Molecular Improvement of Bacterial Mosquito Larvicides

描述（由申请人提供）：我们研究的总体目标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。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苏云金亚种的重组细菌。Israelensis（Bti）和球形芽孢杆菌（Bs），其比目前用作媒介控制的杀蚊幼虫剂的这些物种的野生型菌株显著更有效。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B的主要问题。sphaericus制剂为了支持细菌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，（1）继续我们在Bti中内毒素合成和伴胞体组装的基础研究，（2）加强我们对Cyt 1A蛋白的分子生物学和遗传学及其延迟蚊子抗性的能力的知识，以及（3）测试最近的假设，即以亚致死剂量使用细菌杀幼虫剂可能能够缩短雌蚊的成年寿命，从而显著降低其传病能力。内毒素合成和伴胞体组装的研究主要旨在阐明杀蚊蛋白被运输到Bti的伴胞体包膜并沉积在其中的分子机制，以及试图定义参与PB包膜组装的关键基因。我们将使用重组DNA技术和突变体分析的组合来鉴定参与转运的内毒素蛋白的关键蛋白和/或区域，以及对包膜组装重要的蛋白。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伴孢体。我们对Cyt 1A、其他内毒素和耐药遗传学的额外研究应该为进一步改进耐药管理计划提供有价值的信息。Bti对成虫寿命影响的研究将产生一个模型，可用于测试是否可以使用任何杀幼虫剂来减少媒介寿命，从而减少病原体传播。综合起来，这些研究将加强使用杀幼虫剂作为控制最重要的病媒属（即按蚊、伊蚊和库蚊）物种的病媒综合管理方案的组成部分的基础。 公共卫生相关性：进展报告中描述的我们的研究结果清楚地表明，可以构建比野生型细菌有效得多的重组细菌，并且重要的是，它们更不容易进化出耐药性。基于Bti作为宿主细胞的重组杀蚊细菌的进一步改进和商业开发应使得这些能够在发达国家以及发展中国家中成本有效地用作许多综合病媒控制程序的组分。在某些栖息地，在一年中的某些时候，这些重组细菌可能是IVC计划的主要组成部分。除了提高功效外，使用这些菌株还应大大减少对合成化学杀虫剂的需求。此外，这些新的杀幼虫剂应该是有用的IVC管理程序，即使在战略，以基因工程载体，以减少病原体传播的开发或其他策略开发。由于这些新型细菌是重组生物，它们需要比野生型灭蚊细菌更多的安全测试。然而，我们已经获得美国环境保护署以及加州和佛罗里达的相应州机构的批准，可以进行现场试验。在今后两年内，在收到资金和批准进行此类研究之前，我们预计将在非洲，可能是肯尼亚、坦桑尼亚或尼日利亚进行针对当地主要疟疾媒介按蚊的试验。因为我们现有的重组体的发酵，特别是产生Bs Bin毒素的Bti菌株，已经看起来非常有希望，这是一项有待监管部门批准的技术，可以在未来五年内用于疟疾控制。对于正在开发中的任何其他病媒控制和减少疾病的分子技术来说，情况不太可能如此。

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. PUBLIC HEALTH RELEVANCE: The results of our studies described in the Progress Report show clearly that recombinant bacteria can be constructed that are much more efficacious than wild type bacteria, and importantly are much less prone to the evolution of resistance. Further improvement and commercial development of recombinant mosquitocidal bacteria based on Bti as a host cell should enable these to be used cost- effectively as components of many integrated vector control programs, both in developed countries as well as developing countries. In some habitats, at certain times of the year, these recombinant bacteria could be a major component of IVC programs. Aside from improved efficacy, use of these strains should greatly reduce the need for synthetic chemical insecticides. Moreover, these novel larvicides should be useful in IVC management programs even after strategies to genetically engineer vectors to reduce pathogen transmission are developed or other strategies are developed. As these new types of bacteria are recombinant organisms, they will require more safety testing than wild type mosquitocidal bacteria. However, we already have approval from the U.S. Environmental Protection Agency, and the corresponding state agencies in California and Florida to proceed with field trial. Within the next two years, pending receipt of funding and approval for such studies, we anticipate trials in Africa, likely Kenya, Tanzania, or Nigeria, against major local anopheline vectors of malaria. Because fermentations of our existing recombinants, especially the Bti strain that produces the Bs Bin toxin already look very promising, this is a technology, pending regulatory approvals, that could be operational for malaria control within the next five years. This is unlikely to be true for any of the other molecular technologies for vector control and disease reduction under development.