scholarly journals Binding of Cyt1Aa and Cry11Aa Toxins of Bacillus thuringiensis Serovar israelensis to Brush Border Membrane Vesicles of Tipula paludosa (Diptera: Nematocera) and Subsequent Pore Formation

2007 ◽  
Vol 73 (11) ◽  
pp. 3623-3629 ◽  
Author(s):  
Jesko Oestergaard ◽  
Ralf-Udo Ehlers ◽  
Amparo C. Martínez-Ramírez ◽  
Maria Dolores Real
Keyword(s):  
Bacillus Thuringiensis ◽  
Brush Border ◽  
Pore Formation ◽  
Brush Border Membrane ◽  
Synergistic Effects ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Proteinase K ◽  
Toxic Protein ◽  
Key Steps

ABSTRACT Bacillus thuringiensis serovar israelensis (B. thuringiensis subsp. israelensis) produces four insecticidal crystal proteins (ICPs) (Cry4A, Cry4B, Cry11A, and Cyt1A). Toxicity of recombinant B. thuringiensis subsp. israelensis strains expressing only one of the toxins was determined with first instars of Tipula paludosa (Diptera: Nematocera). Cyt1A was the most toxic protein, whereas Cry4A, Cry4B, and Cry11A were virtually nontoxic. Synergistic effects were recorded when Cry4A and/or Cry4B was combined with Cyt1A but not with Cry11A. The binding and pore formation are key steps in the mode of action of B. thuringiensis subsp. israelensis ICPs. Binding and pore-forming activity of Cry11Aa, which is the most toxic protein against mosquitoes, and Cyt1Aa to brush border membrane vesicles (BBMVs) of T. paludosa were analyzed. Solubilization of Cry11Aa resulted in two fragments, with apparent molecular masses of 32 and 36 kDa. No binding of the 36-kDa fragment to T. paludosa BBMVs was detected, whereas the 32-kDa fragment bound to T. paludosa BBMVs. Only a partial reduction of binding of this fragment was observed in competition experiments, indicating a low specificity of the binding. In contrast to results for mosquitoes, the Cyt1Aa protein bound specifically to the BBMVs of T. paludosa, suggesting an insecticidal mechanism based on a receptor-mediated action, as described for Cry proteins. Cry11Aa and Cyt1Aa toxins were both able to produce pores in T. paludosa BBMVs. Protease treatment with trypsin and proteinase K, previously reported to activate Cry11Aa and Cyt1Aa toxins, respectively, had the opposite effect. A higher efficiency in pore formation was observed when Cyt1A was proteinase K treated, while the activity of trypsin-treated Cry11Aa was reduced. Results on binding and pore formation are consistent with results on ICP toxicity and synergistic effect with Cyt1Aa in T. paludosa.

scholarly journals Mutations in Domain I Interhelical Loops Affect the Rate of Pore Formation by the Bacillus thuringiensis Cry1Aa Toxin in Insect Midgut Brush Border Membrane Vesicles

2009 ◽  
Vol 75 (12) ◽  
pp. 3842-3850 ◽  
Author(s):  
Geneviève Lebel ◽  
Vincent Vachon ◽  
Gabrielle Préfontaine ◽  
Frédéric Girard ◽  
Luke Masson ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Conformational Changes ◽  
Brush Border ◽  
Pore Formation ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Thiol Group ◽  
Brush Border Membrane Vesicles ◽  
Site Directed Mutagenesis ◽  
Domain I

ABSTRACT Pore formation in the apical membrane of the midgut epithelial cells of susceptible insects constitutes a key step in the mode of action of Bacillus thuringiensis insecticidal toxins. In order to study the mechanism of toxin insertion into the membrane, at least one residue in each of the pore-forming-domain (domain I) interhelical loops of Cry1Aa was replaced individually by cysteine, an amino acid which is normally absent from the activated Cry1Aa toxin, using site-directed mutagenesis. The toxicity of most mutants to Manduca sexta neonate larvae was comparable to that of Cry1Aa. The ability of each of the activated mutant toxins to permeabilize M. sexta midgut brush border membrane vesicles was examined with an osmotic swelling assay. Following a 1-h preincubation, all mutants except the V150C mutant were able to form pores at pH 7.5, although the W182C mutant had a weaker activity than the other toxins. Increasing the pH to 10.5, a procedure which introduces a negative charge on the thiol group of the cysteine residues, caused a significant reduction in the pore-forming abilities of most mutants without affecting those of Cry1Aa or the I88C, T122C, Y153C, or S252C mutant. The rate of pore formation was significantly lower for the F50C, Q151C, Y153C, W182C, and S252C mutants than for Cry1Aa at pH 7.5. At the higher pH, all mutants formed pores significantly more slowly than Cry1Aa, except the I88C mutant, which formed pores significantly faster, and the T122C mutant. These results indicate that domain I interhelical loop residues play an important role in the conformational changes leading to toxin insertion and pore formation.

scholarly journals Protease Inhibitors Fail To Prevent Pore Formation by the Activated Bacillus thuringiensis Toxin Cry1Aa in Insect Brush Border Membrane Vesicles

2006 ◽  
Vol 72 (1) ◽  
pp. 506-515 ◽  
Author(s):  
Martin Kirouac ◽  
Vincent Vachon ◽  
Delphine Quievy ◽  
Jean-Louis Schwartz ◽  
Raynald Laprade
Keyword(s):  
Bacillus Thuringiensis ◽  
Protease Inhibitors ◽  
Brush Border ◽  
Pore Formation ◽  
Brush Border Membrane ◽  
Divalent Cations ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Metalloprotease Inhibitors ◽  
The Stability

ABSTRACT To investigate whether membrane proteases are involved in the activity of Bacillus thuringiensis insecticidal toxins, the rate of pore formation by trypsin-activated Cry1Aa was monitored in the presence of a variety of protease inhibitors with Manduca sexta midgut brush border membrane vesicles and by a light-scattering assay. Most of the inhibitors tested had no effect on the pore-forming ability of the toxin. However, phenylmethylsulfonyl fluoride, a serine protease inhibitor, promoted pore formation, although this stimulation only occurred at higher inhibitor concentrations than those commonly used to inhibit proteases. Among the metalloprotease inhibitors, o-phenanthroline had no significant effect; EDTA and EGTA reduced the rate of pore formation at pH 10.5, but only EDTA was inhibitory at pH 7.5. Neither chelator affected the properties of the pores already formed after incubation of the vesicles with the toxin. Taken together, these results indicate that, once activated, Cry1Aa is completely functional and does not require further proteolysis. The effect of EDTA and EGTA is probably better explained by their ability to chelate divalent cations that could be necessary for the stability of the toxin's receptors or involved elsewhere in the mechanism of pore formation.

scholarly journals Pore-forming properties of the Bacillus thuringiensis toxin Cry9Ca in Manduca sexta brush border membrane vesicles

2010 ◽  
Vol 1798 (6) ◽  
pp. 1111-1118 ◽  
Author(s):  
Jean-Frédéric Brunet ◽  
Vincent Vachon ◽  
Marc Juteau ◽  
Jeroen Van Rie ◽  
Geneviève Larouche ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Manduca Sexta ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Bacillus Thuringiensis Toxin

scholarly journals Changes in Permeability of Brush Border Membrane Vesicles fromSpodoptera littoralis Midgut Induced by Insecticidal Crystal Proteins from Bacillus thuringiensis

1998 ◽  
Vol 64 (4) ◽  
pp. 1563-1565 ◽  
Author(s):  
B. Escriche ◽  
N. De Decker ◽  
J. Van Rie ◽  
S. Jansens ◽  
E. Van Kerkhove
Keyword(s):  
Bacillus Thuringiensis ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Osmotic Swelling ◽  
Midgut Cell ◽  
Crystal Proteins ◽  
Light Scattering Technique ◽  
Insecticidal Crystal Proteins

ABSTRACT Bacillus thuringiensis insecticidal crystal proteins (ICPs) are thought to induce pore formation in midgut cell membranes of susceptible insects. Cry1Ca, which is significantly active inSpodoptera littoralis, made brush border membrane vesicles permeable to KCl (osmotic swelling was monitored by the light scattering technique); the marginally active ICPs Cry1Aa, Cry1Ab, and Cry1Ac did not.

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