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.

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 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 Knockout of Two Cry-Binding Aminopeptidase N Isoforms Does Not Change Susceptibility of Aedes aegypti Larvae to Bacillus thuringiensis subsp. israelensis Cry4Ba and Cry11Aa Toxins

Insects ◽  
2021 ◽  
Vol 12 (3) ◽  
pp. 223
Author(s):  
Junxiang Wang ◽  
Xiaozhen Yang ◽  
Huan He ◽  
Jingru Chen ◽  
Yuanyuan Liu ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Reverse Genetics ◽  
Membrane Vesicles ◽  
Molecular Evidence ◽  
Bacillus Thuringiensis Subsp ◽  
Membrane Bound ◽  
Homozygous Strain ◽  
High Binding Affinity ◽  
Single Knockout

The insecticidal Cry4Ba and Cry11Aa crystal proteins from Bacillus thuringiensis subsp. israelensis (Bti) are highly toxic to Ae. aegypti larvae. The glycosylphosphatidylinositol (GPI)-anchored APN was identified as an important membrane-bound receptor for multiple Cry toxins in numerous Lepidoptera, Coleoptera, and Diptera insects. However, there is no direct molecular evidence to link APN of Ae. aegypti to Bti toxicity in vivo. In this study, two Cry4Ba/Cry11Aa-binding Ae. aegypti GPI-APN isoforms (AeAPN1 and AeAPN2) were individually knocked-out using CRISPR/Cas9 mutagenesis, and the AeAPN1/AeAPN2 double-mutant homozygous strain was generated using the reverse genetics approach. ELISA assays showed that the high binding affinity of Cry4Ba and Cry11Aa protoxins to the midgut brush border membrane vesicles (BBMVs) from these APN knockouts was similar to the background from the wild-type (WT) strain. Likewise, the bioassay results showed that neither the single knockout of AeAPN1 or AeAPN2, nor the simultaneous disruption of AeAPN1 and AeAPN2 resulted in significant changes in susceptibility of Ae. aegypti larvae to Cry4Ba and Cry11Aa toxins. Accordingly, our results suggest that AeAPN1 and AeAPN2 may not mediate Bti Cry4Ba and Cry11Aa toxicity in Ae. aegypti larvae as their binding proteins.

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 Helix α4 of the Bacillus thuringiensis Cry1Aa Toxin Plays a Critical Role in the Postbinding Steps of Pore Formation

2008 ◽  
Vol 75 (2) ◽  
pp. 359-365 ◽  
Author(s):  
Frédéric Girard ◽  
Vincent Vachon ◽  
Gabrielle Préfontaine ◽  
Lucie Marceau ◽  
Jean-Louis Schwartz ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Disulfide Bonds ◽  
Pore Formation ◽  
Brush Border Membrane ◽  
Critical Role ◽  
Membrane Vesicles ◽  
Amino Acid Residues ◽  
Osmotic Swelling ◽  
Structural Differences ◽  
Intermolecular Disulfide Bonds

ABSTRACT Helix α4 of Bacillus thuringiensis Cry toxins is thought to play a critical role in the toxins' mode of action. Accordingly, single-site substitutions of many Cry1Aa helix α4 amino acid residues have previously been shown to cause substantial reductions in the protein's pore-forming activity. Changes in protein structure and formation of intermolecular disulfide bonds were investigated as possible factors responsible for the inactivity of these mutants. Incubation of each mutant with trypsin and chymotrypsin for 12 h did not reveal overt structural differences with Cry1Aa, although circular dichroism was slightly decreased in the 190- to 210-nm region for the I132C, S139C, and V150C mutants. The addition of dithiothreitol stimulated pore formation by the E128C, I132C, S139C, T142C, I145C, P146C, and V150C mutants. However, in the presence of these mutants, the membrane permeability never reached that measured for Cry1Aa, indicating that the formation of disulfide bridges could only partially explain their loss of activity. The ability of a number of inactive mutants to compete with wild-type Cry1Aa for pore formation in brush border membrane vesicles isolated from Manduca sexta was also investigated with an osmotic swelling assay. With the exception of the L147C mutant, all mutants tested could inhibit the formation of pores by Cry1Aa, indicating that they retained receptor binding ability. These results strongly suggest that helix α4 is involved mainly in the postbinding steps of pore formation.

scholarly journals Bacillus thuringiensis Cry1Ac Toxin-Binding and Pore-Forming Activity in Brush Border Membrane Vesicles Prepared from Anterior and Posterior Midgut Regions of Lepidopteran Larvae

2008 ◽  
Vol 74 (6) ◽  
pp. 1710-1716 ◽  
Author(s):  
Ana Rodrigo-Simón ◽  
Silvia Caccia ◽  
Juan Ferré
Keyword(s):  
Bacillus Thuringiensis ◽  
Brush Border ◽  
Mode Of Action ◽  
Pore Formation ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Insect Species ◽  
Binding Parameters ◽  
Larval Midgut ◽  
Posterior Midgut

ABSTRACT It is generally accepted that Bacillus thuringiensis Cry toxins insert into the apical membrane of the larval midgut after binding to specific receptors, and there is evidence that the distribution of binding molecules along the midgut is not uniform. By use of the voltage-sensitive dye DiSC3(5) and 125I-labeled Cry1Ac, we have measured the effect of Cry1Ac in terms of permeabilization capacity and of binding parameters on brush border membrane vesicles (BBMV) prepared from the anterior and the posterior regions of the larval midgut from two insect species, Manduca sexta and Helicoverpa armigera. The permeabilizing activity was significantly higher with BBMV from the posterior region than with the one observed in the anterior region in both insect species. Instead, 125I-Cry1Ac bound specifically to BBMV from the two midgut regions, with no significant differences in the binding parameters between the anterior and posterior regions within an insect species. N-acetylgalactosamine inhibition patterns on pore formation and binding differed between anterior and posterior midgut regions and between species, providing evidence of a multifaceted involvement of the sugar in the Cry1Ac mode of action. The analysis of binding and pore formation in different midgut regions could be an effective method to study differences in the mode of action of Cry1Ac toxin in different species.

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