A rapid and sensitive in vitro assay for the activity of Bacillus thuringiensis delta-endotoxins (original) (raw)
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Applied and environmental microbiology, 1997
To test whether the ability of Bacillus thuringiensis toxins to form pores in the midgut epithelial cell membrane of susceptible insects correlates with their in vivo toxicity, we measured the effects of different toxins on the electrical potential of the apical membrane of freshly isolated midguts from gypsy moth (Lymantria dispar) and silkworm (Bombyx mori) larvae. In the absence of toxin, the membrane potential, measured with a conventional glass microelectrode, was stable for up to 30 min. It was sensitive to the K+ concentration and the oxygenation of the external medium. Addition of toxins to which L. dispar is highly [CryIA(a) and CryIA(b)] or only slightly [CryIA(c) and CryIC] sensitive caused a rapid, irreversible, and dose-dependent depolarization of the membrane. CryIF, whose toxicity towards L. dispar is unknown, and CryIE, which is at best poorly active in vivo, were also active in vitro. In contrast, CryIB and CryIIIA, a coleopteran-specific toxin, had no significant e...
Journal of Invertebrate Pathology, 1997
Epithelial tissue wholemounts were produced after enzymatic removal of basal lamina and connective tissue from midguts of Trichoplusia ni larvae. Wholemounts were nourished in artificial hemolymph and tissue viability was assessed for up to 24 hr using the vital dyes trypan blue, acridine orange (AO), propidium iodide (PI), and 48,6-diamidino-2-phenylindole (DAPI). Peritrophic membrane synthesis and modification of Bacillus thuringiensis Cry1Ac protoxin to active toxin confirmed some normal epithelial function. Vital staining using the combination of AO and PI, or DAPI revealed altered membrane permeability in columnar epithelial and regenerative cells of tissues treated with activated Cry1Ac toxin while feeding and oral inoculation bioassays verified Cry1Ac toxicity. DAPI was selected to identify target cells in a rapid and highly sensitive assay.
Comparative Biochemistry and Physiology Part C: Comparative Pharmacology, 1990
1. The effect of two recombinant Bacillus thuringiensis delta-endotoxins on brush border membrane vesicles of Manduca sexta midgut was investigated using an in vitro assay system, based on ion-amino acid cotransport.2. A CryIA(b)-toxin provoked an increase in the permeability of the vesicles.3. A CryIB-toxin, not toxic to M. sexta larvae in vivo, had no effect in our assay.4. In contrast to earlier reports, the increase in permeability was found to be neither selective for K+ nor specifically inhibited by Ca2+ or Ba2+.5. Our data support the hypothesis that B. thuringiensis delta-endotoxins create non-specific pores.
FEBS Letters, 1986
The luminal -membrane of larval midgut cells is the site of action of insecticidal delta-endotoxin from Bacillus thuringiensis. At concentrations that correspond to normal effective doses in vivo, the toxin inhibits the uptake of amino acids by brush border membrane vesicles prepared from midguts of Pieris brassicae larvae. The toxin does not interact with the K+-amino acid symport but rather increases the K+ permeability of the membrane. The toxin does not increase the permeability of lepidopteran midgut brush border membrane to either Na+ or H+ nor does it increase the K+ nermeabilitv of brush border membrane vesicles prepared from mammalian smaliintestine.
Enzyme and Microbial Technology, 2002
Delta-endotoxin production capacity of several Bacillus thuringiensis (Bt) strains exhibiting various larvicidal activities towards either lepidoptera or diptera was investigated in gruel and fish meal media. Diptera-specific strains produced less delta-endotoxins (1246-1998 mg l −1 ) than lepidoptera-specific ones (3060-3301 mg l −1 ). Carbon catabolite repression regulated delta-endotoxins synthesis for all strains, but no relationship existed between the specificity of delta-endotoxins and their sensitivity to such repressive regulation. The production of proteolytic activities by the isolate BNS3 and the commercial strain HD1, was reduced in 0.5 g l −1 sodium chloride and 0.1% Tween-80 but not by other strains. Moreover, in the case of the diptera-specific strain BUPM98, there was a need for additional nitrogen sources and to some extent, several amino acids from yeast extract, leading to an increase of 22% in delta-endotoxin production. Sporulation of BUPM98 was never reached in the rich Luria Broth (LB) medium, compared with that of the lepidoptera-specific strain BNS3. In shake flask cultures at 10 g l −1 , sodium acetate notably increased (38-79%) delta-endotoxins production by the diptera-specific strains, with a strong decrease in proteolytic activities. Similar delta-endotoxin production and high proteolytic activities were obtained with or without 10 g l −1 sodium acetate with an excess of aeration in a 2-l fermenter. In a less aerated medium, a strong effect of acetate was observed, decreasing production of proteolytic activities and exhibiting high catabolite repression on delta-endotoxin synthesis. The effect of sodium acetate was not directly related to sporulation and protease synthesis but to acetate metabolism. Culture conditions were defined for the screening of Bt strains producing high counts of delta-endotoxins specific to either lepidoptera or diptera.
Interaction of Two Bacillus thuringiensis d-Endotoxins with the Digestive System of Lygus hesperus
Current Microbiology, 2004
The active-toxin form of Cry1Ac (65 kDa) or Cry2Ab was fed to a non-susceptible insect, Lygus hesperus, in an artificial diet. Biochemical and immunocytochemical methods were used to determine the distribution of ingested toxin. The toxins did not elicit a feeding deterrent response. Cry1Ac and Cry2Ab were ingested; small amounts were absorbed into the hemolymph as holoproteins, but most was excreted. SDS-PAGE analysis of Cry1Ac and Cry2Ab incubations with salivary gland homogenate showed a small decrease in the molecular weight of the active toxins. Proteolytic processing of the toxins also occurred in vivo, within the digestive system of L. hesperus. Excreted Cry1Ac and Cry2Ab retained activity toward lepidopteran larvae. Immunocytochemical in vivo localization studies showed negligible association of Cry1Ac with L. hesperus tissues. In contrast, strong extracellular association of Cry2Ab was observed with L. hesperus midgut brush border microvilli and basement membrane, as well as with cellular outlines within the hemolymph and fat body.