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 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.

Biological Activity of Bacillus thuringiensis and Associated Toxins against the Asian Longhorned Beetle (Coleoptera: Cerambycidae)

2004 ◽  
Vol 39 (3) ◽  
pp. 318-324 ◽  
Author(s):  
Vincent D'Amico ◽  
John D. Podgwaite ◽  
Sara Duke
Keyword(s):  
Bacillus Thuringiensis ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
In Vitro Assays ◽  
Asian Longhorned Beetle ◽  
Negative Effects ◽  
In Vivo Tests ◽  
Longhorned Beetles

Bacillus thuringiensis Berliner var. tenebrionis and B. thuringiensis toxins were assayed against larval and adult Asian longhorned beetles, Anoplophora glabripennis (A. glabripennis). Preliminary in vitro assays showed some toxins to be active on whole midgut preparations in voltage clamp assays and in assays on brush border membrane vesicles formed from midgut epithelial cells. For in vivo tests, a commercially-available product (Novodor®) was incorporated into artificial diet, upon which larvae were allowed to feed ad lib. In other tests, droplets of solubilized B. thuringiensis toxins were fed to larval and adult beetles using a micropipette. None of the in vivo assays showed significant negative effects on either larvae or adults. We believe that some aspect of A. glabripennis midgut chemistry may be incompatible with toxin activation or mode of action.

Proteolytic release of membrane-bound endo-(1,4)-β-glucanase activity associated with cell wall softening inAchlya ambisexualis

2002 ◽  
Vol 48 (1) ◽  
pp. 93-98 ◽  
Author(s):  
Terry W Hill ◽  
Darlene M Loprete ◽  
Kim N Vu ◽  
Susan P. Bayat Mokhtari ◽  
L Vanessa Hardin
Keyword(s):  
Cell Wall ◽  
Membrane Vesicles ◽  
Cysteine Proteases ◽  
Proteolytic Processing ◽  
Cysteine Protease Inhibitor ◽  
Glucanase Activity ◽  
Molecular Masses ◽  
Detergent Treatment ◽  
Membrane Bound

Branching and other cell wall softening events in fungi and oomycetes are thought to involve the activity of secreted enzymes, which are packaged in membrane vesicles and delivered to sites of cell expansion, there to work in a carefully regulated manner upon the structure of the wall. Here we demonstrate a latent endo-(1,4)-β-glucanase activity in a mixed membrane fraction of the oomycete Achlya ambisexualis, which can be released by cysteine proteases with an increase of apparent activity. In addition, a similar endogenous process is strongly inhibited by the cysteine protease inhibitor iodoacetamide, while inhibitors of other types of proteases have a much smaller effect. Detergent treatment of membranes releases two glucanases detectable by electrophoretic activity staining, with apparent molecular masses of about 164 and 35 kDa. Proteolysis produces several activity bands, with major species having apparent molecular masses of about 149, 133, 48, 35, and 25 kDa. The ca. 35- and 25-kDa bands migrate in parallel with glucanases secreted during wall softening in vivo. We propose that the initiation of wall softening in Achlya involves the proteolytic processing and solubilization of at least some secreted endoglucanases. We also propose that the solubilization component of this process functions not just to provide the enzymes with access to wall matrix substrates but also may provide a mechanism for the eventual termination of their biological function.Key words: apical growth, hyphal branching, proteases, cell walls, protein secretion.

scholarly journals Aedes aegypti cadherin serves as a putative receptor of the Cry11Aa toxin from Bacillus thuringiensis subsp. israelensis

2009 ◽  
Vol 424 (2) ◽  
pp. 191-200 ◽  
Author(s):  
Jianwu Chen ◽  
Karlygash G. Aimanova ◽  
Luisa E. Fernandez ◽  
Alejandra Bravo ◽  
Mario Soberon ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Aedes Aegypti ◽  
Membrane Vesicles ◽  
Toxin Binding ◽  
Bacillus Thuringiensis Subsp ◽  
Posterior Midgut ◽  
Cry Toxin ◽  
Lepidopteran Insects ◽  
Toxin Receptor ◽  
Apical Membranes

Cry11Aa of Bacillus thuringiensis subsp. israelensis is the most active toxin to Aedes aegypti in this strain. We previously reported that, in addition to a 65 kDa GPI (glycosylphosphatidylinositol)-anchored ALP (alkaline phosphatase), the toxin also binds a 250 kDa membrane protein. Since this protein is the same size as cadherin, which in lepidopteran insects is an important Cry toxin receptor, we developed an anti-AaeCad antibody. This antibody detects a 250 kDa protein in immunoblots of larval BBMVs (brush border membrane vesicles). The antibody inhibits Cry11Aa toxin binding to BBMVs and immunolocalizes the cadherin protein to apical membranes of distal and proximal caecae and posterior midgut epithelial cells. This localization is consistent with areas to which Cry11Aa toxin binds and causes pathogenicity. Therefore, the full-length Aedes cadherin cDNA was isolated from Aedes larvae and partial overlapping fragments that covered the entire protein were expressed in Escherichia coli. Using toxin overlay assays, we showed that one cadherin fragment, which contains CR7–11 (cadherin repeats 7–11), bound Cry11Aa and this binding was primarily through toxin domain II loops α8 and 2. Cadherin repeats CR8–11 but not CR7 bound Cry11Aa under non-denaturing conditions. Cry11Aa bound the cadherin fragment with high affinity with an apparent Kd of 16.7 nM. Finally we showed that this Cry11Aa-binding site could also be competed by Cry11Ba and Cry4Aa but not Cry4Ba. These results indicate that Aedes cadherin is possibly a receptor for Cry11A and, together with its ability to bind an ALP, suggest a similar mechanism of toxin action as previously proposed for lepidopteran insects.

scholarly journals Bacillus thuringiensis subsp. israelensis Cyt1Aa synergizes Cry11Aa toxin by functioning as a membrane-bound receptor

2005 ◽  
Vol 102 (51) ◽  
pp. 18303-18308 ◽  
Author(s):  
C. Perez ◽  
L. E. Fernandez ◽  
J. Sun ◽  
J. L. Folch ◽  
S. S. Gill ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Bacillus Thuringiensis Subsp ◽  
Membrane Bound

Selective inhibition of binding of Bacillus thuringiensis Cry1Ab toxin to cadherin-like and aminopeptidase proteins in brush-border membranes and dissociated epithelial cells from Bombyx mori

2007 ◽  
Vol 409 (1) ◽  
pp. 215-221 ◽  
Author(s):  
M. Sales Ibiza-Palacios ◽  
Juan Ferré ◽  
Satoshi Higurashi ◽  
Kazuhisa Miyamoto ◽  
Ryoichi Sato ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Epithelial Cells ◽  
Bombyx Mori ◽  
Brush Border ◽  
Membrane Vesicles ◽  
Inhibitory Effect ◽  
Irreversible Binding ◽  
Toxin Binding ◽  
Cry1ab Toxin

Binding analyses with denatured epithelial membrane proteins from Bt (Bacillus thuringiensis) demonstrated at least two kinds of proteins, APNs (aminopeptidases N) and cadherin-like proteins, as possible receptors for the Cry1A class of Bt toxins. Two alternative models have been proposed, both based on initial toxin binding to a cadherin-like protein, but one involving APN and the other not. We have used two Bombyx mori strains (J65 and Kin), which are highly susceptible to Cry1Ab, to study the role of these two types of receptors on Cry1Ab toxin binding and cytotoxicity by means of the inhibitory effect of antibodies. BBMVs (brush-border membrane vesicles) of strain J65 incubated with labelled 125I-Cry1Ab revealed a marked reduction in reversible and irreversible binding when anti-BtR175 (a cadherin-like protein) was used for BBMV pre-treatment. By contrast, the anti-APN1 antibody specifically affected the irreversible binding, while the reversible binding component was not affected. This is the first time that binding of Cry1Ab to APN1 and to a cadherin-like protein from BBMVs in solution has been shown. Dissociated epithelial cells from the Kin strain were used to test the inhibitory effect of the antibodies on the cytotoxicity of Cry1Ab. Pre-incubation of the cells with the anti-BtR175 antibody conferred protection against Cry1Ab, but not the anti-APN1 antibody. Therefore our results seem to support the two models of the mode of action of Cry1Ab in Lepidoptera, depending on whether BBMVs or intact dissociated cells are used, suggesting that both pathways may co-operate for the toxicity of Cry1A toxins in vivo.

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