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.
|Cheong, H; Gill, S S (1997) Cloning and characterization of a cytolytic and mosquitocidal delta-endotoxin from Bacillus thuringiensis subsp. jegathesan. Appl Environ Microbiol 63:3254-60|
|Lee, H K; Gill, S S (1997) Molecular cloning and characterization of a novel mosquitocidal protein gene from Bacillus thuringiensis subsp. fukuokaensis. Appl Environ Microbiol 63:4664-70|
|Cheong, H; Dhesi, R K; Gill, S S (1997) Marginal cross-resistance to mosquitocidal Bacillus thuringiensis strains in Cry11A-resistant larvae: presence of Cry11A-like toxins in these strains. FEMS Microbiol Lett 153:419-24|
|Gill, S S; Cowles, E A; Francis, V (1995) Identification, isolation, and cloning of a Bacillus thuringiensis CryIAc toxin-binding protein from the midgut of the lepidopteran insect Heliothis virescens. J Biol Chem 270:27277-82|
|Kawalek, M D; Benjamin, S; Lee, H L et al. (1995) Isolation and Identification of novel toxins from a new mosquitocidal isolate from Malaysia, Bacillus thuringiensis subsp. jegathesan. Appl Environ Microbiol 61:2965-9|
|Gill, S S (1995) Mechanism of action of Bacillus thuringiensis toxins. Mem Inst Oswaldo Cruz 90:69-74|
|Cowles, E A; Yunovitz, H; Charles, J F et al. (1995) Comparison of toxin overlay and solid-phase binding assays to identify diverse CryIA(c) toxin-binding proteins in Heliothis virescens midgut. Appl Environ Microbiol 61:2738-44|
|Dai, S M; Gill, S S (1993) In vitro and in vivo proteolysis of the Bacillus thuringiensis subsp. israelensis CryIVD protein by Culex quinquefasciatus larval midgut proteases. Insect Biochem Mol Biol 23:273-83|
|Chang, C; Yu, Y M; Dai, S M et al. (1993) High-level cryIVD and cytA gene expression in Bacillus thuringiensis does not require the 20-kilodalton protein, and the coexpressed gene products are synergistic in their toxicity to mosquitoes. Appl Environ Microbiol 59:815-21|
|Gill, S S; Cowles, E A; Pietrantonio, P V (1992) The mode of action of Bacillus thuringiensis endotoxins. Annu Rev Entomol 37:615-36|
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