Mosquitocidal Avtive Clostridium Toxins

杀蚊活性梭状芽胞杆菌毒素

• 批准号: 7244010
• 负责人: Sarjeet S Gill
• 金额: $ 18.21万
• 依托单位: UNIVERSITY OF CALIFORNIA RIVERSIDE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-06-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7244010
• 关键词: Aedes; Africa; Agriculture; Anopheles Genus; Attenuated; Bacillus thuringiensis; Bacteria; Biological; Biological Assay; California; Clostridium; Clostridium bifermentans; Cosmids; Culex (Genus); Culicidae; DNA; Data; Dengue; Developed Countries; Developing Countries; Disease; Disease Vectors; Drug Formulations; Funding; Genes; Genetic Recombination; Genomics; Gills; Goals; Hemolysin; Hour; Incidence; Insect Control; Larva; Lead; Malaria; Malaysia; Molecular; Molecular Target; Monitor; Mutagenesis; Numbers; Onchocerciasis; Pathogenicity; Process; Proteins; Proteomics; Public Health; Reproduction spores; Research; Research Personnel; Resistance development; Role; Seeds; Simulium genus; Staining method; Stains; Symptoms; Techniques; Technology; Toxic effect; Toxin; Universities; Virus Diseases; West Nile virus; Work; base; gene cloning; human disease; mosquitocidal; novel; programs; success; tool; vector; vector mosquito

DESCRIPTION (provided by applicant): Formulations of bacterial strains with insecticidal activity have been used for the control of insect pests in public health for more than two decade. For example, Bacillus thuringiensis subsp. israelensis has been used with remarkable success for the control of Simulium vectors in West Africa, resulting in the reduction of onchocerciasis, a disease of critical importance in that region. Similarly, this bacterium together with B. sphaericus has been used successfully against both Aedes and Culex in many developed and developing countries, thereby attenuating diseases, such as dengue and West Nile. However, the use of both these bacteria against Anopheles vectors of malaria has been much more limited, in part due to their lower activity against mosquitoes of this genus. In contrast Clostridium bifermentans subsp malaysia, another Gram positive spore forming strain isolated from Malaysia, has the highest toxicity to a number of Anopheles spp. Preliminary evidence supports our hypotheses, which are: a) that the mosquitocidal activity of this strain is due to toxins produced by the bacteria, and b) that toxins from this strain are novel. The focus of this R21proposal is therefore to identify the genes and isolate the toxic proteins using mosquito bioassays to drive both processes, and to characterize their activity against Anopheles, Aedes and Culex mosquitoes. This objective lays the ground work for our long-term objective of characterizing the molecular basis of selective activity of these toxins to different mosquito species. To achieve the objective proposed we will use both genomic and proteomic approaches. In the former approach, we have identified cosmid clones, prepared from genomic DNA, that are toxic to An. Stephensi larvae, and in this proposal we will characterize the critical gene(s) involved in toxicity using transposon mutagenesis and cosmid sequencing. As an additional approach we will purify the toxins involved using classical purification techniques, in conjunction with assays using larval mosquitoes, which will allow for monitoring purification of toxic proteins. Finally the toxins will be expressed to confirm their role in mosquitocidal toxicity. The proposed research will lead to the identification of new and novel mosquitocidal proteins. These protein toxins will provide additional tools that can be used to delay resistance development in mosquito vectors of human diseases and also, provide tools for optimization of mosquitocidal toxins that could be generated by modern biotechnological tools.

描述（由申请人提供）：具有杀虫活性的细菌菌株制剂在公共卫生领域用于控制害虫已有二十多年的历史。例如，苏云金芽孢杆菌亚种。 israelensis在西非用于控制蝽病媒方面取得了显著成功，从而减少了盘尾丝虫病，这是该地区至关重要的疾病。同样，这种细菌与球形芽孢杆菌一起已在许多发达国家和发展中国家成功用于对抗伊蚊和库蚊，从而减轻了登革热和西尼罗河等疾病。然而，这两种细菌在对抗疟疾按蚊媒介方面的应用受到了很大的限制，部分原因是它们对抗该属蚊子的活性较低。相比之下，从马来西亚分离出的另一种革兰氏阳性芽孢形成菌株双发酵梭菌马来西亚亚种，对多种按蚊属具有最高的毒性。初步证据支持我们的假设，即：a）该菌株的杀蚊活性是由于细菌产生的毒素，b）该菌株的毒素是新的。因此，R21 提案的重点是使用蚊子生物测定来识别基因并分离有毒蛋白质，以驱动这两个过程，并表征它们针对按蚊、伊蚊和库蚊的活性。这一目标为我们的长期目标奠定了基础，即表征这些毒素对不同蚊子种类的选择性活性的分子基础。为了实现所提出的目标，我们将使用基因组学和蛋白质组学方法。在前一种方法中，我们鉴定了由基因组 DNA 制备的对 An 有毒的粘粒克隆。 Stephensi 幼虫，在本提案中，我们将使用转座子诱变和粘粒测序来表征与毒性有关的关键基因。作为另一种方法，我们将使用经典纯化技术结合使用幼虫蚊子的测定来纯化所涉及的毒素，这将允许监测有毒蛋白质的纯化。最后将表达毒素以确认其在杀蚊毒性中的作用。拟议的研究将导致新型杀蚊蛋白的鉴定。这些蛋白质毒素将提供额外的工具，可用于延迟人类疾病蚊媒的抗药性发展，并且还提供优化现代生物技术工具产生的杀蚊毒素的工具。