (68% lysis) than CryIAc (32% lysis) at optimum toxin levels (2.5–5 μg). Also, the SEUCR1A cells were more sensitive (2.3-fold) to CryIC (70% lysis) than CryIAc (30% lysis) at optimum toxin levels of 5–10 μg. The SF21AE cells, however, were twofold less sensitive to CryIC (30% lysis) than SEUCR1A cells and response to CryIAc and CryIC was similar. Immunoblot analysis of either Spodoptera sp. MEC or brush border membrane vesicles (BBMV) identified seven CryIC binding proteins with molecular mass of 137, 120, 115, 68, 65, 63, and 45 kDa. Occasionally, a 148-kDa protein band was observed. The CryIAc toxin bound to two proteins on MEC and BBMV with molecular mass of 137 and 120 kDa.     

Enhanced production of insecticidal proteins in Bacillus thuringiensis strains carrying an additional crystal protein gene in their chromosomes.

A two-step procedure was used to place a cryIC crystal protein gene from Bacillus thuringiensis subsp. aizawai into the chromosomes of two B. thuringiensis subsp. kurstaki strains containing multiple crystal protein genes. The B. thuringiensis aizawai cryIC gene, which encodes an insecticidal protein highly specific to Spodoptera exigua (beet armyworm), has not been found in any B. thuringiensis subsp. kurstaki strains. The cryIC gene was cloned into an integration vector which contained a B. thuringiensis chromosomal fragment encoding a phosphatidylinositol-specific phospholipase C, allowing the B. thuringiensis subsp. aizawai cryIC to be targeted to the homologous region of the B. thuringiensis subsp. kurstaki chromosome. First, to minimize the possibility of homologous recombination between cryIC and the resident crystal protein genes, B. thuringiensis subsp. kurstaki HD73, which contained only one crystal gene, was chosen as a recipient and transformed by electroporation. Second, a generalized transducing bacteriophage, CP-51, was used to transfer the integrated cryIC gene from HD73 to two other B. thuringiensis subsp. kurstaki stains. The integrated cryIC gene was expressed at a significant level in all three host strains, and the expression of cryIC did not appear to reduce the expression of the endogenous crystal protein genes. Because of the newly acquired ability to produce the CryIC protein, the recombinant strains showed a higher level of activity against S. exigua than did the parent strains. This two-step procedure should therefore be generally useful for the introduction of an additional crystal protein gene into B. thuringiensis strains which have multiple crystal protein genes and which show a low level of transformation efficiency.     

Chitinolytic activities in Bacillus thuringiensis and their synergistic effects on larvicidal activity

AIMS: To investigate the distribution of chitinase in Bacillus thuringiensis strains, and the enhancing effects of the chitinase-producing B. thuringiensis strains on insecticidal toxicity of active B. thuringiensis strain against Spodoptera exigua larvae. METHODS AND RESULTS: The chitinolytic activities of B.thuringiensis strains representing the 70 serotypes were investigated by the whitish opaque halo and the colorimetric method. Thirty-eight strains produced different levels of chitinase at pH 7.0, and so did 17 strains at pH 10.0. The strain T04A001 exhibited the highest production, reaching a specific activity of 355 U ml(-1) in liquid medium. SDS-PAGE and Western blotting showed that the chitinase produced by some B. thuringiensis strains had a molecular weight of about 61 kDa. The bioassay results indicated that the chitinase-producing B. thuringiensis strains could enhance the insecticidal activity of B. thuringiensis strain DL5789 against S. exigua larvae, with an enhancing ratio of 2.35-fold. CONCLUSION: This study demonstrated that chitinase was widely produced in B. thuringiensis strains and some of the strains could enhance the toxicity of active B. thuringiensis strain. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first investigation devoted exclusively to analyse the distribution of chitinase in B. thuringiensis. It infers that the chitinase produced by B. thuringiensis might play a role in the activity of the biopesticide.     

Altered binding of the Cry1Ac toxin to larval membranes but not to the toxin-binding protein in Plodia interpunctella selected for resistance to different Bacillus thuringiensis isolates.

Immunoblotting and cytochemical procedures were used to determine whether toxin binding was altered in strains of the Indianmeal moth, Plodia interpunctella, selected for resistance to various strains of Bacillus thuringiensis. Each of these B. thuringiensis subspecies produces a mixture of protoxins, primarily Cry1 types, and the greatest insect resistance is to the Cry1A protoxins. In several cases, however, there was also resistance to toxins not present in the B. thuringiensis strains used for selection. The Cry1Ab and Cry1Ac toxins bound equally well over a range of toxin concentrations and times of incubation to a single protein of ca. 80-kDa in immunoblots of larval membrane extracts from all of the colonies. This binding protein is essential for toxicity since a mutant Cry1Ac toxin known to be defective in binding and thus less toxic bound poorly to the 80-kDa protein. This binding protein differed in size from the major aminopeptidase N antigens implicated in toxin binding in other insects. Binding of fluorescently labeled Cry1Ac or Cry1Ab toxin to larval sections was found at the tips of the brush border membrane prepared from the susceptible but not from any of the resistant P. interpunctella. Accessibility of a major Cry1A-binding protein appears to be altered in resistant larvae and could account for their broad resistance to several B. thuringiensis toxins.     

Study of the Bacillus thuringiensis Vip3Aa16 histopathological effects and determination of its putative binding proteins in the midgut of Spodoptera littoralis

The bacterium Bacillus thuringiensis produces, at the vegetative stage of its growth, Vip3A proteins with activity against a broad spectrum of lepidopteran insects. The Egyptian cotton leaf worm (Spodoptera littoralis) is an important agricultural pest that is susceptible to the Vip3Aa16 protein of Bacillus thuringiensis kurstaki strain BUPM95. The midgut histopathology of Vip3Aa fed larvae showed vacuolization of the cytoplasm, brush border membrane destruction, vesicle formation in the apical region and cellular disintegration. Biotinylated Vip3Aa toxin bound proteins of 55- and 100-kDa on blots of S. littoralis brush border membrane preparations. These binding proteins differ in molecular size from those recognized by Cry1C, one of the very few Cry proteins active against the polyphagous S. littoralis. This result supports the use of Vip3Aa16 proteins as insecticidal agent, especially in case of Cry-resistance management.     

Identification and characterization of Heliothis virescens midgut membrane proteins binding Bacillus thuringiensis delta-endotoxins.

To investigate the specificity of Bacillus thuringiensis var. kurstaki strain HD1 insecticidal crystal proteins (ICP), we used membrane preparations obtained from the midgut of Heliothis virescens larvae to perform separate ligand-blot experiments with the three activated CryIA toxins. The CryIA(a) and the CryIA(b) toxins bind the same 170-kDa protein, but most likely at two different binding sites. The CryIA(c) toxin binds two proteins of molecular masses 140 kDa and 120 kDa. We also demonstrate that the binding proteins for each of the B. thuringiensis toxins are not part of a covalent complex. Although the 170-kDa protein is a glycoprotein, endoglycosidase treatment does not prevent the binding of the CryIA(a) or CryIA(b) toxin. This indicates that the sugars are not important for the binding of these toxins. A model for a protein complex binding the B. thuringiensis HD1 ICPs is presented. Our results support the idea that binding proteins on membranes of the gut epithelial cells of H. virescens larvea are important for the specificity of the bacterial toxins.     

Insecticidal Activity of the Toxins from Bacillus thuringiensis subspecies kurstaki and tenebrionis Adsorbed and Bound on Pure and Soil Clays

The release of transgenic plants and microorganisms expressing truncated genes from various subspecies of Bacillus thuringiensis that encode active insecticidal toxins rather than inactive protoxins could result in the accumulation of these active proteins in soil, especially when bound on clays and other soil particles. Toxins from B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. tenebrionis, either free or adsorbed at equilibrium or bound on pure clay minerals (montmorillonite or kaolinite) or on the clay size fraction of soil, were toxic to larvae of the tobacco hornworm (Manduca sexta) and the Colorado potato beetle (Leptinotarsa decemlineata), respectively. The 50% lethal concentrations (LC(inf50)) of free toxins from B. thuringiensis subsp. kurstaki were higher than those of both bound and adsorbed complexes of these toxins with clays, indicating that adsorption and binding of these toxins on clays increase their toxicity in diet bioassays. The LC(inf50) of the toxin from B. thuringiensis subsp. tenebrionis that was either free or adsorbed on montmorillonite were comparable, whereas the toxin bound on this clay had higher LC(inf50) and the toxin bound on kaolinite had lower LC(inf50) than when adsorbed on this clay. Results obtained with the clay size fraction separated from unamended soil or soil amended with montmorillonite or kaolinite were similar to those obtained with the respective pure clay minerals. Therefore, insecticidal activity of these toxins is retained and sometimes enhanced by adsorption and binding on clays.     

A 106-kDa aminopeptidase is a putative receptor for Bacillus thuringiensis Cry11Ba toxin in the mosquito Anopheles gambiae

Bacillus thuringiensis (Bt) insecticidal toxins bind to receptors on midgut epithelial cells of susceptible insects, and binding triggers biochemical events that lead to insect mortality. Recently, a 100-kDa aminopeptidase N (APN) was isolated from brush border membrane vesicles (BBMV) of Anopheles quadrimaculatus and shown to bind Cry11Ba toxin with surface plasmon resonance (SPR) detection [Abdullah et al. (2006) BMC Biochem. 7, 16]. In our study, a 106-kDa APN, called AgAPN2, released by phosphatidylinositol-specific phospholipase C (PI-PLC) from Anopheles gambiae BBMV was extracted by Cry11Ba bound to beads. The AgAPN2 cDNA was cloned, and analysis of the predicted AgAPN2 protein revealed a zinc-binding motif (HEIAH), three potential N-glycosylation sites, and a predicted glycosylphosphatidylinositol (GPI) anchor site. Immunohistochemistry localized AgAPN2 to the microvilli of the posterior midgut. A 70-kDa fragment of the 106-kDa APN was expressed in Escherichia coli. When purified, it competitively displaced 125I-Cry11Ba binding to An. gambiae BBMV and bound Cry11Ba on dot blot and microtiter plate binding assays with a calculated K d of 6.4 nM. Notably, this truncated peptide inhibited Cry11Ba toxicity to An. gambiae larvae. These results are evidence that the 106-kDa GPI-anchored APN is a specific binding protein, and a putative midgut receptor, for Bt Cry11Ba toxin.     

Aminopeptidase N purified from gypsy moth brush border membrane vesicles is a specific receptor for Bacillus thuringiensis CryIAc toxin.

We have evaluated the binding of Bacillus thuringiensis Cry toxins to aminopeptidase N (APN) purified from Lymantria dispar (gypsy moth) brush border membrane vesicle (BBMV). CryIAc toxin bound strongly to APN, while either the structurally related CryIAa and CryIAb toxins or CryIC, CryIIA, and CryIIIA toxins showed weak binding to APN. An in vitro competition binding study demonstrated that the binding of CryIAc to L. dispar BBMV was inhibited by APN. Inhibition of short circuit current for CryIAc, measured by voltage clamping of whole L. dispar midgut, was substantially reduced by addition of phosphatidylinositol-specific phospholipase C, which is known to release APN from the midgut membrane. In contrast, addition of phosphatidylinositol-specific phospholipase C had only a marginal effect on the inhibition of short circuit current for CryIAa. These data suggest that APN is the major functional receptor for CryIAc in L. dispar BBMV. A ligand blotting experiment demonstrated that CryIAc recognized a 120-kDa peptide (APN), while CryIAa and CryIAb recognized a 210-kDa molecule in L. dispar BBMV. In contrast, CryIAa and CryIAb bound to both the 120- and 210-kDa molecules in Manduca sexta BBMV, while CryIAc recognized only the 120-kDa peptide. The 120-kDa peptide (APN) in L. dispar BBMV reacted with soybean agglutinin, indicating that N-acetylgalactosamine is a component of this glycoprotein.     

Binding of Bacillus thuringiensis Cry1Ac Toxin to Aminopeptidase in Susceptible and Resistant Diamondback Moths (Plutella xylostella)

Bacillus thuringiensis Cry1Ac toxin bound to a 120-kDa protein isolated from the brush border membranes of both susceptible and resistant larvae of Plutella xylostella, the diamondback moth. The 120-kDa protein was purified by Cry1Ac toxin affinity chromatography. Like Cry1Ac-binding aminopeptidase N (EC 3.4.11.2) from other insects, this protein was eluted from the affinity column with 200 mM N-acetylgalactosamine. The purified protein had aminopeptidase activity and bound Cry1Ac toxin on ligand blots. Purified aminopeptidase was recognized by antibodies to the cross-reacting determinant found on phosphatidylinositol-specific phospholipase C-solubilized proteins. The results show that the presence of Cry1Ac-binding aminopeptidase in the brush border membrane is not sufficient to confer susceptibility to Cry1Ac. Furthermore, the results do not support the hypothesis that resistance to Cry1Ac was caused by lack of a Cry1Ac-binding aminopeptidase.