Functional Domains of Bacillus thuringiensis Endotoxins

描述(申请人提供)：苏云金芽孢杆菌是一种微生物杀虫剂，广泛用于控制昆虫，包括蚊子和黑蝇。该项目的长期目标是研究几种灭蚊蛋白对关键食疫蚊种冈比亚按蚊、埃及伊蚊和致倦库蚊的结合作用和作用机制。主要的灭蚊毒素是苏云金杆菌毒素。Israelensis(Bti)、Cry4Aa、Cry4Ab和Cry11Aa。其他杀蚊毒素Cry11BA、Cry19Aa和Cry2Aa也将被调查。 需要检验的假设是，这些毒素与蚊子中肠上的特定受体结合，这是模型昆虫毒素研究(鳞翅目毒素范例)中所识别的；即，一系列蚊子中肠蛋白(类钙粘素和氨基肽酶)和糖蛋白与毒素结合；并且，这些毒素的结构域II和I11是相互作用的结合表位。一个推论是，蚊子对Bti产生抵抗力的能力下降是由于毒素(Cry4Aa、Cry4Ab和Cry11Aa)的组合，每一种毒素都与一个独特的受体或非竞争结合部位结合。 该方案的具体目的是：(1)检测Cry4Aa、4BA、11Aa、11BA、19AA和2AA毒素与蚊虫BBMV及其纯化受体的竞争、饱和和不可逆结合。在Cry4BA(库蚊活性)和19AA(伊蚊活性)中引入了新的灭蚊活性。将对这些新活动的机制基础进行调查。(2)与鳞翅目毒素范式比较，研究这些毒素的杀蚊作用机制；具体地说，确定这些毒素与埃及伊蚊、冈比亚按蚊和致倦库蚊边界膜小泡(BBMV)的结合表位。(3)结合鳞翅目受体模式研究蚊虫中肠受体的性质；具体地说，确定这些杀蚊Cry蛋白与这些蚊子的氨基肽酶、类钙粘素蛋白、糖蛋白和其他潜在受体的特异性结合。

DESCRIPTION (provided by the applicant): Bacillus thuringiensis is a microbial insecticide that is widely used to control insects, including mosquitoes and black flies. The long-range goal of this project is to investigate the binding and mechanism of action of several mosquitocidal proteins against key pestiferous mosquito species, Anopheles gambiae, Aedes aegypti and Culex quinquefaciatus. The main mosquitocidal toxins of interest are the toxins of B. thuringiensis var. israelensis (Bti), Cry4Aa, Cry4Ab and Cry11Aa. Other mosquitocidal toxins, Cry11Ba, Cry19Aa and Cry2Aa, will also be investigated. The hypothesis to be tested is that these toxins bind to specific receptors on the mosquito midgut as recognized in model insect-toxin studies (the Lepidoptera-toxin paradigm); i.e., an array of mosquito midgut proteins (cadherin-like and aminopeptidases) and glycoproteins bind the toxins; and, that domains II and Ill of the are the interacting binding epitopes. A corollary hypothesis is that the reduced ability of mosquitoes to develop resistance to Bti is due to a combination of toxins (Cry4Aa, Cry4Ab and Cry11Aa) each of which binds to a unique receptor or non-competing binding site. The specific aims of the proposal are: (1) Test the competition, saturation and irreversible binding of Cry4Aa, 4Ba, 11Aa, 11Ba, 19Aa and 2Aa toxins to mosquito BBMV and purified receptors. New mosquitocidal activity has been introduced into Cry4Ba (Culex activity) and 19Aa (Aedes activity). The mechanistic basis for these new activities will be investigated. (2) Examine the mechanism of action of mosquitocidal of these toxins, in comparison to the Lepidoptera toxins paradigm; specifically, to define the binding epitopes of these toxins to brush border membrane vesicles (BBMV) of the mosquitoes Aedes aegypti, Anopheles gambiae and Culex quinquefasciatus. (3) Examine the nature of mosquito midgut receptors in relation to what has been learned from Lepidoptera receptor paradigm; specifically, to identify the specific binding of these mosquitocidal Cry proteins on aminopeptidases, cadherin-like proteins, glycoproteins and other potential receptors of these mosquitoes.