• BMB Reports
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• 제36권5호
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• pp.508-513
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• 2003

The bacterium Bacillus thuringiensis produces toxin inclusions that are deleterious to target insect larvae. These toxins are believed to interact with a specific receptor protein(s) that is present on the gut epithelial cells of the larvae. In various insect species (in particular those belonging to the lepidopteran class), aminopeptidase N (APN) is one of the two receptor proteins that are considered to be involved in toxin-receptor interactions. However, in mosquitoes, the nature and identity of the receptor protein is unknown. Here, using RT-PCR, we identified two isoforms of the APN transcripts in the Aedes aegypti mosquito larval midgut. These results are congruent with a previous report of multiple isoforms of the APN gene expression in lepidopteran larvae. Which of the two isoforms (or other yet unidentified receptor proteins) is involved in the killing of mosquito larvae remains to be elucidated.

• BMB Reports
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• 제40권5호
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• pp.783-790
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• 2007

Receptor binding plays an important role in determining host specificity of the Bacillus thuringiensis Cry $\delta$-endotoxins. Mutations in domains II and III have suggested the participation of certain residues in receptor recognition and insect specificity. In the present study, we expressed the cloned domain II-III fragment of Cry4Ba and examined its binding characteristics to mosquito-larval midgut proteins. The 43-kDa Cry4Ba-domain II-III protein over-expressed in Escherichia coli as inclusion bodies was only soluble when carbonate buffer, pH 10.0 was supplemented with 4M urea. After renaturation via stepwise dialysis and subsequent purification, the refolded domain II-III protein, which specifically reacts with anti Cry4Ba-domain III monoclonal antibody, predominantly exists as a $\beta$-sheet structure determined by circular dichroism spectroscopy. In vitro binding analysis to both histological midgut tissue sections and brush border membrane proteins prepared from susceptible Aedes aegypti mosquito-larvae revealed that the isolated Cry4Ba-domain II-III protein showed binding functionality comparable to the 65-kDa full-length active toxin. Altogether, the data present the 43-kDa Cry4Ba fragment comprising domains II and III that was produced in isolation was able to retain its receptor-binding characteristics to the target larval midgut proteins.

• BMB Reports
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• 제36권3호
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• pp.294-298
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• 2003

The pathological effect of the Bacillus thuringiensis Cry $\delta$-endotoxins on susceptible insect larvae had extensive damage on the midgut epithelial cells. In this study, an ex vivo assay was devised for assessing the insecticidal potency of the cloned Cry4B mosquito-larvicidal protein that is expressed in Escherichia coli. Determination of toxicity was carried out by using a cell viability assay on the midguts that were dissected from 5-day old Aedes aegypti mosquito larvae. After incubation with the toxin proteins, the number of viable epithelial cells was determined photometrically by monitoring the quantity of the bioreduced formazan product at 490 nm. The results showed that the 65-kDa trypsin-activated Cry4B toxin exhibited toxic potency ca. 3.5 times higher than the 130-kDa Cry4B protoxin. However, the trypsin-treated products of the non-bioactive Cry4B mutant (R158A) and the lepidopteran-specific Cry1Aa toxin displayed relatively no ex vivo activity on the mosquito-larval midguts. The ex vivo cytotoxicity studies presented here confirms data that was obtained in bioassays.