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 Cadherin binding is not a limiting step for Bacillus thuringiensis subsp. israelensis Cry4Ba toxicity to Aedes aegypti larvae

2012 ◽  
Vol 443 (3) ◽  
pp. 711-717 ◽  
Author(s):  
Claudia Rodríguez-Almazán ◽  
Esmeralda Z. Reyes ◽  
Fernando Zúñiga-Navarrete ◽  
Carlos Muñoz-Garay ◽  
Isabel Gómez ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Aedes Aegypti ◽  
Lipid Bilayers ◽  
Insect Pests ◽  
Cry Toxins ◽  
Bacillus Thuringiensis Subsp ◽  
Limiting Step ◽  
Cry Toxin ◽  
Receptor Fragment

Bacillus thuringiensis subsp. israelensis produces three Cry toxins (Cry4Aa, Cry4Ba and Cry11Aa) that are active against Aedes aegypti larvae. The identification of the rate-limiting binding steps of Cry toxins that are used for insect control in the field, such as those of B. thuringiensis subsp. israelensis, should provide targets for improving insecticides against important insect pests. Previous studies showed that Cry11Aa binds to cadherin receptor fragment CR7–11 (cadherin repeats 7–11) with high affinity. Binding to cadherin has been proposed to facilitate Cry toxin oligomer formation. In the present study, we show that Cry4Ba binds to CR7–11 with 9-fold lower binding affinity compared with Cry11Aa. Oligomerization assays showed that Cry4Ba is capable of forming oligomers when proteolytically activated in vitro in the absence of the CR7–11 fragment in contrast with Cry11Aa that formed oligomers only in the presence of CR7–11. Pore-formation assays in planar lipid bilayers showed that Cry4Ba oligomers were proficient in opening ion channels. Finally, silencing the cadherin gene by dsRNA (double-stranded RNA) showed that silenced larvae were more tolerant to Cry11Aa in contrast with Cry4Ba, which showed similar toxic levels to those of control larvae. These findings show that cadherin binding is not a limiting step for Cry4Ba toxicity to A. aegypti larvae.

scholarly journals Denaturation of Either Manduca sexta Aminopeptidase N or Bacillus thuringiensis Cry1A Toxins Exposes Binding Epitopes Hidden under Nondenaturing Conditions

2002 ◽  
Vol 68 (5) ◽  
pp. 2106-2112 ◽  
Author(s):  
Anu Daniel ◽  
Sreedhara Sangadala ◽  
Donald H. Dean ◽  
Michael J. Adang
Keyword(s):  
Bacillus Thuringiensis ◽  
Manduca Sexta ◽  
Brush Border ◽  
Binding Proteins ◽  
Membrane Vesicles ◽  
Aminopeptidase N ◽  
Toxin Binding ◽  
Cry Toxin ◽  
Domain I ◽  
Ligand Blot

ABSTRACT The effect of polypeptide denaturation of Bacillus thuringiensis Cry1A toxins or purified Manduca sexta 120-kDa aminopeptidase N on the specificities of their interactions was investigated. Ligand and dot blotting experiments were conducted with 125I-labeled Cry1Ac, Cry1Ac mutant 509QNR-AAA511 (QNR-AAA), or 120-kDa aminopeptidase N as the probe. Mutant QNR-AAA does not bind the N-acetylgalactosamine moiety on the 120-kDa aminopeptidase. Both 125I-Cry1Ac and 125I-QNR-AAA bound to 210- and 120-kDa proteins from M. sexta brush border membrane vesicles and purified 120-kDa aminopeptidase N on ligand blots. However, on dot blots 125I-QNR-AAA bound brush border vesicles but did not bind purified aminopeptidase except when aminopeptidase was denatured. In the reciprocal experiment, 125I-aminopeptidase bound Cry1Ac but did not bind QNR-AAA. 125I-aminopeptidase bound Cry1Ab to a limited extent but not the Cry1Ab domain I mutant Y153D or Cry1Ca. However, denatured 125I-aminopeptidase detected each Cry1A toxin and mutant but not Cry1Ca on dot blots. The same pattern of recognition occurred with native (nondenatured) 125I-aminopeptidase probe and denatured toxins as the targets. The broader pattern of toxin-binding protein interaction is probably due to peptide sequences being exposed upon denaturation. Putative Cry toxin-binding proteins identified by the ligand blot technique need to be investigated under native conditions early in the process of identifying binding proteins that may serve as functional toxin receptors.

scholarly journals Cloning and Expression of Two Crystal Protein Genes, cry30Ba1 and cry44Aa1, Obtained from a Highly Mosquitocidal Strain, Bacillus thuringiensis subsp. entomocidus INA288

2006 ◽  
Vol 72 (8) ◽  
pp. 5673-5676 ◽  
Author(s):  
Takeshi Ito ◽  
Tomonori Ikeya ◽  
Ken Sahara ◽  
Hisanori Bando ◽  
Shin-ichiro Asano
Keyword(s):  
Bacillus Thuringiensis ◽  
Aedes Aegypti ◽  
Culex Pipiens ◽  
Crystal Protein ◽  
Cloning And Expression ◽  
Culex Pipiens Pallens ◽  
Bacillus Thuringiensis Subsp ◽  
Pipiens Pallens ◽  
Crystal Protein Genes

ABSTRACT Two novel crystal protein genes, cry30Ba and cry44Aa, were cloned from Bacillus thuringiensis subsp. entomocidus INA288 and expressed in an acrystalliferous strain. Cry44Aa crystals were highly toxic to second-instar Culex pipiens pallens (50% mortality concentration [LC50] = 6 ng/ml) and Aedes aegypti (LC50 = 12 ng/ml); however, Cry30Ba crystals were not toxic.

scholarly journals Effects of two formulations based on Bacillus thuringiensis subsp. israelensis on Aedes aegypti mosquito larvae under laboratory conditions

2008 ◽  
Vol 23 (4) ◽  
pp. 251-258
Author(s):  
Milenka Peric ◽  
Mirjana Prijovic ◽  
Goran Andric
Keyword(s):  
Bacillus Thuringiensis ◽  
Aedes Aegypti ◽  
Aqueous Suspension ◽  
Pure Water ◽  
Clay Content ◽  
Experimental Period ◽  
Mosquito Larvae ◽  
Water Dispersible ◽  
Bacillus Thuringiensis Subsp ◽  
Zero Time

Toxicity and persistence of two formulations based on Bacillus thuringiensis subsp. israelensis applied to Aedes aegypti mosquito larvae were tested under laborabory conditions. The formulations were: a) water dispersible granules (product VectoBac WDG), and b) aqueous suspension (product VectoBac 12AS). The effects of both products on mosquito larvae were tested immediately after their dilution in pure water (zero time) and in 1-, 2-, 8- and 13-day old solutions. The two products were also tested in mixtures of water and clay at a rate of 0.5 g clay/150 ml water immediately after product dilution, and in one-day old solutions containing 0.1 and 0.05 g of clay in the same amount of water. The product VectoBac WDG was persistent and highly effective against Ae. aegypti larvae in pure water after the longest experimental period of 13 days, and significantly more effective than VectoBac 12AS at equal rates of application. The effectiveness of VectoBac 12AS weakened significantly after 8 and 13 days of treatment, compared to the effects at zero time and in 1- and 2-day old solutions. High clay content in water significantly reduced the larvicidal effectiveness of both products, indicating its possible compromising role during product application in practice.

Characterization of Bacillus thuringiensis Cry toxin binding novel GPI anchored aminopeptidase from fat body of the moth Spodoptera litura

2007 ◽  
Vol 29 (11) ◽  
pp. 1651-1657 ◽  
Author(s):  
Madhusudhan Budatha ◽  
Gargi Meur ◽  
P. B. Kirti ◽  
Aparna Dutta Gupta
Keyword(s):  
Bacillus Thuringiensis ◽  
Spodoptera Litura ◽  
Fat Body ◽  
Toxin Binding ◽  
Cry Toxin

scholarly journals Voltage-dependent blockade of muscle Na+ channels by guanidinium toxins.

1984 ◽  
Vol 84 (5) ◽  
pp. 687-704 ◽  
Author(s):  
E Moczydlowski ◽  
S Hall ◽  
S S Garber ◽  
G S Strichartz ◽  
C Miller
Keyword(s):  
Voltage Dependence ◽  
Single Channel ◽  
Membrane Vesicles ◽  
Hydroxyl Groups ◽  
Binding Affinities ◽  
Na Channels ◽  
Plasma Membrane Vesicles ◽  
Toxin Binding ◽  
Voltage Dependent ◽  
Toxin Receptor

Na+ channels from rat muscle plasma membrane vesicles were inserted into neutral planar phospholipid bilayers and were activated by batrachotoxin. Single channel blocking events induced by the addition of various guanidinium toxins were analyzed to derive the rates of channel-toxin association and dissociation. Blocking by tetrodotoxin, saxitoxin, and six natural saxitoxin derivatives containing sulfate or hydroxyl groups were studied. Although the binding affinities vary over 2,000-fold, all of the toxins exhibit identical voltage dependence of the blocking reactions, regardless of the toxin's net charge. The results suggest that the voltage dependence of toxin binding is due to a voltage-dependent conformational equilibrium of the toxin receptor, rather than to direct entry of the charged toxin molecule into the applied transmembrane electric field.

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