Mosquitocidal Avtive Clostridium Toxins

描述（由申请人提供）：具有杀虫活性的菌株制剂已在公共卫生领域用于害虫控制超过二十年。例如，苏云金芽孢杆菌亚种。在西非，将以色列虫用于控制血吸虫病媒取得了显著成功，从而减少了盘尾丝虫病，这是该区域极为重要的一种疾病。同样，在许多发达国家和发展中国家，这种细菌与球形芽孢杆菌一起成功地用于对付伊蚊和库蚊，从而减轻了登革热和西尼罗河等疾病。然而，使用这两种细菌对抗疟疾媒介按蚊的效果要有限得多，部分原因是它们对该属蚊子的活性较低。相比之下，马来西亚双歧梭菌（Clostridium bifermentans subsp malaysia），另一种从马来西亚分离出来的革兰氏阳性孢子形成菌株，对许多按蚊具有最高的毒性。初步证据支持我们的假设，即：a)该菌株的杀蚊活性是由于该细菌产生的毒素，b)该菌株的毒素是新的。因此，本r21提案的重点是利用蚊子生物测定来鉴定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。这一目标为我们表征这些毒素对不同蚊子选择性活性的分子基础的长期目标奠定了基础。为了实现提出的目标，我们将使用基因组学和蛋白质组学方法。在前一种方法中，我们已经确定了从基因组DNA制备的宇宙克隆，它们对An有毒。在这个建议中，我们将利用转座子诱变和cosmid测序来表征涉及毒性的关键基因。作为一种额外的方法，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进行检测，这将允许监测有毒蛋白质的纯化。最后将毒素进行表达，以证实其在杀蚊毒性中的作用。这项研究将导致新的和新的杀蚊蛋白的鉴定。这些蛋白毒素将提供额外的工具，可用于延缓人类疾病的蚊子媒介的耐药性发展，并为优化现代生物技术工具可产生的杀蚊毒素提供工具。

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.