Mosquitocidal Avtive Clostridium Toxins

描述(申请人提供)：二十多年来，具有杀虫活性的细菌菌株配方已用于控制公共卫生中的害虫。如苏云金芽孢杆菌亚种。在西非，以色列利用以色列成功地控制了西门子病媒，从而减少了盘尾丝虫病，这是该地区的一种至关重要的疾病。同样，这种细菌与球形芽孢杆菌一起在许多发达国家和发展中国家成功地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，这两种细菌对疟疾媒介按蚊的使用要有限得多，部分原因是它们对该属蚊子的活性较低。而马来西亚分离的另一株革兰氏阳性芽胞形成菌马来西亚双发酵梭菌对多种按蚊的毒力最高。初步证据支持我们的假设，即：a)该菌株的灭蚊活性是由于细菌产生的毒素所致，以及b)该菌株的毒素是新的。因此，R21提案的重点是利用蚊子生物测试来识别基因和分离有毒蛋白，以驱动这两个过程，并表征它们对按蚊、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物种选择性活性的分子基础。为了实现所提出的目标，我们将同时使用基因组和蛋白质组方法。在前一种方法中，我们已经鉴定了从基因组DNA中制备的粘粒克隆，它们对AN是有毒的。在这个建议中，我们将利用转座子突变和粘粒测序来鉴定与毒性有关的关键基因(S)。作为另一种方法，我们将使用经典的纯化技术来纯化所涉及的毒素，并结合使用幼虫蚊子的分析，这将允许监测有毒蛋白质的纯化。最后，将表达这些毒素，以确认它们在灭蚊毒性中的作用。这项拟议的研究将导致新的和新的灭蚊蛋白的鉴定。这些蛋白质毒素将提供额外的工具，可用于延缓人类疾病蚊子媒介的耐药性发展，还将提供工具，优化现代生物技术工具可能产生的杀蚊毒素。

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.