scholarly journals Investigation of physicochemical condition to stabilize phosphatidylcholine-liposome enclosing fluorescent calcein and its exploitation for analysis of pore formation with Cry1A toxins of Bacillus thuringiensis

2010 ◽  
Vol 45 (3) ◽  
pp. 477-488 ◽  
Author(s):  
Kohsuke Haginoya ◽  
Vaijayanthi Thangavel ◽  
Ganesh N. Pandian ◽  
Kazuya Tomimoto ◽  
Yasuyuki Shitomi ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Pore Formation ◽  
Physicochemical Condition ◽  
Phosphatidylcholine Liposome

scholarly journals Monitoring of Bacillus Thuringiensis Cry3Aa Toxin Pore Formation using Artificial Bilayer Array with Fused Brush Border Membrane Vesicles from Colorado Potato Beetle Larvae

2017 ◽  
Vol 112 (3) ◽  
pp. 523a
Author(s):  
Ekaterina Zaitseva ◽  
Gerhard Baaken ◽  
Victor M. Ruiz-Arroyo ◽  
Inmaculada García-Robles ◽  
Camila Ochoa-Campuzano ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Colorado Potato Beetle ◽  
Brush Border ◽  
Pore Formation ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Potato Beetle

Lipid-induced Pore Formation of the Bacillus thuringiensis Cry1Aa Insecticidal Toxin

2001 ◽  
Vol 180 (3) ◽  
pp. 195-203 ◽  
Author(s):  
V. Vié ◽  
N. Van Mau ◽  
P. Pomarède ◽  
C. Dance ◽  
J.L. Schwartz ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Pore Formation ◽  
Insecticidal Toxin

scholarly journals Investigation of the pore-forming mechanism of a cytolytic δ-endotoxin from Bacillus thuringiensis

2003 ◽  
Vol 374 (1) ◽  
pp. 255-259 ◽  
Author(s):  
Boonhiang PROMDONKOY ◽  
David J. ELLAR
Keyword(s):  
Bacillus Thuringiensis ◽  
Blood Cells ◽  
Pore Formation ◽  
Response Curve ◽  
Effect Of Temperature ◽  
Membrane Insertion ◽  
Free Solution ◽  
Forming Mechanism ◽  
Close Proximity ◽  
Membrane Bound

Cyt2Aa1 is a cytolytic protein produced by Bacillus thuringiensis subsp. kyushuensis. Penetration of the toxin into membranes has been studied to learn more about membrane-insertion mechanisms and transmembrane-pore formation. The haemolysis assay of Cyt2Aa1 showed a steep and sigmoidal dose–response curve, indicating that toxin aggregation or oligomerization is required for pore formation. Studies of the effect of temperature on pore formation and fluorimetric studies of acrylodan-labelled toxin suggest that toxin inserts into the membrane before oligomerizing to form a pore. Low temperature neither inhibited membrane binding nor closed pores that have been formed, but markedly inhibited oligomerization of the toxin molecules. When toxin-treated red blood cells at 4 °C were transferred to a toxin-free solution at 37 °C, no significant increase in haemolysis was observed. This result suggests that membrane-bound toxin could not diffuse laterally and interact with other molecules to form a pore. From these results, we propose that Cyt2Aa1 binds and inserts into the membrane as a monomer. Oligomerization occurs when toxin molecules have bound in close proximity to each other and pores are formed from large oligomers.

scholarly journals Cysteine Scanning Mutagenesis of α4, a Putative Pore-Lining Helix of the Bacillus thuringiensis Insecticidal Toxin Cry1Aa

2008 ◽  
Vol 74 (9) ◽  
pp. 2565-2572 ◽  
Author(s):  
Frédéric Girard ◽  
Vincent Vachon ◽  
Gabrielle Préfontaine ◽  
Lucie Marceau ◽  
Yanhui Su ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Pore Formation ◽  
Membrane Vesicles ◽  
Significant Activity ◽  
Amino Acid Residues ◽  
Insect Larvae ◽  
Insecticidal Toxin ◽  
Cysteine Scanning ◽  
Cysteine Scanning Mutagenesis ◽  
Pore Lining

ABSTRACT Helix α4 of Bacillus thuringiensis Cry toxins is thought to line the lumen of the pores they form in the midgut epithelial cells of susceptible insect larvae. To define its functional role in pore formation, most of the α4 amino acid residues were replaced individually by a cysteine in the Cry1Aa toxin. The toxicities and pore-forming abilities of the mutated toxins were examined, respectively, by bioassays using neonate Manduca sexta larvae and by a light-scattering assay using midgut brush border membrane vesicles isolated from M. sexta. A majority of these mutants had considerably reduced toxicities and pore-forming abilities. Most mutations causing substantial or complete loss of activity map on the hydrophilic face of the helix, while most of those having little or only relatively minor effects map on its hydrophobic face. The properties of the pores formed by mutants that retain significant activity appear similar to those of the pores formed by the wild-type toxin, suggesting that mutations resulting in a loss of activity interfere mainly with pore formation.

scholarly journals In vivo nanoscale analysis of the dynamic synergistic interaction of Bacillus thuringiensis Cry11Aa and Cyt1Aa toxins in Aedes aegypti

2021 ◽  
Vol 17 (1) ◽  
pp. e1009199
Author(s):  
Samira López-Molina ◽  
Nathaly Alexandre do Nascimento ◽  
Maria Helena Neves Lobo Silva-Filha ◽  
Adán Guerrero ◽  
Jorge Sánchez ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Pore Formation ◽  
Lower Layer ◽  
Larval Mortality ◽  
Membrane Insertion ◽  
Cell Cytoplasm ◽  
Mutant Proteins ◽  
Posterior Midgut ◽  
High Larval Mortality

The insecticidal Cry11Aa and Cyt1Aa proteins are produced by Bacillus thuringiensis as crystal inclusions. They work synergistically inducing high toxicity against mosquito larvae. It was proposed that these crystal inclusions are rapidly solubilized and activated in the gut lumen, followed by pore formation in midgut cells killing the larvae. In addition, Cyt1Aa functions as a Cry11Aa binding receptor, inducing Cry11Aa oligomerization and membrane insertion. Here, we used fluorescent labeled crystals, protoxins or activated toxins for in vivo localization at nano-scale resolution. We show that after larvae were fed solubilized proteins, these proteins were not accumulated inside the gut and larvae were not killed. In contrast, if larvae were fed soluble non-toxic mutant proteins, these proteins were found inside the gut bound to gut-microvilli. Only feeding with crystal inclusions resulted in high larval mortality, suggesting that they have a role for an optimal intoxication process. At the macroscopic level, Cry11Aa completely degraded the gastric caeca structure and, in the presence of Cyt1Aa, this effect was observed at lower toxin-concentrations and at shorter periods. The labeled Cry11Aa crystal protein, after midgut processing, binds to the gastric caeca and posterior midgut regions, and also to anterior and medium regions where it is internalized in ordered “net like” structures, leading finally to cell break down. During synergism both Cry11Aa and Cyt1Aa toxins showed a dynamic layered array at the surface of apical microvilli, where Cry11Aa is localized in the lower layer closer to the cell cytoplasm, and Cyt1Aa is layered over Cry11Aa. This array depends on the pore formation activity of Cry11Aa, since the non-toxic mutant Cry11Aa-E97A, which is unable to oligomerize, inverted this array. Internalization of Cry11Aa was also observed during synergism. These data indicate that the mechanism of action of Cry11Aa is more complex than previously anticipated, and may involve additional steps besides pore-formation activity.

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 The Cyt1Aa toxin from Bacillus thuringiensis inserts into target membranes via different mechanisms in insects, red blood cells, and lipid liposomes

2020 ◽  
Vol 295 (28) ◽  
pp. 9606-9617 ◽  
Author(s):  
Janette Onofre ◽  
Sabino Pacheco ◽  
Mary Carmen Torres-Quintero ◽  
Sarjeet S. Gill ◽  
Mario Soberon ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Pore Formation ◽  
Membrane Vesicles ◽  
Membrane System ◽  
Bound State ◽  
Cysteine Residue ◽  
Formation Model ◽  
Small Unilamellar Vesicle ◽  
Membrane Bound ◽  
Detergent Effect

Bacillus thuringiensis subsp. israelensis produces crystal inclusions composed of three-domain Cry proteins and cytolytic Cyt toxins, which are toxic to different mosquito larvae. A key component is the Cyt toxin, which synergizes the activity of the other Cry toxins, thereby resulting in high toxicity. The precise mechanism of action of Cyt toxins is still debated, and two models have been proposed: the pore formation model and the detergent effect. Here, we performed a systematic structural characterization of the Cyt toxin interaction with different membranes, including in Aedes aegypti larval brush border membrane vesicles, small unilamellar vesicle liposomes, and rabbit erythrocytes. We examined Cyt1Aa insertion into these membranes by analyzing fluorescence quenching in solution and in the membrane-bound state. For this purpose, we constructed several Cyt1Aa variants having substitutions with a single cysteine residue in different secondary structures, enabling Cys labeling with Alexa Fluor 488 for quenching analysis using I-soluble quencher in solution and in the membrane-bound state. We identified the Cyt1Aa residues exposed to the solvent upon membrane insertion, predicting a possible topology of the membrane-inserted toxin in the different membranes. Moreover, toxicity assays with these variants revealed that Cyt1Aa exerts its insecticidal activity and hemolysis through different mechanisms. We found that Cyt1Aa exhibits variable interactions with each membrane system, with deeper insertion into mosquito larva membranes, supporting the pore formation model, whereas in the case of erythrocytes and small unilamellar vesicles, Cyt1Aa's insertion was more superficial, supporting the notion that a detergent effect underlies its hemolytic activity.

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