Mosquitocidal Action of Bacillus thuringiensis Toxins

苏云金芽孢杆菌毒素的杀蚊作用

• 批准号: 9300824
• 负责人: Sarjeet S Gill
• 金额: $ 50.97万
• 依托单位: UNIVERSITY OF CALIFORNIA RIVERSIDE
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9300824
• 关键词: Address; Aedes; Affect; Affinity; Africa; Asia; Bacillus thuringiensis; Bacterial Toxins; Binding; Binding Proteins; Biological; Cadherins; California; Chemicals; Collaborations; Complex; Country; Culicidae; Data; Dengue; Disease; Disease Outbreaks; Disease Vectors; Epithelial; Europe; Genes; Gills; Goals; Health; Human; Insect Vectors; Insecticides; Intercellular Junctions; Knowledge; Laboratories; Larva; MAP Kinase Gene; MAP4K4 gene; MAPK Signaling Pathway Pathway; MAPK8 gene; Membrane; Membrane Lipids; Midgut; Mosquito Control; N-Cadherin; Onchocerciasis; Pathway interactions; Pattern; Play; Protein Binding Domain; Proteins; Recruitment Activity; Reporting; Research; Resistance; Resources; Risk; Role; Signal Transduction; Simuliidae; Structure; Testing; Toxic effect; Toxin; Transcript; Virus; Virus Diseases; Work; alpha helix; avermectin; chikungunya; climate change; defense response; filaria; human disease; in vivo; man; mosquitocidal; mutant; novel; overexpression; pathogenic bacteria; programs; protein expression; receptor; resistant strain; response; success; synergism; transcriptome sequencing; vector; vector control

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.

项目摘要 生物杀虫剂在世界范围内用于控制人类疾病媒介。其中包括芽孢杆菌， ringiensis israelensis（Bti），其已用于蚊子和黑蝇控制数十年， 田间抗性，主要是由于三种Cry和两种Cyt毒素的存在。我们的长期目标是-- 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持久使用Bti控制重要的昆虫媒介， 男人的疾病但是，我们对Bti行动的理解存在重大的知识差距。我们计划解决 这三个知识缺口。 首先，我们的初步数据表明，Bti Cry 11 A毒素以高亲和力结合到一个新的靶点，一个N- 钙粘蛋白，其在Cry 11 A抗性菌株中下调。我们将测试Cry 11 Aa不存在的假设- 通过结合然后破坏细胞间连接来破坏中肠细胞间连接。因此N-Cad蛋白 将鉴定结合Cry 11 Aa毒素的结构域，并评估它们是否也可以结合Cry 4Aa和Cry 4 Ba 毒素我们将评估N-Cad体内沉默对Cry 11 Aa毒性的影响，并最终评估N-Cad体内沉默对Cry 11 Aa毒性的影响。 分析该蛋白在中肠连接处的表达。这种新的机制将表明Bti毒素可以破坏- 通过先前未鉴定的机制破坏中肠功能，即细胞旁转运。确认 这一机制表明，Bti的作用比目前所了解的更为复杂， 病原菌及其毒素利用这些连接的破坏来绕过中肠上皮杆， 更容易发挥其毒性。第二，细胞色素毒素插入细胞膜的精确机制， 协同毒性是未知的。我们将检验Cyt 1A α螺旋1和3是关键的假设 对于毒素寡聚化和膜插入，而其他结构域对于协同作用是必需的。我们将 确定这些螺旋突变体寡聚化和协同毒性的能力，然后分析 Cry 11 A是一种插入膜中的结构。第三，我们发现蚊子的细胞防御 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测序 来自暴露于Cry 11 A毒素的幼虫和Cry 11 A抗性品系的数据显示，在许多改变的基因中， MAPK信号通路中的数目。因此，我们将测试的假设，诱导这一途径en- 使幼虫能够进行防御，从而减少对Bti的敏感性。 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， 实验室（Gill、Bravo和Soberon），以最大限度地利用每个实验室的可用资源和专业知识。我们已经- 我请来了一位生物信息学家（吉克）来帮助我们的分析。