C. elegans as a model for ricin intoxication

描述（由申请人提供）：蓖麻毒素是一种由蓖麻（Ricinus communis）产生的植物毒素，对哺乳动物细胞具有极大毒性，被美国卫生与公众服务部（HHS）列为精选剂，被疾病控制中心（CDC）列为B类优先病原体。虽然已尝试生产一种有效的蓖麻毒素疫苗，但尚未批准用于人类的重组疫苗，也没有针对蓖麻毒素暴露的有效治疗措施。因此，迫切需要治疗方法来保护蓖麻毒素暴露个体。为了进一步了解蓖麻毒素诱导细胞死亡的机制，我们将利用秀丽隐杆线虫独特的实验特征，使其成为几乎所有生物学研究领域的主要模式生物。我们的主要目标是建立秀丽隐杆线虫作为一个新的完整的动物模型来研究蓖麻毒素中毒，并将我们的发现转化为哺乳动物。在初步研究中，我们用表达蓖麻毒素a链（ricin A-chain， RTA）的大肠杆菌或从大肠杆菌中纯化的重组RTA喂养秀丽隐杆线虫，并证明RTA在极低剂量下对秀丽隐杆线虫是致命的。与哺乳动物数据一致，我们发现蓖麻毒素通过网格蛋白和筏独立的内吞途径进入秀丽隐杆线虫肠上皮细胞，进入早期内体对细胞毒性至关重要。我们确定了两种主要的吞噬/吞噬途径在秀丽隐杆线虫肠道中介导蓖麻毒素敏感性的新作用。这些结果为秀丽隐杆线虫作为蓖麻毒素转运和细胞死亡机制的分子遗传学研究提供了新的多细胞模型。我们将检查运输蓖麻毒素在蠕虫肠，一个典型的极化上皮，在一个活的动物的背景下。在细胞内运输途径中存在缺陷的秀丽隐杆线虫突变体将被用于验证秀丽隐杆线虫作为蓖麻毒素摄取和运输机制分析的相关模型，并确定导致细胞毒性的蓖麻毒素运输的关键步骤。我们将利用秀丽隐杆线虫遗传学的强大功能，分离出新的蓖麻毒素中毒缺陷突变体。这些研究将有助于阐明蓖麻毒素转运的分子细节，并为鉴定能够防止蓖麻毒素诱导的细胞死亡的抑制剂奠定基础。公共卫生相关性：我们在这项应用中的主要目标是建立秀丽隐杆线虫作为研究蓖麻毒素中毒机制的新模型，并将我们的发现转化为人类。这些研究意义重大，因为目前没有针对蓖麻毒素接触的疫苗、解毒剂或任何其他治疗措施。这些研究将为确定能够保护蓖麻毒素暴露个体的抑制剂奠定基础。

DESCRIPTION (provided by applicant): Ricin is a plant toxin produced by the castor bean (Ricinus communis) that is extremely toxic to mammalian cells and is classified as a select agent by the US Department of Health and Human Services (HHS) and a category B priority pathogen by the Center for Disease Control (CDC). Although there have been attempts to generate an effective vaccine against ricin, no recombinant vaccine has yet been approved for human use and there are no effective treatment measures against ricin exposure. Therefore there is an urgent need for therapeutics to protect ricin-exposed individuals. To gain understanding into the mechanism of ricin induced cell death, we will take advantage of the unique experimental features of C. elegans that have made it a leading model organism in nearly all areas of biological research. Our primary objective in this proposal is to establish C. elegans as a new whole animal model to study ricin intoxication and to translate our findings to mammals. In preliminary studies, we fed C. elegans with either E. coli expressing ricin A-chain (RTA) or recombinant RTA purified from E. coli and demonstrated that RTA is lethal to C. elegans at extremely low doses. In agreement with mammalian data, we showed that ricin uses a clathrin and a raft independent endocytosis pathway to enter the C. elegans intestinal epithelial cells and entry to the early endosome is critical for cytotoxicity. We identified a novel role for the two major phagocytosis/engulfment pathways in mediating ricin sensitivity in the C. elegans intestine. These results provided support for the development of C. elegans as a new multicellular model for molecular genetic dissection of the mechanisms of ricin transport and cell death. We will examine transport of ricin in the worm intestine, a classic polarized epithelium, in the context of a living animal. C. elegans mutants defective in intracellular trafficking pathways will be used to validate C. elegans as a relevant model for mechanistic analysis of ricin uptake and transport and to identify critical steps in ricin transport that lead to cytotoxicity. We will exploit powerful features of C. elegans genetics to isolate new mutants defective in ricin intoxication. The proposed studies will lead to elucidation of the molecular details of ricin transport and will lay the foundation for identification of inhibitors that can prevent ricin induced cell death. PUBLIC HEALTH RELEVANCE: Our primary objective in this application is to establish C. elegans as a new model to study the mechanism of ricin intoxication and to translate our findings to humans. These studies are significant because currently there are no vaccines, antidotes or any other treatment measures against ricin exposure. These studies will lay the foundation for identification of inhibitors that can protect ricin exposed individuals.