Molecular Improvement of Bacterial Mosquito Larvicides

描述(申请人提供)：我们研究的总体目标是开发更有效和对环境安全的细菌，以控制疟疾、丝虫病、登革热和病毒性脑炎等重大人类疾病的蚊媒。在本申请中，我们建议继续我们的基础研究，这些研究已经使我们能够开发基于苏云金芽孢杆菌亚种的重组细菌。以色列和球形芽孢杆菌(Bti)和球形芽孢杆菌(Bs)比目前用作蚊子控制的蚊子幼虫的野生型菌株要有效得多。此外，正如我们的进展报告所示，我们的重组细菌具有显著延迟耐药性的能力，这一直是球形芽孢杆菌制剂的主要问题。为了支持杀幼虫剂在媒介控制中的可持续使用，我们提出了三个主要目标，以(1)继续我们在Bti中内毒素合成和伴胞体内组装的基础研究，(2)加强我们对Cyt1A蛋白的分子生物学和遗传学及其延缓蚊子抗性的能力的了解，以及(3)检验最近的假设，即亚致死剂量的杀幼虫剂可能能够缩短雌性蚊子的成虫寿命，从而显著降低其媒介能力。内毒素合成和伴孢子体组装的研究主要是为了阐明灭蚊蛋白运输和沉积在Bti伴孢子膜上的分子机制，以及试图确定参与PB囊膜组装的关键基因。我们将使用重组DNA技术和突变体分析相结合的方法来鉴定参与转运的关键蛋白和/或内毒素蛋白区域，以及对包膜组装重要的蛋白质。这些研究的结果应该使我们能够进一步改进已经开发的重组细菌，例如通过将BS Bin蛋白靶向Bti伴孢子体。我们对Cyt1A、其他内毒素和耐药性遗传学的进一步研究将为进一步改进耐药性管理计划提供有价值的信息。对Bti对成虫寿命影响的研究将产生一个模型，可以用来测试是否有任何杀虫剂可以用来降低媒介寿命，从而减少病原体的传播。总而言之，这些研究将加强使用杀虫剂作为综合病媒管理方案的组成部分的基础，以控制属于最重要的病媒属的物种，即按蚊、伊蚊和库蚊。

DESCRIPTION (provided by applicant): The overall objective our 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. In the present application, we propose to continue our basic studies that have already enabled us to develop recombinant bacteria based on Bacillus thuringiensis subsp. israelensis (Bti) and Bacillus sphaericus (Bs) that are significantly more efficacious than the wild type strains of these species currently used as mosquito larvicides for vector control. In addition, as shown in our progress report, our recombinant bacteria have the capacity to delay resistance significantly, which has been a major problem with B. sphaericus preparations. To support the sustainable use of bacterial larvicides in vector control, we propose three major objectives, to (1) continue our basic studies of endotoxin synthesis and parasporal body assembly in Bti, (2) strengthen our knowledge of the molecular biology and genetics of the Cyt1A protein and its ability to delay mosquito resistance, and (3) test the recent hypothesis that bacterial larvicides used at sublethal doses may be capable of reducing the adult life span of female mosquitoes, thereby reducing their vectorial capacity significantly. The studies of endotoxin synthesis and parasporal body assembly are aimed primarily at elucidating the molecular mechanisms by which mosquitocidal proteins are trafficked to and deposited in the parasporal envelope of Bti, as well as attempting to define key genes involved in assembly of the PB envelope. We will use a combination of recombinant DNA technology and analysis of mutants to identify both key proteins and/or regions of the endotoxin proteins involved in transport, as well as the proteins important to envelope assembly. The results of these studies should enable us to further improve upon the recombinant bacteria already developed, for example by targeting the Bs Bin protein to the Bti parasporal body. Our additional studies of Cyt1A, other endotoxins, and the genetics of resistance should produce valuable information for further improving resistance management programs. And the studies of the effect of Bti on adult longevity will yield a model that can be used to test whether any larvicide can be used to decrease vector longevity and thus pathogen transmission. Combined, these studies will strengthen the basis for using larvicides as component of integrated vector management programs for controlling species belonging to the most important vector genera, namely, Anopheles, Aedes, and Culex.