Characterization and partial purification of a plasma membrane receptor for Bacillus thuringiensis var. kurstaki lepidopteran-specific delta-endotoxin

1986 ◽  
Vol 83 (1) ◽  
pp. 89-101
Author(s):  
B.H. Knowles ◽  
D.J. Ellar
Keyword(s):  
Plasma Membrane ◽  
Bacillus Thuringiensis ◽  
Choristoneura Fumiferana ◽  
Galactose Oxidase ◽  
Partial Purification ◽  
Wide Range ◽  
Delta Endotoxin ◽  
Plasma Membrane Receptor ◽  
Insect Gut

The lepidopteran-specific P1 delta-endotoxin of Bacillus thuringiensis var. kurstaki HD-1 was activated in vitro using insect gut proteases and found to be highly specific for the lepidopteran cell line Choristoneura fumiferana CF1 among a wide range of lepidopteran and dipteran cell lines tested. The toxicity of P1 against CF1 cells is inhibited by N-acetylgalactosamine (GalNAc), and the lectins soybean agglutinin (SBA) and wheat-germ agglutinin. Protein blotting was used to identify a glycoprotein of 146 X 10(3) Mr in the plasma membrane of CF1 cells, capable of binding both the toxin and SBA, which is specific for GalNAc. This glycoprotein was labelled using galactose oxidase and sodium boro-[3H]hydride and solubilized in Triton X-100 before partial purification by affinity chromatography on SBA-agarose. We propose that this glycoprotein is a good candidate for the cellular receptor of the lepidopteran-specific P1 delta-endotoxin of B. thuringiensis var. kurstaki HD-1.

Bacillus thuringiensis var israelensis crystal delta-endotoxin: effects on insect and mammalian cells in vitro and in vivo

1983 ◽  
Vol 60 (1) ◽  
pp. 181-197 ◽  
Author(s):  
W.E. Thomas ◽  
D.J. Ellar
Keyword(s):  
Body Weight ◽  
Bacillus Thuringiensis ◽  
Mammalian Cells ◽  
Choristoneura Fumiferana ◽  
Similar Response ◽  
Parasporal Crystal ◽  
Discontinuous Sucrose Gradient ◽  
Delta Endotoxin

Bacillus thuringiensis var israelensis parasporal crystal delta-endotoxin was purified by ultracentrifugation on a discontinuous sucrose gradient. Native delta-endotoxin crystals showed no detectable toxicity in the vitro and in vivo systems that are described. By contrast alkali-solubilized crystal delta-endotoxin caused rapid cytological and cytopathological changes in Aedes albopictus, Choristoneura fumiferana 63 CF1, Spodoptera frugiperda and Trichoplusia ni cell lines as observed by phase-contrast microscopy and vital staining. Mouse fibroblasts, primary pig lymphocytes and three mouse epithelial carcinoma cell types showed a similar response to the alkali-soluble crystal delta-endotoxin. In addition the soluble crystal delta-endotoxin protein caused haemolysis of rat, mouse, sheep, horse and human erythrocytes. Intravenous administration of the alkali-soluble crystal delta-endotoxin to Balb. c mice at a dose rate of 15–30 micrograms of protein per gram body weight resulted in rapid paralysis followed by death within 12h. Subcutaneous inoculation of 15–30 micrograms of protein per gram body weight resulted in death of suckling mice in 2–3 h. The alkali-solubilized crystal delta-endotoxin was not toxic however, when administered per os. A comparison is made with a similar alkali-soluble fraction from the parasporal crystal delta-endotoxin of B. thuringiensis var kurstaki. With the exception of the Lepidopteran cell line, Choristoneura fumiferana 63 CF1, this soluble crystal delta-endotoxin protein showed no in vitro or in vivo toxicity, and no haemolytic activity.

scholarly journals Effects of Bacillus thuringiensis δ-Endotoxin on Insect and Mammalian Cells In Vitro

1980 ◽  
Vol 15 (2) ◽  
pp. 133-139 ◽  
Author(s):  
Junko NISHITSUTSUJI-UWO ◽  
Yasuhisa ENDO ◽  
Michio HIMENO
Keyword(s):  
Bacillus Thuringiensis ◽  
Mammalian Cells ◽  
Delta Endotoxin

scholarly journals In vitro release of physically separable factors from monocytes that exert opposing effects on erythropoiesis

Blood ◽  
1986 ◽  
Vol 67 (5) ◽  
pp. 1454-1459 ◽  
Author(s):  
L Feldman ◽  
CM Cohen ◽  
N Dainiak
Keyword(s):  
Plasma Membrane ◽  
Membrane Vesicle ◽  
In Vitro Release ◽  
Membrane Vesicles ◽  
Plasma Membrane Vesicles ◽  
Wide Range ◽  
Dose Dependent Fashion ◽  
Membrane Bound ◽  
Opposing Effects

Abstract In order to investigate the capacity of monocytes to release erythroid burst-promoting activity (BPA), we added media conditioned by homologous monocytes to both serum-free human and serum-restricted murine marrow culture. We found that soluble, membrane vesicle-free culture medium is a potent source of the growth factor. On the other hand, monocyte membranes or exfoliated plasma membrane vesicles elaborate a factor that inhibits erythroid burst formation by up to 100%. Inhibitory activity is expressed in a dose-dependent fashion over a wide range of concentrations (0.001 to 10 micrograms/mL) tested. Experiments with antilymphocyte plasma membrane IgG, which has been shown to neutralize both soluble and membrane-bound lymphocyte-derived BPA in human marrow culture, indicate that the expression of soluble BPA by monocytes is unaffected by these antibodies. Furthermore, while antimembrane IgG is capable of absorbing BPA from LCM supernatants, these antibodies are ineffective in removing BPA from MCM supernatants, suggesting that these two soluble growth factors may be antigenically distinct. Our findings indicate that while monocytes release soluble BPA, they are also a source of membrane-associated factors that exert inhibitory effects on erythropoiesis in vitro.

scholarly journals In vitro release of physically separable factors from monocytes that exert opposing effects on erythropoiesis

Blood ◽  
1986 ◽  
Vol 67 (5) ◽  
pp. 1454-1459
Author(s):  
L Feldman ◽  
CM Cohen ◽  
N Dainiak
Keyword(s):  
Plasma Membrane ◽  
Membrane Vesicle ◽  
In Vitro Release ◽  
Membrane Vesicles ◽  
Plasma Membrane Vesicles ◽  
Wide Range ◽  
Dose Dependent Fashion ◽  
Membrane Bound ◽  
Opposing Effects

In order to investigate the capacity of monocytes to release erythroid burst-promoting activity (BPA), we added media conditioned by homologous monocytes to both serum-free human and serum-restricted murine marrow culture. We found that soluble, membrane vesicle-free culture medium is a potent source of the growth factor. On the other hand, monocyte membranes or exfoliated plasma membrane vesicles elaborate a factor that inhibits erythroid burst formation by up to 100%. Inhibitory activity is expressed in a dose-dependent fashion over a wide range of concentrations (0.001 to 10 micrograms/mL) tested. Experiments with antilymphocyte plasma membrane IgG, which has been shown to neutralize both soluble and membrane-bound lymphocyte-derived BPA in human marrow culture, indicate that the expression of soluble BPA by monocytes is unaffected by these antibodies. Furthermore, while antimembrane IgG is capable of absorbing BPA from LCM supernatants, these antibodies are ineffective in removing BPA from MCM supernatants, suggesting that these two soluble growth factors may be antigenically distinct. Our findings indicate that while monocytes release soluble BPA, they are also a source of membrane-associated factors that exert inhibitory effects on erythropoiesis in vitro.

scholarly journals Mutagenesis of two surface-exposed loops of the Bacillus thuringiensis CryIC δ-endotoxin affects insecticidal specificity

1994 ◽  
Vol 302 (2) ◽  
pp. 611-616 ◽  
Author(s):  
G P Smith ◽  
D J Ellar
Keyword(s):  
Bacillus Thuringiensis ◽  
Aedes Aegypti ◽  
Time Dependence ◽  
Time Course ◽  
Site Directed Mutagenesis ◽  
Sf9 Cells ◽  
Degenerate Oligonucleotide ◽  
Delta Endotoxin ◽  
A Cell

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.

scholarly journals Comparative analysis of the individual protoxin components in P1 crystals of Bacillus thuringiensis subsp. kurstaki isolates NRD-12 and HD-1

1990 ◽  
Vol 269 (2) ◽  
pp. 507-512 ◽  
Author(s):  
L Masson ◽  
G Préfontaine ◽  
L Péloquin ◽  
P C K Lau ◽  
R Brousseau
Keyword(s):  
Bacillus Thuringiensis ◽  
Choristoneura Fumiferana ◽  
Insect Cell Line ◽  
Gene Products ◽  
Bacillus Thuringiensis Subsp ◽  
Independent Verification ◽  
Lepidopteran Larvae ◽  
Bacterium Bacillus ◽  
The Individual

Two commercially important strains (NRD-12 and HD-1) of the entomopathogenic bacterium Bacillus thuringiensis subsp. kurstaki each contain three genes of partially identical sequence coding for three classes of 130-135 kDa protoxins (termed the 4.5, 5.3 and 6.6 protoxins) that display toxicity towards various lepidopteran larvae. These gene products combine to form the intracellular bipyramidal P1 crystal. Each of the genes from both strains was cloned and expressed in Escherichia coli. Analysis of the cloned genes at the restriction-endonuclease level revealed no detectable differences among genes within a particular gene class. The composition of the P1 crystal from both strains was quantitatively analysed by CNBr cleavage of the purified P1 crystal, with the purified recombinant gene products as reference proteins. Independent verification of the presence of high 6.6-protoxin gene product in the P1 crystal was provided by a rapid in vitro lawn cell toxicity assay directed against a Choristoneura fumiferana (CF-1) insect cell line. The results indicate that, although all three gene products are represented within the P1 crystal of either NRD-12 or HD-1, only the contents of the 4.5 and 5.3 protoxins vary between the two crystals, whereas the 6.6 protoxin contents are similar and represent approximately one-third of the P1 crystal in either strain.

scholarly journals Solubilization, Activation, and Insecticidal Activity of Bacillus thuringiensis Serovar thompsoni HD542 Crystal Proteins

2008 ◽  
Vol 74 (23) ◽  
pp. 7145-7151 ◽  
Author(s):  
Samir Naimov ◽  
Rumyana Boncheva ◽  
Rumyana Karlova ◽  
Stefan Dukiandjiev ◽  
Ivan Minkov ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Pieris Rapae ◽  
Insect Pest ◽  
Polyacrylamide Gel Electrophoresis ◽  
Cydia Pomonella ◽  
Sodium Dodecyl ◽  
Crystal Proteins ◽  
Stable Product ◽  
Insect Gut

ABSTRACT Cry15Aa protein, produced by Bacillus thuringiensis serovar thompsoni HD542 in a crystal together with a 40-kDa accompanying protein, is one of a small group of nontypical, less well-studied members of the Cry family of insecticidal proteins and may provide an alternative for the more commonly used Cry proteins in insect pest management. In this paper, we describe the characterization of the Cry15Aa and 40-kDa protein's biochemical and insecticidal properties and the mode of action. Both proteins were solubilized above pH 10 in vitro. Incubation of solubilized crystal proteins with trypsin or insect midgut extracts rapidly processed the 40-kDa protein to fragments too small to be detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas the Cry15 protein yielded a stable product of approximately 30 kDa. Protein N-terminal sequencing showed that Cry15 processing occurs exclusively at the C-terminal end. Cry15 protein showed in vitro hemolytic activity, which was greatly enhanced by preincubation with trypsin or insect gut extract. Larvae of the lepidopteran insects Manduca sexta, Cydia pomonella, and Pieris rapae were susceptible to crystals, and presolubilization of the crystals enhanced activity to P. rapae. Activity for all three species was enhanced by preincubation with trypsin. Larvae of Helicoverpa armigera and Spodoptera exigua were relatively insensitive to crystals, and activity against these insects was not enhanced by prior solubilization or trypsin treatment. The 40-kDa crystal protein showed no activity in the insects tested, nor did its addition or coexpression in Escherichia coli increase the activity of Cry15 in insecticidal and hemolytic assays.

scholarly journals Proteolytic processing of a coleopteran-specific δ-endotoxin produced by Bacillus thuringiensis var. tenebrionis

1989 ◽  
Vol 261 (1) ◽  
pp. 99-105 ◽  
Author(s):  
J Carroll ◽  
J Li ◽  
D J Ellar
Keyword(s):  
Amino Acid ◽  
Bacillus Thuringiensis ◽  
Amino Acid Sequence ◽  
Proteinase Inhibitors ◽  
Cleavage Product ◽  
Proteolytic Processing ◽  
N Terminus ◽  
Delta Endotoxin ◽  
Insect Gut

Insecticidal protein delta-endotoxin crystals harvested from sporulated cultures of Bacillus thuringiensis var. tenebrionis contain a major polypeptide of 67 kDa and minor polypeptides of 73, 72, 55 and 46 kDa. During sporulation, only the 73 kDa polypeptide could be detected at stage I. The 67 kDa polypeptide was first detected at stage II and increased in concentration throughout the later stages of sporulation and after crystal release, with a concomitant decrease in the 73 kDa polypeptide. This change could be blocked by the addition of proteinase inhibitors. Trypsin or insect-gut-extract treatment of the delta-endotoxin crystals after solubilization resulted in a cleavage product of 55 kDa with asparagine-159 of the deduced amino acid sequence of the toxin [Höfte, Seurinck, van Houtven & Vaeck (1987) Nucleic Acids Res. 15, 71-83; Sekar, Thompson, Maroney, Bookland & Adang (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 7036-7040; McPherson, Perlak, Fuchs, Marrone, Lavrik & Fischhoff (1988) Biotechnology 6, 61-66] at the N-terminus. This polypeptide was found to be as toxic in vivo as native delta-endotoxin.

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