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 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 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.

scholarly journals A Strain of Bacillus thuringiensis Containing a Novel cry7Aa2 Gene that Is Toxic to Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae)

Insects ◽  
2019 ◽  
Vol 10 (9) ◽  
pp. 259 ◽  
Author(s):  
Mikel Domínguez-Arrizabalaga ◽  
Maite Villanueva ◽  
Ana Beatriz Fernandez ◽  
Primitivo Caballero
Keyword(s):  
Amino Acid ◽  
Bacillus Thuringiensis ◽  
Molecular Mass ◽  
Leptinotarsa Decemlineata ◽  
Insect Pests ◽  
Amino Acid Residues ◽  
Cry Protein ◽  
Lc50 Value ◽  
Leptinotarsa Decemlineata Say

The genome of the Bacillus thuringiensis BM311.1 strain was sequenced and assembled in 359 contigs containing a total of 6,390,221 bp. The plasmidic ORF of a putative cry gene from this strain was identified as a potential novel Cry protein of 1138 amino acid residues with a 98% identity compared to Cry7Aa1 and a predicted molecular mass of 129.4 kDa. The primary structure of Cry7Aa2, which had eight conserved blocks and the classical structure of three domains, differed in 28 amino acid residues from that of Cry7Aa1. The cry7Aa2 gene was amplified by PCR and then expressed in the acrystalliferous strain BMB171. SDS-PAGE analysis confirmed the predicted molecular mass for the Cry7Aa2 protein and revealed that after in vitro trypsin incubation, the protein was degraded to a toxin of 62 kDa. However, when treated with digestive fluids from Leptinotarsa decemlineata larvae, one major proteinase-resistant fragment of slightly smaller size was produced. The spore and crystal mixture produced by the wild-type BM311.1 strain against L. decemlineata neonate larvae resulted in a LC50 value of 18.8 μg/mL, which was statistically similar to the estimated LC50 of 20.8 μg/mL for the recombinant BMB17-Cry7Aa2 strain. In addition, when this novel toxin was activated in vitro with commercial trypsin, the LC50 value was reduced 3.8-fold to LC50 = 4.9 μg/mL. The potential advantages of Cry7Aa2 protoxin compared to Cry7Aa1 protoxin when used in the control of insect pests are discussed.

scholarly journals Coassembly of SecYEG and SecA Fully Restores the Properties of the Native Translocon

2018 ◽  
Vol 201 (1) ◽  
Author(s):  
Priya Bariya ◽  
Linda L. Randall
Keyword(s):  
Lipid Bilayers ◽  
Rate Constant ◽  
Apparent Rate Constant ◽  
Membrane Vesicles ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Secretory System ◽  
Rate Limiting

ABSTRACTIn all cells, a highly conserved channel transports proteins across membranes. InEscherichia coli, that channel is SecYEG. Many investigations of this protein complex have used purified SecYEG reconstituted into proteoliposomes. How faithfully do activities of reconstituted systems reflect the properties of SecYEG in the native membrane environment? We investigated by comparing threein vitrosystems: the native membrane environment of inner membrane vesicles and two methods of reconstitution. One method was the widely used reconstitution of SecYEG alone into lipid bilayers. The other was our method of coassembly of SecYEG with SecA, the ATPase of the translocase. For nine different precursor species we assessed parameters that characterize translocation: maximal amplitude of competent precursor translocated, coupling of energy to transfer, and apparent rate constant. In addition, we investigated translocation in the presence and absence of chaperone SecB. For all nine precursors, SecYEG coassembled with SecA was as active as SecYEG in native membrane for each of the parameters studied. Effects of SecB on transport of precursors faithfully mimicked observations madein vivo. From investigation of the nine different precursors, we conclude that the apparent rate constant, which reflects the step that limits the rate of translocation, is dependent on interactions with the translocon of portions of the precursors other than the leader. In addition, in some cases the rate-limiting step is altered by the presence of SecB. Candidates for the rate-limiting step that are consistent with our data are discussed.IMPORTANCEThis work presents a comprehensive quantification of the parameters of transport by the Sec general secretory system in the threein vitrosystems. The standard reconstitution used by most investigators can be enhanced to yield six times as many active translocons simply by adding SecA to SecYEG during reconstitution. This robust system faithfully reflects the properties of translocation in native membrane vesicles. We have expanded the number of precursors studied to nine. This has allowed us to conclude that the rate constant for translocation varies with precursor species.

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