Transformation of C. elegans with a novel schistosome calcium channel subunit

描述（由申请人提供）：血吸虫属扁虫是血吸虫病的病原体，是一种热带寄生虫病，全世界有超过2亿人感染。吡喹酮（PZQ）是目前抗血吸虫病的首选药物，但其作用机制仍然不明确，甚至在其发现几十年后。我们的实验室已经表明，PZQ可能与血吸虫电压门控Ca2+ (Cav)通道的分子组分相互作用，特别是结构和功能上不典型的Cav通道b亚基。迄今为止，这种b亚单位亚型仅在扁形蠕虫中发现；PZQ对线虫无效，并且线虫基因组中不包含这种变异的Cav通道b亚基。这个探索性的R21提案描述了一个高风险/高潜力的项目，通过将这种变异血吸虫Cav通道b亚基转化为秀丽隐杆线虫，将PZQ敏感性转移到线虫。我们推测，在适当的秀丽隐杆线虫细胞类型中表达变型血吸虫b亚基将导致对PZQ敏感的线虫Cav通道的形成。因此，我们将把血吸虫b亚基置于几个不同的组织和细胞特异性秀丽隐杆线虫启动子的控制之下。我们还将使用条件启动子来控制基因的表达。如果成功，这些实验将进一步提供令人信服的证据，证明血吸虫变体b亚基确实是PZQ的靶标，并将提供机会获得关于这种独特的b亚基所起生理作用的重要信息。此外，该转基因模型的开发可以为进一步的实验（作为R01项目）铺平道路，通过利用更易于处理和强大的秀丽隐杆线虫模型系统，获得PZQ作用模式的细节，包括识别相互作用因素。该项目的具体目标是：1。在多种启动子的控制下，产生表达血吸虫SmCavbvar通道亚基的转基因秀丽隐杆线虫。2. 测试表达bvar的转基因秀丽隐杆线虫对PZQ的敏感性，无论是在整个蠕虫还是在特定组织中。公共卫生相关性：血吸虫病是一种主要的热带疾病，由称为血吸虫的寄生扁形虫引起。虽然有一种有效的抗血吸虫病药物，但其作用机制尚不清楚。通过了解这一机制，有可能获得影响相同分子靶标或与该靶标相互作用的分子的药物。在这个项目中，我们正在使用一种创新的方法来确定这种药物的分子靶点，未来的目标是确定可能与这种药物靶点相互作用的其他因素。

DESCRIPTION (provided by applicant): Trematode flatworms of the genus Schistosoma are the causative agents of schistosomiasis, a tropical parasitic disease with over 200 million people infected worldwide. Praziquantel (PZQ) is the current drug of choice against schistosomiasis, but the mechanism by which PZQ acts remains poorly defined, even several decades following its discovery. Our laboratory has shown that PZQ is likely interacting with a molecular component of schistosome voltage-gated Ca2+ (Cav) channels, specifically a structurally and functionally atypical Cav channel b subunit. This b subunit subtype has been found to date only in platyhelminths; PZQ is not effective against nematodes, and nematode genomes do not contain this variant Cav channel b subunit. This exploratory R21 proposal describes a high-risk/high-potential payoff project to transfer PZQ sensitivity to nematodes by transforming C. elegans with this variant schistosome Cav channel b subunit. We hypothesize that expression of the variant schistosome b subunit in appropriate C. elegans cell types will result in the formation of nematode Cav channels that are sensitive to PZQ. As such, we will place the schistosome b subunit under the control of several different tissue- and cell-specific C. elegans promoters. We will also use conditional promoters to control expression of the gene. If successful, these experiments will offer further compelling evidence that the schistosome variant b subunit is indeed targeted by PZQ, and will also provide the opportunity to acquire important information about the physiological role played by this unique b subunit. Additionally, development of this transgenic model could pave the way for further experiments (as an R01 project) to obtain details of the mode of action of PZQ, including the identification of interacting factors, by exploiting the far more tractable and powerful C. elegans model system. The specific aims of the project are to: 1. Produce transgenic C. elegans lines expressing the schistosome SmCavbvar Cav channel subunit under the control of various promoters. 2. Test transgenic C. elegans lines expressing bvar for sensitivity to PZQ, either in whole worms or in specific tissues. PUBLIC HEALTH RELEVANCE: Schistosomiasis is a major tropical disease caused by parasitic flatworms called schistosomes. Although there is an effective drug against schistosomiasis, the mechanism by which it works is unclear. By understanding that mechanism, it may be possible to obtain drugs that affect the same molecular target, or molecules that interact with that target. In this project, we are using an innovative approach to define the molecular target of this drug with a future goal of identifying other factors that might interact with this drug target.