Mosquitocidal Action of Bacillus thuringensis Toxins

描述（申请人提供）：苏云金芽孢杆菌亚种。以色列螟（Bti）已在田间使用了二十多年，未在任何目标昆虫中产生抗性。相比之下，在许多国家的野外已观察到蚊子对球形白僵菌的抗性。对两种杀蚊芽孢杆菌菌株产生耐药性的倾向存在显著差异，可能是由于Bti中存在多种毒素。然而，蚊子能够迅速对该菌株的个别毒素产生抗药性。蚊子显然无法对Bti产生耐药性的一个主要原因是，这种和其他杀蚊菌株中存在细胞溶解（Cyt）毒素。在上一个资助期间，我们表明蚊子对Bti缺乏抵抗力的关键是该菌株中的两种毒素，即Cry11A和Cyt1A毒素如何相互作用。我们发现Cyt1A可以作为Cry11Aa的替代受体。重要的是，Cyt1Aa通过Cry11A环域结合，Cry11A环域也参与结合内源性埃及伊蚊受体蛋白。我们的研究发现伊蚊受体蛋白包括钙粘蛋白、碱性磷酸酶（ALP）和氨基肽酶（apn）。与鳞翅目中只有钙粘蛋白具有高亲和力的结合不同，这三种类都以相对高的亲和力结合灭蚊毒素。这表明蚊子的APN和ALP可能是主要受体，而不是像飞蛾那样是次要受体，这表明杀蚊剂和鳞翅目Cry毒素的作用方式可能不同。然而，我们相信，Bt Cry毒素的作用模式是守恒的。因此，我们假设在蚊子中发生的作用模式与鳞翅目动物相似。在本提案中，我们计划检验这一假设。因此，我们推测：(1)钙粘蛋白是介导与杀蚊毒素初始结合的关键蛋白，对幼虫的毒性至关重要；ii) ALPs（和apn）作为次级受体，允许毒素靶向细胞膜。在之前的研究中，我们发现Cyt1A在作为Cry11Aa的替代受体中起着关键作用。因此，我们假设（iii） Cyt1A也是Bti中其他Cry毒素的受体，并协同杀蚊Cry毒素的毒性。我们将验证这一假设，并阐明细胞毒素插入细胞膜作为替代受体的机制。该项目还建议在三个不同的研究人员之间继续成功合作，以最好地利用每个实验室的专业知识。

DESCRIPTION (provided by applicant): Bacillus thuringiensis subsp. israelensis (Bti) has been used in the field for over twenty years without resistance development in any target insect. In contrast, mosquito resistance to B. sphaericus has been observed in the field in many countries. This remarkable difference in the propensity to develop resistance to two mosquitocidal Bacillus strains is likely due to the presence of multiple toxins in Bti. However, mosquitoes are able to rapidly develop resistance to individual toxins from this strain. A major reason for the apparent inability of mosquitoes to develop resistance to Bti is the presence of cytolytic (Cyt) toxins in this and other mosquitocidal strains. During the last grant period we showed that key to the lack of mosquito resistance to Bti was how two toxins in this strain, namely Cry11A and Cyt1A toxins interact. We showed Cyt1A acts as a surrogate receptor for Cry11Aa. Importantly Cyt1Aa binds through Cry11A loop domains that are also involved in binding endogenous Aedes aegypti receptor proteins. Our research showed Aedes receptor proteins include cadherin, alkaline phosphatases (ALP), and aminopeptidases (APNs). All three classes bind mosquitocidal toxins with relatively high affinity, unlike in lepidopterans where only cadherin binds with high affinity. This implies both APN and ALP of mosquitoes could act as primary rather than as secondary receptors as in moths, suggesting a possible difference in the mode of action of mosquitocidal and lepidopteran Cry toxins. We believe however, there is conservation in the mode of action of Bt Cry toxins. Therefore we hypothesize a similar mode of action of action occurs in mosquitoes as in lepidopterans. In this proposal we plan to test this hypothesis. Consequently, we hypothesize that: (i) cadherin is a key protein which mediates initial binding to mosquitocidal Cry toxins and is essential for larval toxicity; ii) ALPs (and APNs) act as secondary receptors that allow toxin targeting to the cell membrane. Also in the previous proposal we showed Cyt1A plays a critical role in acting as a surrogate receptor for Cry11Aa. We therefore hypothesize that (iii) Cyt1A also is a receptor for other Cry toxins in Bti, and synergizes the toxicity of mosquitocidal Cry toxins. We will test this hypothesis and also elucidate the mechanism by which Cyt toxins insert into the membrane to act as surrogate receptors. This project is also a proposal to continue a successful collaboration between three different investigators to best use the expertise of each laboratory. PUBLIC HEALTH RELEVANCE: B. thuringiensis subsp. israelensis are used for control of human disease vectors, such as species of Simulium, Aedes, Culex, and Anopheles. Elucidating mechanisms of Cry toxin action in aids our understanding of how mosquito and black fly control is achieved and also enables us to determine mechanisms by which resistance can occur. This mode of Cyt1A toxin action investigation also illuminates mechanism of synergism between mosquitocidal Cry and Cyt toxins. Finally, elucidation of mechanisms by which the Cyt1Aa toxin acts also will help us understand how other cytolytic toxins of human health significance cause toxicity.