Molecular Improvement of Bacterial Mosquito Larvicides

描述（由申请人提供）：拟议研究的目的是开发更有效和环境安全的细菌，用于控制主要人类疾病（包括疟疾、丝虫病、登革热和病毒性脑炎）的蚊子媒介。这些细菌将比苏云金芽孢杆菌亚种更具成本效益。以色列芽孢杆菌（Bti）和球形芽孢杆菌（Bs），这两种目前用于病媒控制的物种，并且将不太容易诱导蚊子抗性。为了支持在病媒控制中可持续地使用细菌杀幼虫剂，将使用重组DNA技术在单个菌株中产生杀虫蛋白的新组合。将对这些疫苗的效力和抗药性管理特性进行评价，目的是控制属于最重要的病媒属的物种，即按蚊、伊蚊和库蚊。这些新细菌的开发和使用将通过完成研究来加强，这些研究的重点是提高对Cyt1A蛋白延迟载体群体对细菌内毒素耐药性的高毒性和能力的协同作用机制的认识。这些目标将通过一项综合研究计划来实现，该计划包括以下三个具体目标：（1）完成重组细菌的构建，重点是球形芽孢杆菌作为宿主细胞，并增加Bti和Bs伴孢组装的知识，（2）评估最佳Bti和Bs重组体的抗性管理特性，（3）完成Cyt1A协同内毒素和延迟抗性的一般机制的研究。新的表达和染色体整合策略将用于构建Bs重组体，而参与伴胞体组装的非内毒素基因将主要通过细菌突变体和基因敲除来鉴定。将通过实验室选择和遗传杂交评估抗性管理特性及其潜在的孟德尔基础。Cyt1A作用机制的研究将采用多种遗传学和组织学技术。通过我们的研究开发的细菌杀幼虫剂应改善病媒控制和疾病减少，同时减少合成化学杀虫剂的使用对健康有益。此外，鉴定出的优化抗性管理的杀虫蛋白组合将为可能工程化田间细菌种群以进行病媒控制提供模型，并且也可能证明对Bt转基因作物的抗性管理有用。

DESCRIPTION (provided by applicant): The objective of the proposed research is to develop more effective and environmentally safe bacteria for controlling the mosquito vectors of major human diseases including malaria, filariasis, dengue, and the viral encephalitides. These bacteria will be much more cost-effective than Bacillus thuringiensis subsp. israelensis (Bti) and Bacillus sphaericus (Bs), the two species currently used in vector control, and will be much less prone to induce mosquito resistance. To support sustainable use of bacterial larvicides in vector control, recombinant DNA technology will be used to create novel combinations of insecticidal proteins in individual strains. These will be evaluated for efficacy and resistance management properties aimed at controlling species belonging to the most important vector genera, namely, Anopheles, Aedes, and Culex. Development and use of these new bacteria will be enhanced by completion of studies focusing on improving knowledge of mechanisms underlying the synergism responsible for the high toxicity and capacity of the Cyt1A protein to delay resistance to bacterial endotoxins in vector populations. These objectives will be achieved through a comprehensive research program consisting of the following three specific aims: (1) Complete construction of recombinant bacteria with emphasis on Bacillus sphaericus as a host cell and increase knowledge of Bti and Bs parasporal assembly, (2) Assess the resistance management properties of the best Bti and Bs recombinants, and (3) Complete studies of the general mechanism by which Cyt1A synergizes endotoxins and delays resistance. New expression and chromosome-integration strategies will be used to construct Bs recombinants, and non-endotoxin genes involved in parasporal body assembly will be identified primarily by making bacterial mutants and gene knockouts. Resistant management properties and their underlying Mendelian basis will be evaluated through laboratory selections and genetic crosses. Studies of Cyt1A's mechanism of action will employ a variety of genetic and histological techniques. Bacterial larvicides developed through our studies should improve vector control and disease reduction, with concomitant health benefits accruing from reduced use of synthetic chemical insecticides. Moreover, insecticidal protein combinations identified to optimize resistance management will provide models for possibly engineering field populations of bacteria for vector control, and may also prove useful for resistance management in Bt-transgenic crops.