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

描述（由申请人提供）：血吸虫属的吸虫扁虫是血吸虫病的病原体，血吸虫病是一种热带寄生虫病，全球感染人数超过2亿。吡喹酮（PZQ）是目前治疗血吸虫病的首选药物，但PZQ的作用机制仍然不清楚，即使在发现几十年后。我们的实验室已经表明，PZQ可能是相互作用的一个分子组成部分的一些电压门控钙（Cav）通道，特别是结构和功能的非典型Cav通道B亚基。这种B亚基亚型迄今为止仅在扁形动物中发现; PZQ对线虫无效，并且线虫基因组不包含这种变体Cav通道B亚基。这个探索性的R21提案描述了一个高风险/高潜力的回报项目，通过转化C. elegans中含有这种变异的双链体Cav通道B亚基。我们推测，在合适的C. elegans细胞类型将导致对PZQ敏感的线虫Cav通道的形成。因此，我们将把染色体B亚单位置于几种不同的组织和细胞特异性C的控制下。elegans启动子。我们还将使用条件启动子来控制基因的表达。如果成功的话，这些实验将提供进一步令人信服的证据，证明PZQ确实靶向了染色体变异体B亚基，并且还将提供获得关于这种独特的B亚基所发挥的生理作用的重要信息的机会。此外，这种转基因模型的开发可以为进一步的实验（作为R 01项目）铺平道路，以获得PZQ作用模式的细节，包括相互作用因子的鉴定，通过利用更易处理和强大的C。elegans模型系统。该项目的具体目标是：1.产生转基因C.在各种启动子的控制下表达杆状病毒SmCavbvar Cav通道亚基的线虫系。2.测试转基因C.表达bvar的线虫品系对PZQ的敏感性，无论是在整个蠕虫还是在特定组织中。与公共卫生的关系：血吸虫病是一种主要的热带疾病，由寄生的扁形虫引起。虽然有一种有效的药物可以治疗血吸虫病，但其作用机制尚不清楚。通过了解这种机制，可能会获得影响相同分子靶点的药物，或与该靶点相互作用的分子。在这个项目中，我们正在使用一种创新的方法来定义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.