Mosquitocidal Action of Bacillus thuringiensis Toxins

项目摘要 全世界都在使用生物杀虫剂来控制人类病媒。其中就有清华芽孢杆菌-- 以色列林芝(Bti)，几十年来一直用于控制蚊子和黑蝇，没有 场抗性，主要是由于存在三种Cry和两种Cyt毒素。我们的长期目标是- 了解这些Cry和Cyt毒素的确切作用机制，以及蚊子对此的反应 细菌剂。这些知识将有助于Bti的持久使用，以控制重要的媒介昆虫--胡蜂。 人类疾病。但是，在我们对BTI行动的理解上存在着重大的知识差距。我们计划解决 这项提案中的三个知识缺口。 首先，我们的初步数据显示，Bti Cry11A毒素与一个新的靶标-N-N-具有高亲和力。 钙粘蛋白，它在抗Cry11A的品系中下调。我们将测试Cry11Aa dis- 通过结合然后破坏细胞间连接来中断中肠细胞间连接。因此N-Cad蛋白 将识别与Cry11Aa毒素结合的结构域，并评估它们是否也能结合Cry4Aa和Cry4BA 毒素。我们将评估N-Cad体内沉默对Cry11Aa毒性的影响，并最终 对该蛋白在中肠连接中的表达进行了分析。这一新的机制将表明Bti毒素可以消除 通过一种以前未知的机制，即细胞旁运输，破坏中肠功能。确认 这种机制将表明，Bti操作比目前所理解的更复杂，并且像其他 病原菌及其毒素利用这些连接的破坏来绕过中肠上皮条- 更容易发挥它们的毒性。第二，细胞毒素插入细胞膜的确切机制和 协同作用的毒性尚不清楚。我们将检验Cyt1aα螺旋1和3是关键的假设 对于毒素齐聚和膜插入，而其他结构域对于协同作用是必不可少的。我们会 确定这些螺旋突变体的寡聚和协同毒性的能力，然后分析 Cry11Aa插入膜中的结构。第三，我们展示了蚊子的细胞防御系统 对细菌毒素的反应。但这种情况发生的机制尚不清楚。我们最近的RNA序列 暴露在Cry11A毒素下的幼虫和抗Cry11A品系的数据表明，在许多基因改变中， 许多都在MAPK信号通路中。因此，我们将检验这一假设，即这条通路的诱导- 使幼虫能够建立防御，从而不太容易受到Bti的影响。 值得注意的是，该项目延续了三个不同实验室之间非常成功的长期合作- 托利党(吉尔、布拉沃和索伯伦)最好地利用每个实验室的可用资源和专业知识。我们已经重新- 一位生物信息学家(Girke)帮助我们进行了分析。

Project Summary Biological insecticides are used worldwide for control of human disease vectors. Among these is Bacillus thu- ringiensis israelensis (Bti), which has been used for many decades for mosquito and blackfly control with no field resistance, primarily due to the presence of three Cry and two Cyt toxins. Our long-term goal is to under- stand the precise mechanisms of action of these Cry and Cyt toxins, and how the mosquito responds to this bacterial agent. Such knowledge will aid in the lasting use of Bti for control of important insect vectors of hu- man diseases. But there are significant knowledge gaps in our understanding of Bti action. We plan to address three of these knowledge gaps in this proposal. First, our preliminary data shows that the Bti Cry11A toxin binds with high affinity to a novel target, an N- cadherin, which is down regulated in a Cry11A-resistant strain. We will test the hypothesis that Cry11Aa dis- rupts midgut intercellular junctions by binding and then disrupting intercellular junctions. Hence N-Cad protein domains that bind Cry11Aa toxin will be identified, and evaluated if they can also bind the Cry4Aa and Cry4Ba toxins. We will evaluate the effect of N-Cad in vivo silencing on Cry11Aa toxicity will be evaluated and finally the expression of this protein in midgut junctions analyzed. This novel mechanism will show Bti toxins can dis- rupt midgut function through a previously unidentified mechanism, i.e. paracellular transport. Confirmation of this mechanism would suggest that Bti action is even more complex than currently understood, and like other pathogenic bacteria and their toxins utilize disruption of these junctions to circumvent the midgut epithelial bar- rier to exert their toxicity. Second, the precise mechanism by which the Cyt toxin inserts into membranes and synergizes toxicity is not known. We will test the hypothesis that the Cyt1A alpha helices 1 and 3, are critical for toxin oligomerization and membrane insertion, and while other domains are essential for synergism. We will determine the ability of these helix mutants to oligomerize and synergize toxicity, and then analyze the Cry11Aa structure that inserts into the membrane. Third, we show that the mosquito mounts a cellular defense response against bacterial toxins. But the mechanism by which this occurs is not known. Our recent RNA seq data from larvae exposed to Cry11A toxin and of a Cry11A-resistant line show that of many genes altered, a number are in the MAPK signaling pathway. Thus we will test the hypothesis that induction of this pathway en- ables larvae to mount a defense thereby being less susceptible to Bti. Significantly this project continues a highly successful long-term collaboration between three different labora- tories (Gill, Bravo and Soberon) to best use available resources and expertise of each laboratory. We have re- cruited a bioinformaticist (Girke) to aid our analyses.