Ex vivo cytotoxicity of the Bacillus thuringiensis Cry4B delta-endotoxin to isolated midguts of Aedes aegypti larvae.
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.
Barusrux
Sahawat
S
Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus, Nakornpathom, 73170 Thailand.
Sramala
Issara
I
Katzenmeier
Gerd
G
Bunyaratvej
Ahnond
A
Panyim
Sakol
S
Angsuthanasombat
Chanan
C
D000330:Aedes / Q000166:cytology / Q000187:drug effects*; D000818:Animals; D001413:Bacillus thuringiensis / Q000737:chemistry; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism / Q000633:toxicity*; D001427:Bacterial Toxins / Q000633:toxicity; D002470:Cell Survival / Q000187:drug effects; D003465:Culex / Q000166:cytology / Q000187:drug effects; D004064:Digestive System / Q000166:cytology / Q000187:drug effects*; D004591:Electrophoresis, Polyacrylamide Gel; D004731:Endotoxins / Q000235:genetics / Q000378:metabolism / Q000633:toxicity*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006460:Hemolysin Proteins; D066298:In Vitro Techniques; D007814:Larva / Q000166:cytology / Q000187:drug effects; D009154:Mutation; D013997:Time Factors; D014357:Trypsin / Q000378:metabolism
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D014357:Trypsin