Vector biology-Using a mosquito pathogen as a delivery system for anti-malarial a

描述(申请人提供)：约46%的世界人口生活在疟疾、丝虫病、病毒性脑炎、登革热和黄热病等蚊媒疾病流行的地区。最近证实，昆虫致病真菌金龟子绿僵菌有可能在城市环境中控制成年蚊子，但前提是它的效力要增加。我们的目标是生产一种或多种真菌产品，这些产品可以消耗按蚊和疟原虫种群，从而显著降低疟疾流行率。我们已经证明，金龟子绿僵菌是一种非常有效的昆虫选择性蝎子毒素AAIT的递送系统，表达AAIT可以使抗蚊子的有效孢子剂量减少9倍。这是非常重要的，但杀戮时间仍然太慢，无法提供足够的保护。在这一应用中，我们建议进行实验，以比较几种优化金黄色葡萄球菌减少疾病传播能力的策略。感染真菌的蚊子唾液腺上的子孢子数量显著减少，但其机制尚不清楚。我们将对疟原虫、蚊子和金黄色葡萄球菌之间的相互作用进行详细的分析。这将包括测试一种能引起高度免疫反应的金黄色葡萄球菌减毒株，以确定金黄色葡萄球菌是否可以用来免疫蚊子免受疟疾的侵袭。此外，我们将比较感染了表达杀虫和抗疟原虫蛋白不同组合的绿僵菌菌株的蚊子的死亡率和子孢子流行情况。将确定是否可以协同使用这些技术，以实现有效降低传输潜力。基于这些结果，我们还将测试使用绿僵菌表达合成多功能基因的有效性，这些基因是不同活性的杂交基因，例如可以同时针对昆虫和疟原虫。目前的提议将：1)探索蚊子免疫系统；2)开发有可能极大降低疟疾流行的工具和基因工程真菌；3)开发绿僵菌作为一种易于处理的模型系统，可用于筛选新的效应者。我们预计，经过筛选，最有效的效应物可以通过在金黄色葡萄球菌和/或替代病原体、共生体或转基因蚊子中表达来对抗蚊子或疟原虫。该项目旨在设计、构建和评估针对成年蚊子和疟疾寄生虫的重组真菌病原体。生产优化的真菌病原体的最重要的可能结果将是由于阻断目标寄生虫的传播而减少人类疾病。

DESCRIPTION (provided by applicant): Around 46% of the World's population lives in areas where mosquito-borne diseases including malaria, filariasis, viral encephalitides, dengue and yellow fever are endemic. It was recently established that the insect pathogenic fungus Metarhizium anisopliae has the potential to control adult mosquitoes in an urban setting, but only if its potency is increased. Our goal is to produce one or more fungal products which can deplete anopheline and Plasmodium populations to the extent that marked reductions in malaria prevalence are achieved. We have already shown that M. anisopliae is a very effective delivery system for the insect-selective scorpion toxin AaIT, and that expressing AaIT produced a 9-fold reduction in effective spore doses against mosquitoes. This was very significant but kill times remain too slow for adequate protection. In this application, we propose experiments to compare several strategies for optimizing M. anisopliae's ability to curtail disease transmission. Mosquitoes infected with fungi showed a significant reduction in the number of sporozoites on salivary glands, but the mechanism responsible is unknown. We will carry out a detailed analysis of the interactions between Plasmodium, mosquitoes and M. anisopliae. This will include testing an attenuated strain of M. anisopliae that elicits a hyperimmune response to determine whether M. anisopliae can be used to immunize mosquitoes from Plasmodium. Further, we will compare mortality and sporozoite prevalence in mosquitoes infected with M. anisopliae strains expressing different combinations of insecticidal and anti-plasmodial proteins. It will be determined if these can be used synergistically to achieve effective reductions in transmission potential. Based on these results, we will also test the efficacy of using M. anisopliae to express synthetic multifunctional genes that are hybrids of different activities and that could, for example, target both the insect and the Plasmodium. The current proposal will: 1) explore the mosquito immune system; 2) develop tools and genetically engineered fungi that have the potential to greatly reduce malaria prevalence, and 3) develop M. anisopliae as a tractable model system that can be used to screen novel effectors. We envisage that after screening, the most potent effectors could be delivered against mosquitoes or Plasmodium by expression in M. anisopliae and/or in alternative pathogens, commensals or via transgenic mosquitoes. PUBLIC HEALTH RELEVANCE This project aims to design, construct and evaluate recombinant fungal pathogens that target adult mosquitoes and the malaria parasite. The most significant possible outcome of producing an optimized fungal pathogen will be a reduction of human disease as a result of interrupting transmission of the target parasite.