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保的细菌，以控制主要人类疾病的蚊子媒介，包括疟疾、丝虫病、登革热和病毒性脑炎。在目前的申请中，我们建议继续我们的基础研究，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。以色列芽孢杆菌（Bti）和球形芽孢杆菌（Bs），这些菌株的野生型菌株明显比这些物种的野生型菌株更有效，目前用作病媒控制的杀幼虫剂。此外，正如我们的进展报告所示，我们的重组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。为了支持细菌杀幼虫剂在病媒控制中的可持续使用，我们提出了三个主要目标：(1)继续我们对Bti内毒素合成和副孢子体组装的基础研究；(2)加强我们对Cyt1A蛋白的分子生物学和遗传学及其延迟蚊子抗性的能力的了解；(3)验证最近的假设，即使用亚致死剂量的细菌杀幼虫剂可能能够缩短雌蚊的成年寿命。从而大大降低了它们的矢量容量。内毒素合成和副孢子体组装的研究主要是为了阐明杀蚊蛋白转运到Bti副孢子包膜并沉积的分子机制，并试图确定参与PB包膜组装的关键基因。我们将结合重组DNA技术和突变体分析来鉴定参与运输的内毒素蛋白的关键蛋白质和/或区域，以及对包膜组装重要的蛋白质。这些研究的结果将使我们能够进一步改进已经开发的重组细菌，例如将Bs Bin蛋白靶向Bti副孢子体。我们对Cyt1A、其他内毒素和耐药基因的进一步研究将为进一步改善耐药管理方案提供有价值的信息。对Bti对成虫寿命影响的研究将产生一种模型，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。综上所述，这些研究将加强将杀幼虫剂作为病媒综合管理方案的组成部分的基础，以控制最重要的病媒属，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. 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.