The long-term objective is to understand the mechanism(s) by which the endotoxins of Bacillus thuringiensis (B.t.) exert their toxic effect. The endotoxin, although bacterial in origin is a mechanicl insecticide. Its use in insect control programs is increasing annually, however, inspite of this we do not have reliable information on its mode of action and the basis for its selective toxicity both between insects and mammals, and between insect species.
The specific aims of this proposal are: I. Purify and characterize the protoxin and endotoxin of B.t., and develop antibodies and selective immunossays to endotoxin. II. Determine the toxicity of B.t. toxins. The toxicity of crystal, protoxin and endotoxin will be compared in both cell culture (insect, mammalian) and mouse. In addition, the effect of proteases (insect, mammalian, bacterial) on endotoxin structure, and the effect of chemical modification of endotoxin on its toxicity will be evaluated. III. Determine the mode of action of the endotoxin. The effect of protoxin and endotoxin on cell membranes, and on adenylate cyclase and protein synthesis will be determined. In addition, cellular uptake of the endotoxin will be monitored and initial experiments on characterizing cell membrane receptors for the endotoxin will be performed. Purification of the protoxin and endotoxin will be performed by a variety of biochemical techniques including ion exchange, hydrophobic and gel filtration chromatography, and polyacrylamide gel electrophoresis (PAGE) and preparative isoelectricfocusing (IEF). Antibodies will be raised in rabbits and enzymelinked immunosorbent assays developed. The endotoxin will be characterized by a number of biochemical and chemical methods. Assays will be developed using cell culture and radiolabeling techniques for evaluating endotoxin toxicity before and after chemical modification. Effect of proteases on endotoxin will be monitored by cytotoxicity, PAGE and IEF assays. Effect of endotoxin on cell membranes will be evaluated by electro-physiological techniques and ion channel probes. Cellular uptake and receptor binding will be monitored by radiolabeled ligands, while endotoxin effects on protein synthesis and adenylate cyclase will be determined by amino acid incorporation and enzyme assays, respectively. The research proposed will provide a better understanding of B.t. endotoxin structure, mode of action and basis of selective toxicity. Such an understanding will allow us to evaluate whether B.t. endotoxins can be used as selective membrane probes which may be useful in targeting immunotoxins in the treatment of disease.
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