Characterization of the toxicity and cytopathic specificity of a cloned Bacillus thuringiensis crystal protein using insect cell culture

An insecticidal protein gene from Bacillus thuringiensis var. aizawal was cloned in Escherichia coli. The cloned gene expressed at a high level and the synthesized protein appeared as an insoluble, phase-bright inclusion in the cytoplasm. These inclusions were isolated by density gradient centrifugation, the isolated protein was activated in vitro by different proteloytic regimes and the toxicity of the resulting preparations was studied using insect cells grown in tissue culture. The inclusions consisted of a 130 kDa polypeptide which was processed to a protease-resist-ant 55 kDa protein by tryptic digestion. This preparation lysed lepidopteran (Choristoneura fumiferana) CFI ceils but not dipteran (Aedes albopictus) calls. When the crystal protein was activated by sequential treatment, first with trypsin and then with Aedes aegypti gut proteases, the resulting 53 kDa polypeptide was now toxic only to the dipteran cells and not to the lepidopteran cells. Thus the dual specificity of this var. aizawal toxin results from differential proteolytic processing of a single protoxin. The trypsin-activated preparation was weakly active against Spodoptera frugiperda cells. Membrane binding studies of the trypsin-activated toxin revealed a 68 kDa protein in the lepidopteran ceil membranes, which may be the receptor for this toxin.     

Analysis of the molecular basis of insecticidal specificity of Bacillus thuringiensis crystal delta-endotoxin.

The mechanism of action and receptor binding of a dual-specificity Bacillus thuringiensis var. aizawai ICl delta-endotoxin was studied using insect cell culture. The native protoxin was labelled with 125I, proteolytically activated and the affinity of the resulting preparations for insect cell-membrane proteins was studied by blotting. The active preparations obtained by various treatments had characteristic specificity associated with unique polypeptides, and showed affinity for different membrane proteins. The lepidopteran-specific preparation (trypsin-treated protoxin containing 58 and 55 kDa polypeptides) bound to two membrane proteins in the lepidopteran cells but none in the dipteran cells. The dipteran-specific preparation (protoxin treated sequentially with trypsin and Aedes aegypti gut proteases, containing a 53 kDa polypeptide) bound to a 90 kDa membrane protein in the dipteran (A. aegypti) cells but bound to none in the lepidopteran cells or Drosophila melanogaster cells. The toxicity of trypsin-activated delta-endotoxin was completely inhibited by preincubation with D-glucose, suggesting a role for this carbohydrate in toxin-receptor interaction. The toxicity was also decreased by osmotic protectants to an extent proportional to their viscometric radius. These results support a proposal that initial interaction of toxin with a unique receptor determines the specificity of the toxin, following which cell death occurs by a mechanism of colloid osmotic lysis.     

Specificity of Bacillus thuringiensis var. colmeri insecticidal delta-endotoxin is determined by differential proteolytic processing of the protoxin by larval gut proteases

The native crystal delta-endotoxin produced by Bacillus thuringiensis var. colmeri, serotype 21, is toxic to both lepidopteran (Pieris brassicae) and dipteran (Aedes aegypti) larvae. Solubilization of the crystal delta-endotoxin in alkaline reducing conditions and activation with trypsin and gut extracts from susceptible insects yielded a preparation whose toxicity could be assayed in vitro against a range of insect cell lines. After activation with Aedes aegypti gut extract the preparation was toxic to all of the mosquito cell lines but only one lepidopteran line (Spodoptera frugiperda), whereas an activated preparation produced by treatment with P. brassicae gut enzymes or trypsin was toxic only to lepidopteran cell lines. These in vitro results were paralleled by the results of in vivo bioassays. Gel electrophoretic analysis of the products of these different activation regimes suggested that a 130-kDa protoxin in the native crystal is converted to a 55-kDa lepidopteran-specific toxin by trypsin or P. brassicae enzymes and to a 52-kDa dipteran toxin by A. aegypti enzymes. Two-step activation of the 130-kDa protoxin by successive treatment with trypsin and A. aegypti enzymes further suggested that the 52-kDa dipteran toxin is derived from the 55-kDa lepidopteran toxin by enzymes specific to the mosquito gut. Confirmation of this suggestion was obtained by peptide mapping of these two polypeptides. The native crystal 130 kDa delta-endotoxin and the two insect-specific toxins all cross-reacted with antiserum to B. thuringiensis var. kurstaki P1 lepidopteran toxin. Preincubation of the two activated colmeri toxins with P1 antiserum neutralized their cytotoxicity to both lepidopteran and dipteran cell lines.     

Comparison of the in vivo and in vitro activity of the delta-endotoxin of Bacillus thuringiensis var. morrisoni (HD-12) and two of its constituent proteins after cloning and expression in Escherichia coli

The insecticidal crystal delta-endotoxin of Bacillus thuringiensis var. morrisoni HD-12 contains at least five polypeptides in the range 126-140 kDa. Immune blotting revealed that individual proteins in this complex share homology with a range of other B. thuringiensis delta-endotoxins. In vivo the native HD-12 crystal killed a lepidopteran larva (Pieris brassicae) and a dipteran larva (Anopheles gambiae), but not the related dipteran Aedes aegypti. In vitro the solubilized activated crystal lysed Choristoneura fumiferana cells (lepidopteran) and dipteran cells derived from Anopheles gambiae and Culex quinquefasciatus but not those from Aedes aegypti. An intragenic probe derived from a B. thuringiensis var. sotto lepidoptera-specific delta-endotoxin gene hybridized with one of six plasmids extracted from HD-12. When cloned into pUC18 two HindIII fragments from this plasmid (pEG1 and pEG2) were shown to encode polypeptides cross-reacting with HD-12 antiserum. Escherichia coli lysates containing pEG2 were toxic in vivo to lepidoptera and diptera larvae and in vitro to a broader range of insect cell lines than the native crystal. E. coli cells containing pEG3, a subclone derived from pEG1, synthesised large amounts of a 140-kDa protein in the cytoplasm as inclusion bodies. The cytotoxicity of the protein encoded by pEG3 was restricted to C. fumiferana and A. gambiae cell lines.     

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.     

Cytotoxicity of a cloned Bacillus thuringiensis subsp. israelensis CryIVB toxin to an Aedes aegypti cell line

A cloned CryIVB toxin was purified from a cured strain of Bacillus thuringiensis (BT) containing the cryIVB gene on the recombinant plasmid Cam135. Solubilized protoxin was treated with Aedes gut extract or trypsin for varying times and tested for toxicity in vitro on three dipteran and one lepidopteran cell line. Treatment with the Aedes extract but not trypsin, produced an active toxin which lysed only Aedes aegypti cells out of those tested. This activation was time-dependent reaching a maximum after 6 h. Both the Aedes extract-treated and trypsin-treated toxin killed A. aegypti larvae, but this toxicity declined rapidly with increasing time of exposure to the proteolytic preparations.     

Cloning and expression in Escherichia coli of an insecticidal crystal protein gene from Bacillus thuringiensis var. aizawai HD-133

Using a gene probe derived from the cloned var. sotto insecticidal crystal protein (ICP) gene, we have cloned a Bacillus thuringiensis var. aizawai HD-133 ICP gene in Escherichia coli. The gene encodes a polypeptide that is toxic to Lepidoptera in vivo and in vitro. The protein is expressed at a level sufficient to produce phase-bright inclusions in recombinant E. coli strains, and these inclusions can be partially purified using discontinuous sucrose density gradients. Immunoblotting shows that the inclusions contain a 135 kDa polypeptide which reacts strongly with antiserum raised against the B. thuringiensis var. kurstaki HD-1 P1 polypeptide.     

Delineation of the toxin coding fragments and an insect-specificity region of a dual toxicity Bacillus thuringiensis crystal protein gene

A series of deletion mutants have been constructed from the dual toxicity Bacillus thuringiensis aizawai IC1 (Bta IC1) crystal protein gene. The mutant toxin genes were expressed in Escherichia coli, their protein products purified and the authenticity of these mutant proteins confirmed immunologically. Analysis of the toxicity spectra of these mutants revealed that lepidopteran toxicity is located on the N-terminal region of the toxin between residues Ile30-Glu595. 3' deletion of a further 37 residues from Glu595 of the lepidopteran-specific toxin abolished lepidopteran toxicity but the resulting protein consisting of residues Ile30-Gly558 was still fully toxic to dipteran larvae and cells. Another mutant crystal protein gene truncated to encode residues between Ile30-Gly563 was toxic only to diptera. These data indicate that the determinants of lepidopteran specificity in the Bta IC1 toxin are located between residues Gly558-Glu595 and that the N-terminal portion of the toxin between Ile30-Gly558 is sufficient to express dipteran toxicity.     

Specificity of cultured insect tissue cells for bioassay of entomocidal protein fromBacillus thuringiensis

Summary Cultured tissue cells from lepidopteran and dipteran sources displayed an order-specific response to entomocidal protein from crystals ofBacillus thuringiensis. Protein isolated from crystals ofB. thuringiensis subsp.kurstaki was effective against cells of the spruce budworm (Choristoneura fumiferana) and the tobacco hornworm (Manduca sexta), but was inactive against both mosquito cell lines tested (Aedes aegypti andAnopheles gambiae). Conversely, protein from inclusion bodies ofB. thuringiensis subsp.israelensis was fully active only against the mosquito cell lines but displayed reduced (four- to seven-fold) toxicity for the lepidopteran cell lines. One exception to this pattern of specificity was observed with aPlodia interpunctella cell line, which failed to respond to either crystal protein preparation. The moth toxin was stable at 4° C for months, whereas the mosquito toxin was susceptible to proteolytic degradation and was unstable for periods longer than 2 wk.     

Comparative toxicity of Bacillus thuringiensis var. israelensis crystal proteins in vivo and in vitro

Bacillus thuringiensis var. israelensis crystal proteins were purified by FPLC on a Mono Q column to yield 130, 65, 28, 53, 30-35 and 25 kDa proteins. All the purified proteins killed Aedes aegypti larvae after citrate precipitation, but the 65 kDa protein was the most toxic. A precipitated mixture of 27 and 130 kDa proteins was almost as toxic as solubilized crystals. In assays against a range of insect cell lines, the activated form (25 kDa) of the 27 kDa protein was generally cytotoxic with the lowest LC50 values in vitro. By contrast, the activated forms of the 130 kDa and 65 kDa protoxins (53 kDa and 30-35 kDa proteins, respectively) were much more specific than the 25 kDa protein in their action on dipteran cells, and each showed a unique toxicity profile which, in the case of the 130 kDa preparation, was restricted to Anopheles and Culex cell lines.     

Two highly related insecticidal crystal proteins of Bacillus thuringiensis subsp. kurstaki possess different host range specificities.

Two genes encoding insecticidal crystal proteins from Bacillus thuringiensis subsp. kurstaki HD-1 were cloned and sequenced. Both genes, designated cryB1 and cryB2, encode polypeptides of 633 amino acids having a molecular mass of ca. 71 kilodaltons (kDa). Despite the fact that these two proteins display 87% identity in amino acid sequence, they exhibit different toxin specificities. The cryB1 gene product is toxic to both dipteran (Aedes aegypti) and lepidopteran (Manduca sexta) larvae, whereas the cryB2 gene product is toxic only to the latter. DNA sequence analysis indicates that cryB1 is the distal gene of an operon which is comprised of three open reading frames (designated orf1, orf2, and cryB1). The proteins encoded by cryB1 and orf2 are components of small cuboidal crystals found in several subspecies and strains of B. thuringiensis; it is not known whether the orf1 or cryB2 gene products are present in cuboidal crystals. The protein encoded by orf2 has an electrophoretic mobility corresponding to a molecular mass of ca. 50 kDa, although the gene has a coding capacity for a polypeptide of ca. 29 kDa. Examination of the deduced amino acid sequence for this protein reveals an unusual structure which may account for its aberrant electrophoretic mobility: it contains a 15-amino-acid motif repeated 11 times in tandem. Escherichia coli extracts prepared from cells expressing only orf1 and orf2 are not toxic to either test insect.     

A broad-spectrum cytolytic toxin from Bacillus thuringiensis var. kyushuensis.

Bacillus thuringiensis (Bt) var. kyushuensis synthesizes a mosquitocidal crystalline inclusion containing several proteins ranging from 140 to 14 kDa. We have identified a 25 kDa protein protoxin in this inclusion which is not cytolytic, but when activated proteolytically to 23-22 kDa products is cytolytic to mosquito, lepidopteran and mammalian cells, can release entrapped glucose from liposomes and forms cation-selective channels in a planar lipid bilayer. This broad-spectrum cytolytic toxin is related antigenically to the 23 kDa toxin from Bt var. darmstadiensis strain 73-E10-2, but not to the 25 kDa CytA toxin of Bt var. israelensis. The cytolytic activity of these Bt var. kyushuensis toxins, like that of the latter two toxins, can be neutralized by incubation with liposomes containing phospholipids.     

Molecular cloning of the delta-endotoxin gene of Bacillus thuringiensis var. israelensis

A transformant of Bacillus megaterium, VB131, was isolated which carries a 6.3-kb XbaI segment of the crystal toxin gene of Bacillus thuringiensis var. israelensis (BTI) cloned in a vector plasmid pBC16 to yield pVB131. The chimeric plasmid DNA from VB131 was introduced into a transformable Bacillus subtilis strain by competence transformation. Both the B. megaterium VB131 strain and the B. subtilis strain harboring the chimeric plasmid produced irregular, parasporal, phase-refractile, crystalline inclusions (Cry+) during sporulation. The sporulated cells as well as the isolated crystal inclusions of the pVB131-containing B. megaterium and B. subtilis strains were highly toxic to the larvae of Aedes aegypti. Also, the solubilized crystal protein preparation from VB131[pVB131] showed clear immuno cross-reaction with antiserum to the BTI crystal toxin. 32P-labeled pVB131 plasmid DNA showed specific hybridization with a 112-kb plasmid DNA of Cry+ strains of BTI, and no hybridization with other plasmid or chromosomal DNA of either Cry+ or Cry- variants. These results are in agreement with our previous findings (González and Carlton, 1984) that the 112-kb plasmid of BTI is associated with the production of the crystal toxin.     

家蚕氨肽酶 (BmAPN5) 与黑胸败血芽孢杆菌伴孢晶体 (PC) 毒素相互作用

氨肽酶N(APN)属于锌金属肽酶M1(Peptidase_M1)家族的成员,不仅参与蛋白水解过程,而且也作为毒素受体参与病原微生物的致病过程。家蚕氨肽酶家族含有16个成员,其中BmAPN4结合黑胸败血芽孢杆菌产生的伴孢晶体(PC)毒素,为研究该基因家族其他成员是否与PC毒素结合,参与其致病过程。本文克隆家蚕中肠特异表达的氨肽酶家族成员BmAPN5基因,全长3 313 bp,编码953个氨基酸,含有1个锌金属肽酶M1和ERAP1_C结构域。构建原核表达载体,表达和纯化获得可溶性GST-BmAPN5重组蛋白。Far-Western blotting、免疫共沉淀和ELISA等实验结果表明BmAPN5和活化的PC毒素相互结合。通过构建BmAPN5细胞转染载体,转染Sf9细胞系,与PC毒素共孵育,导致细胞形态改变和裂解死亡;同时,乳酸脱氢酶含量测定结果 (LDH)表明BmAPN5参与PC毒素致病过程,导致细胞裂解死亡,使细胞培养基中的乳酸脱氢酶升高。上述结果表明BmAPN5作为一种功能性受体,PC毒素与其相互作用,参与了病原物的致病过程,为进一步揭示病原微生物黑胸败血芽孢杆菌与宿主相互作用的致病机制研究奠定了基础。     

Cloning and characterization of an insecticidal crystal protein gene from Bacillus thuringiensis subspecies kenyae

A sporulating culture ofBacillus thuringiensis subsp.kenyae strain HD549 is toxic to larvae of lepidopteran insect species such asSpodoptera litura, Helicoverpa armigera andPhthorimaea operculella, and a dipteran insect,Culex fatigans. A 1.9-kb DNA fragment, PCR-amplified from HD549 using cryII-gene-specific primers, was cloned and expressed inE. coli. The recombinant protein produced 92% mortality in first-instar larvae ofSpodoptera litura and 86% inhibition of adult emergence inPhthorimaea operculella, but showed very low toxicity againstHelicoverpa armigera, and lower mortality against third-instar larvae of dipteran insectsCulex fatigans, Anopheles stephensi andAedes aegypti. The sequence of the cloned crystal protein gene showed almost complete homology with a mosquitocidal toxin gene fromBacillus thuringiensis var.kurstaki, with only five mutations scattered in different regions. Amino acid alignment with different insecticidal crystal proteins using the MUTALIN program suggested presence of the conserved block 3 region in the sequence of this protein. A mutation in codon 409 of this gene that changes a highly conserved phenylalanine residue to serine lies in this block.     

Characterization of the genes encoding the haemolytic toxin and the mosquitocidal delta-endotoxin of Bacillus thuringiensis israelensis

Summary The crystalline parasporal inclusions (crystals) of Bacillus thuringiensis israelensis (Bti), which are specifically toxic to mosquito and black fly larvae, contain three main polypeptides of 28 kDa, 68 kDa and 130 kDa. The genes encoding the 28 kDa protein and the 130 kDa protein have been cloned from a large plasmid of Bti. Escherichiacoli recombinant clones containing the 130 kDa protein gene were highly active against larvae of Aedes aegypti and Culex pipiens, while B. subtilis recombinant cells containing the 28 kDa protein gene were haemolytic for sheep red blood cells. A fragment of the Bti plasmid which is partially homologous to the 130 kDa protein gene was also isolated; it probably corresponds to part of a second type of mosquitocidal toxin gene. Furthermore, restriction enzyme analysis suggested that the 130 kDa protein gene is located on the same Bti EcoRI fragment as another kind of Bti mosquitocidal protein gene cloned by Thorne et al. (1986). Hybridization experiments conducted with the 28 kDa protein gene and the 230 kDa protein gene showed that these two Bti genes are probably present in the plasmid DNA of B. thuringiensis subsp. morrisoni (PG14), which is also highly active against mosquito larvae.     

Binding of the CryIVD Toxin of Bacillus thuringiensis subsp. israelensis to Larval Dipteran Midgut Proteins

Ligand-blotting experiments on dipteran brush border membrane vesicles (BBMVs) showed binding of CryIVD toxin of Bacillus thuringiensis subsp. israelensis to proteins of 148 kDa in Anopheles stephensi and of 78 kDa in Tipula oleracea, both species being susceptible to CryIVD. Binding of CryIVD with BBMVs of A. stephensi resulted in a stronger signal than with BBMVs of T. oleracea. Likewise, larvae of A. stephensi are 10,000-fold more susceptible to the CryIVD toxin than are larvae of T. oleracea. Binding was also found with six proteins ranging in size from 48 to 110 kDa in BBMVs from the lepidopteran species Manduca sexta, but CryIVD was not toxic for M. sexta larvae. No binding of trypsinated CryIVD to BBMV proteins was observed. With the lepidopteran-specific toxin CryIA(b), no binding to dipteran BBMVs was found. Binding of CryIA(b) to nine different BBMV proteins ranging in size from 71 to 240 kDa was observed in M. sexta. The major binding signal was observed with a protein of 240 kDa for CryIA(b).     

Multiplicity of §-endotoxin genes with different insecticidal specificities in Bacillus thuringiensis aizawai 7.29

The hypothesis according to which multiple and different delta-endotoxin genes could determine the host-range specificity of the lepidopteran strains of Bacillus thuringiensis is being checked in the case of strains aizawai 7.29 (serotype 7) and entomocidus 601 (serotype 6). From these strains, several crystal protein genes, belonging to different structural types, have been isolated. One of the cloned genes that is not present in strain entomocidus 601 is duplicated in strain aizawai 7.29. This gene belongs to a previously characterized type of crystal protein gene and encodes a protein preferentially active against Pieris brassicae. Two other genes, of presumed chromosomal location, are present in both strains and each displays a unique physical map. In both strains the two genes are in close proximity and in the same orientation. The first, which belongs to a new type of crystal protein gene, encodes a 130-140 kD protein that is not significantly active against the two insect species tested. The other new type of crystal protein gene directs the synthesis of a polypeptide preferentially active against Spodoptera littoralis.