Biomedical subjects

D J Ellar

Publications and source records attributed to D J Ellar.

At least 37 records · Page 2Linked to original sources

PubMed1997

Intramolecular proteolytic cleavage of Bacillus thuringiensis Cry3A delta-endotoxin may facilitate its coleopteran toxicity.

The Cry3A delta-endotoxin protein inclusion synthesized by Bacillus thuringiensis subsp. tenebrionis is soluble in alkaline and acid buffer solutions but the toxin precipitates when returned to neutral pH conditions. The midgut pH of susceptible beetle larvae is neutral to slightly acidic, a pH environment in which the Cry3A toxin is insoluble. To investigate this paradox we studied the Cry3A toxin after various proteolytic treatments. In many cases the toxin was cleaved into polypeptides that remained associated under non-denaturing conditions. Interestingly a chymotrypsinized Cry3A product was soluble under neutral pH conditions, retained full activity against susceptible beetle larvae, and exhibited specific binding to Leptinotarsa decemlineata midgut membranes.

Animals↗

PubMed1997

Identification of two sequence elements associated with the gene encoding the 24-kDa crystalline component in Bacillus thuringiensis ssp. fukuokaensis: an example of transposable element archaeology.

A 6.5-kb fragment of plasmid DNA from Bacillus thuringiensis (Bt) ssp. fukuokaensis that encodes a 24-kDa crystalline component was analyzed to identify additional open reading frames (orfs). A novel Bt IS240-like element was found upstream of this gene and is considered to be a vestige of a once active insertion sequence due to a stop codon that interrupts the long orf encoding the putative transposase. This element was bounded by 17-bp terminal inverted repeats that defined the length of the insertion sequence as 802 bp. Further upstream of this element two tandem overlapping and out of phase open reading frames (orfX and orfY) were identified which represent the first example of an IS150-like element in Bt containing both orfs. orfX and orfY are not bounded by terminal inverted repeats but are associated with a gene encoding a putative site-specific recombinase of a type found in Staphylococcus aureus Class II transposons but not previously in Bt.

Amino Acid Sequence↗

PubMed1997

The Bacillus thuringiensis Cry1Ac toxin-induced permeability change in Manduca sexta midgut brush border membrane vesicles proceeds by more than one mechanism.

Aminopeptidase N purified from whole Manduca sexta midgut binds the Cry1Ac insecticidal toxin from Bacillus thuringiensis and this binding is inhibited by N-acetylgalactosamine (GalNAc). We have examined the membrane permeabilising activity of the Cry1Ac toxin using brush border membrane vesicles (BBMV) prepared from the anterior (A-BBMV) and posterior (P-BBMV) subregions of the M. sexta midgut. A toxin mixing assay demonstrated a faster rate of toxin activity on P-BBMV than on A-BBMV. In the presence of GalNAc this rapid activity on P-BBMV was reduced to the rate seen with A-BBMV. GalNAc had no effect on the rate of A-BBMV permeabilisation by Cry1Ac. Aminopeptidase N assays of A- and P-BBMV demonstrated that this Cry1Ac binding protein is concentrated in the posterior midgut region of M. sexta. It therefore appears that there are two mechanisms by which Cry1Ac permeabilises the M. sexta midgut membrane: a GalNAc-sensitive mechanism restricted to the posterior midgut region, probably involving aminopeptidase N binding, and a previously undetected mechanism found in both the posterior and anterior regions.

Animals↗

PubMed1996

Structure of the mosquitocidal delta-endotoxin CytB from Bacillus thuringiensis sp. kyushuensis and implications for membrane pore formation.

The delta-endotoxin CytB, found in parasporal inclusions of Bacillus thuringiensis subspecies kyushuensis, is a membrane pore-forming protein which is lethal to the larvae of Dipteran insects and broadly cytolytic in vitro. The crystal structure of CytB in the protoxin form has been determined by isomorphous replacement using heavy-atom derivatives of both the wild-type protein and an engineered cysteine mutant. The atomic model comprising residues 19 to 245 and 28 bound water molecules has been refined at 2.6 angstrom resolution to a crystallographic R-factor of 19.7% and a free R-factor of 26.1%. CytB has a single domain of alpha/beta architecture but a novel connectivity comprising two outer layers of alpha-helix hairpins wrapped around a mixed beta-sheet. In the protoxin form, CytB is a dimer linked by the intertwined N-terminal strands in a continuous, 12-stranded beta-sheet. Proteolytic processing cleaves the intertwined beta-strands to release the active CytB as a monomer, as well as removing the C-terminal tail to uncover the three-layered core. The homologous toxin CytA should show the same fold. Mutations in CytA that inhibit expression map to the dimer contacts and to the tip of helix pair A-B in contact with the sheet, apparently preventing correct folding. Mutations that inhibit toxicity map to the edge of the beta-sheet adjoining the helix pair C-D and to the sheet face, while mutations on the helix surfaces have no effect. Therefore segments forming the sheet, rather than the amphiphilic but short helices, are responsible for membrane binding and pore formation. A conformational change is postulated by which the helix pair C-D peels away from the sheet to lie on the membrane surface, while the sheet region rearranges to form an oligomeric trans-membrane pore.

Amino Acid Sequence↗

PubMed1996

Mutagenesis of three surface-exposed loops of a Bacillus thuringiensis insecticidal toxin reveals residues important for toxicity, receptor recognition and possibly membrane insertion.

Information on the molecular determinants of receptor recognition, membrane insertion and toxin pore-formation was sought by making 42 single and multiple substitutions of residues 312-314 (GYY), 367-370 (YRRP) and 438-441 (SGFS) in the Bacillus thuringiensis insecticidal CrylAc delta-endotoxin by site-directed mutagenesis. These three regions correspond to three putative surface-exposed loops (loops 1, 2 and 3, respectively) in domain II of the delta-endotoxin, forming the molecular apex of the structure. All except mutants GFY (loop 1), YKRA, SRRA, YRKA (loop 2) and TGFS (loop 3) expressed delta-endotoxin protein at wild-type levels which was stable upon activation by Pieris brassicae gut extract or trypsin. Toxicity assays for all the fully stable mutants using Manduca sexta larvae showed that G312, Y367, R368, R369, S438 and G439 are important for activity. Wild-type toxin was then labelled in vivo with [35S]methionine and heterologous competition binding assays were carried out for all the mutants using brush border membrane vesicles prepared from Manduca sexta midgut. Most and least conservative mutations of G439 and least conservative substitutions of Y367, R368 and R369 reduced the ability of the toxin to bind competitively. The most conservative mutation, S441T, gave significantly increased binding. These results suggested that these four residues play a role in the initial receptor binding step in the toxin mechanism. As no significant effect on binding affinity was observed in relatively non-toxic mutants in which residues G312 and S438 were mutated, we suggest that these residues are involved in the subsequent steps of membrane insertion and pore-formation.

Amino Acid Sequence↗

PubMed1996

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.

Aedes↗

PubMed1995

Molecular cloning of an insect aminopeptidase N that serves as a receptor for Bacillus thuringiensis CryIA(c) toxin.

The Bacillus thuringiensis CryIA(c) insecticidal delta-endotoxin binds to a 120-kDa glycoprotein receptor in the larval midgut epithelia of the susceptible insect Manduca sexta. This glycoprotein has recently been purified and identified as aminopeptidase N. We now report the cloning of aminopeptidase N from a M. sexta midgut cDNA library. Two overlapping clones were isolated, and their combined 3095-nucleotide sequence contains an open reading frame encoding a 990-residue pre-pro-protein. The N-terminal amino acid sequence derived from the glycoprotein is present in the open reading frame, immediately following a predicted cleavable signal peptide and a pro-peptide. There are four potential N-linked glycosylation sites. The C-terminal sequence contains a possible glycosylphosphatidylinositol (GPI) anchor signal peptide, which suggests that, unlike most other characterized aminopeptidases, the lepidopteran enzyme is anchored in the membrane by a GPI anchor. This was confirmed by partial release of aminopeptidase N activity from M. sexta midgut brush border membranes by phosphatidylinositol-specific phospholipase C. The deduced amino acid sequence shows significant similarity to the zinc-dependent aminopeptidase gene family, particularly in the region surrounding the consensus zinc-binding motif characteristic of these enzymes.

Amino Acid Sequence↗

PubMed1995

Crystallization of a membrane pore-forming protein with mosquitocidal activity from Bacillus thuringiensis subspecies kyushuensis.

CytB, a membrane pore-forming toxin from Bacillus thuringiensis subspecies kyushuensis, is specifically toxic to dipteran insect larvae but broadly cytolytic in vitro. It has been purified in the protoxin form from a recombinant Escherichia coli source and crystals have been obtained which diffract X-rays to at least 2.6 A resolution. The tendency for CytB to aggregate in solution was overcome by including 50 mM of urea or 8 mM of ethanolamine during crystallization. Mutants designed to add or subtract single cysteine residues for the purpose of heavy atom derivative preparation were similarly purified and crystallized. The crystals are hexagonal bipyramids. They belong to space group P6(1)22 (or P6(5)22) with lattice constants a = b = 67.34 A, c = 170.96 A, and contain one molecule of the CytB protoxin (MW 29235) per asymmetric unit and 27% solvent by volume.

Bacillus thuringiensis↗

PubMed1994

Mutagenesis of two surface-exposed loops of the Bacillus thuringiensis CryIC delta-endotoxin affects insecticidal specificity.

Site-directed mutagenesis was used to determine the role of two surface-exposed loops (Gly-317-Phe-320 and Gln-374-Pro-377) in the insecticidal specificity of the Bacillus thuringiensis CryIC delta-endotoxin. Mutant toxins were generated by PCR using degenerate oligonucleotide primers, and expressed in Escherichia coli. More than 50 mutant toxins were screened for toxicity to the lepidopteran Spodoptera frugiperda Sf9 cell line using an in vitro lawn assay. A panel of these mutant toxins, which included toxic and non-toxic variants from both loops, was further screened for activity towards Aedes aegypti larvae. The activity of these mutants to Sf9 cells was quantified more precisely using a cell lysis assay. Three categories of mutants were identified: (1) those non-toxic to either Sf9 cells or Aedes aegypti larvae; (2) those fully toxic to both genera; and (3) those which were only toxic to Sf9 cells. For the first loop, the differential specificity was not restricted to any single residue. In the second loop, two mutant toxins with a Pro-377-->Ala substitution displayed this phenotype. The time dependence of toxicity towards Sf9 cells was examined using the same panel of mutants. All toxic mutants displayed an identical time course to the wild-type toxin, with the exception of the two Pro-377-->Ala mutants of the second loop. These toxins displayed a lower time dependence, no cell death occurring within the first hour of incubation. These results show that the two loops are important determinants of both the activity and specificity of the CryIC delta-endotoxin.

Aedes↗

PubMed1994

Nucleotide sequence and analysis of an insertion sequence from Bacillus thuringiensis related to IS150.

A 5.8-kb DNA fragment encoding the cryIC gene from Bacillus thuringiensis (Bt) subsp. aizawai HD229 was subcloned into the pMex7 vector for expression in Escherichia coli. In addition to the 135-kDa CryIC delta-endotoxin, this DNA fragment also encoded a 30-kDa polypeptide whose open reading frame (orfX) was located less than 200 bp upstream of cryIC. Nucleotide sequencing showed that orfX was truncated at the 5' end, and full sequence was obtained from a second overlapping clone. Sequence analysis showed that orfX could encode a polypeptide closely related to the putative transposase from IS150. OrfX was flanked by a 17-bp imperfect inverted repeat, defining the length of the element as 998 bp. Southern blot analysis revealed that the novel insertion sequence was present in a single copy and located in an identical position immediately upstream of cryIC in plasmid DNA from both Bt subsp. aizawai and entomocidus.

Amino Acid Sequence↗

PubMed1994

Use of an operon fusion to induce expression and crystallisation of a Bacillus thuringiensis delta-endotoxin encoded by a cryptic gene.

A delta-endotoxin gene previously cloned from Bacillus thuringiensis subsp. galleriae has been shown by a combination of restriction mapping and DNA sequence analysis to be a cryIIB clone; in common with other cryIIB genes it was found to lack a functional promoter. Addition of a promoter resulted in expression of the gene in Bacillus thuringiensis but did not result in the formation of the crystalline inclusions normally associated with such toxins. Inclusion formation was only observed when the gene was incorporated into an operon containing a gene known to be involved in the crystallisation of another delta-endotoxin.

Amino Acid Sequence↗

PubMed1994

Biochemical characterization of Bacillus thuringiensis cytolytic delta-endotoxins.

The entomocidal delta-endotoxins CytA and CytB produced by Bacillus thuringiensis (Bt) subspecies israelensis and kyushuensis respectively showed a similar level of toxicity to mosquito larvae but were not toxic to the larvae of the lepidopteran Manduca sexta. CytA and CytB are also similar in sequence, predicted secondary structure and alpha-helical content, the only obvious difference being a C-terminal fifteen residue 'tail' on CytB. Investigations of the function, if any, of the CytB C-terminal 'tail' showed that this delta-endotoxin is highly expressed and forms inclusions in an acrylstalliferous Bt mutant without the aid of the 20 kDa 'helper' protein from Bt subspecies israelensis which is essential for CytA inclusion formation. After proteinase K treatment, CytA and CytB were processed to virtually the same points in a sequence alignment and were equally haemolytic in vitro. However, the results suggested that unprocessed CytB differs from unprocessed CytA in that the former is not haemolytic.

Amino Acid Sequence↗

PubMed1994

The receptor for Bacillus thuringiensis CrylA(c) delta-endotoxin in the brush border membrane of the lepidopteran Manduca sexta is aminopeptidase N.

A 120 kDa glycoprotein in the larval midgut membrane of the lepidopteran Manduca sexta, previously identified as a putative receptor for Bacillus thuringiensis CrylA(c) delta-endotoxin, has been purified by a combination of protoxin affinity chromatography and anion exchange chromatography. In immunoblotting experiments, the purified glycoprotein has the characteristics predicted of the receptor: it binds CrylA(c) toxin in the presence of GlcNAc but not GalNAc; it binds the lectin SBA; but it does not bind CrylB toxin. N-terminal and internal amino acid sequences obtained from the protein show a high degree of similarity with the enzyme aminopeptidase N (EC 3.4.11.2). When assayed for aminopeptidase activity, purified receptor preparations were enriched 5.3-fold compared to M. sexta brush border membrane vesicles. We propose that the receptor for CrylA(c) toxin in the brush border membrane of the lepidopteran M. sexta is the metalloprotease aminopeptidase N.

Amino Acid Sequence↗

PubMed1993

Characterization of a Bacillus thuringiensis strain which is toxic to the housefly Musca domestica.

A Bacillus thuringiensis isolate has been discovered which is toxic to the common housefly (Musca domestica) as well as other Diptera and Lepidoptera. Crystal delta-endotoxins purified from this isolate killed 50% of Musca larvae at a concentration of 10.2 micrograms/ml, and beta-exotoxin was not detected. Sodium dodecyl polyacrylamide gel electrophoresis of the purified crystals revealed three protein species which were related to CryIA(b), CryIB and CryIIA toxins on the basis of immunoreactivity and amino-terminal sequence determination. Southern blot and DNA restriction analyses suggested that the strain has sequences related to one cryIA(b), one cryIIA, and two cryIIB genes.

Amino Acid Sequence↗

PubMed1993

Identification of a cryptic gene associated with an insertion sequence not previously identified in Bacillus thuringiensis.

After screening several Bt strains with a cryII toxin probe, clones from two strains were found to contain a cryptic cryIIB gene associated with an insertion sequence element belonging to the IS2/IS3 family. The lack of expression of this gene appears to result from mutation of the upstream orf2 gene which has been shown to be necessary for cryII expression.

Amino Acid Sequence↗

PubMed1993

Effects on toxicity of eliminating a cleavage site in a predicted interhelical loop in Bacillus thuringiensis CryIVB delta-endotoxin.

When activated by treatment with mosquito (Aedes aegypti) gut extract, the Bacillus thuringiensis CryIVB delta-endotoxin lysed A. aegypti cells in vitro. SDS-PAGE and N-terminal sequence determination showed that in addition to removal of the C-terminal half of the molecule, the activated toxin had undergone proteolytic cleavage at two internal regions producing 47-48-kDa and 16-18-kDa polypeptides. Aligning the CryIVB protein sequence with the crystallographic structure of the CryIIIA toxin suggested that one set of cleavages occurred in a region before the start of the N-terminal helical bundle and the second cleavage site occurred in a predicted loop between helices 5 and 6 in the bundle at arginine-203. To investigate the suggestion by Li et al. that interhelical proteolysis is important in the cytolytic mechanism of these toxins, arginine-203 was substituted by alanine. The mutated toxin now resisted proteolysis at this position and showed a marked decrease in cytolysis in vitro but an increase in larvicidal activity.

Aedes↗

PubMed1993

An analysis of Bacillus thuringiensis delta-endotoxin action on insect-midgut-membrane permeability using a light-scattering assay.

Changes in the membrane permeability of Manduca sexta midgut brush-border-membrane vesicles (BBMV) after addition of Bacillus thuringiensis delta-endotoxins were studied using osmotic swelling experiments, volume changes being monitored as the change in 90 degrees light scattering. Typically, control BBMV exhibited limited permeability for sucrose and salts (KCl), while being permeable for urea and glucose. The action of delta-endotoxin was examined using proteolytically activated Cry-IA(c) and CryIB toxins. CryIA(c) produced a marked change in the solute permeability of M. sexta BBMV, significant effects being observed at 3.75 pmol/mg BBMV. The permeability change was relatively non-selective with cations, anions and neutral solutes all traversing the membrane to an increased extent in the presence of CryIA(c). In contrast, the CryIB toxin had no effect on BBMV permeability.

Animals↗

PubMed1993

Identification and partial purification of a Bacillus thuringiensis CryIC delta-endotoxin binding protein from Spodoptera littoralis gut membranes.

Immunoblotting experiments were performed using CryIC and CryIA(c) Bacillus thuringiensis delta-endotoxins to detect the presence of specific toxin binding proteins on Spodoptera littoralis brush border membrane vesicles. The CryIC toxin binds two proteins of 40 and 65 kDa and the CryIA(c) binds a protein of 40 kDa. The CryIA(c) toxin also binds faintly to a 120 kDa protein on S. littoralis brush border membrane vesicles as does a polyclonal antiserum raised against a putative CryIA(c) 120 kDa binding protein from Manduca sexta. The 40 kDa CryIC binding protein was partially purified by affinity chromatography and is therefore a strong candidate for in vivo S. littoralis CryIC toxin receptor.

Animals↗