Mosquitocidal Action of Bacillus thuringensis Toxins

描述(申请人提供)：苏云金芽孢杆菌亚种。白纹伊蚊已经在田间使用了20多年，没有对任何目标昆虫产生抗药性。相比之下，在许多国家的现场观察到了蚊子对球形芽孢杆菌的抗药性。这种对两种灭蚊芽孢杆菌菌株产生抗药性的显著差异可能是由于Bti中存在多种毒素所致。然而，蚊子能够迅速对这种菌株的个别毒素产生抵抗力。蚊子显然不能对Bti产生抵抗力的一个主要原因是该菌株和其他杀蚊菌株中存在细胞溶解(Cyt)毒素。在上一次资助期间，我们表明蚊子对Bti缺乏抵抗力的关键是这种菌株中的两种毒素，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环区结合，而Cry11A环区也参与结合内源性埃及伊蚊受体蛋白。我们的研究表明，伊蚊受体蛋白包括钙粘附素、碱性磷酸酶(ALP)和氨基肽酶(APN)。这三个类别都以相对较高的亲和力结合灭蚊毒素，而不像鳞翅目动物那样，只有钙粘附素以高亲和力结合。这表明蚊子的APN和ALP都可以作为初级受体，而不是像飞蛾那样作为次级受体，这表明杀蚊毒素和鳞翅目叫声毒素的作用方式可能不同。然而，我们相信Bt Cry毒素的作用模式是保守的。因此，我们假设在蚊子和鳞翅目动物中也存在类似的作用模式。在这项提议中，我们计划检验这一假设。因此，我们假设：(I)钙粘蛋白是一种关键蛋白，它介导了最初与杀蚊的Cry毒素的结合，并且对幼虫的毒性是必不可少的；ii)Alps(和APns)作为第二受体，允许毒素靶向细胞膜。另外，在前面的研究中，我们发现Cyt1a在作为Cry11Aa的替代受体的过程中起着关键作用。因此，我们假设(Iii)Cyt1A也是Bti中其他Cry毒素的受体，并协同灭蚊Cry毒素的毒性。我们将验证这一假说，并阐明细胞毒素插入细胞膜作为代理受体的机制。该项目还提议继续在三个不同的研究人员之间进行成功的合作，以最大限度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.