Can drug targets from parasitic nematodes be usefully expressed in C. elegans?

描述（由申请人提供）：寄生线虫引起许多最普遍的被忽视的贫困感染和被忽视的热带病。消除和控制这些感染的方案依赖于大规模施用驱虫药，但耐药性可能成为一个问题，并且替代品供应不足。目前的实验方法，研究抗蠕虫药的遗传学，并筛选潜在的新药，是不够的，由于在这些生物体的工作固有的困难。寄生虫基因在自由生活的物种秀丽隐杆线虫中的功能表达具有解决这些问题的潜力，但药物靶点的复杂性可能会造成困难。大多数驱虫药靶向配体门控离子通道，例如配体门控离子通道（LGIC）。LGIC是由一个大的基因家族编码的多亚基蛋白。线虫，并依赖于许多其他基因产物的正确组装和运输。此外，基因家族的大小和组成在线虫物种之间变化很大。我们将集中在两个特点鲜明的LGIC，广泛保守的线虫-谷氨酸门控氯离子通道（GluCl）形成的AVR-14 B基因产物和左旋咪唑敏感的受体发现在神经肌肉接头。我们以前已经证明，我们可以表达AVR-14 B从寄生线虫在C。elegans，现在将优化这样做的方法。我们将尝试拯救C. elegans unc-29和unc-38基因，其编码左旋咪唑受体的亚基，通过在C. elegans启动子。使用unc-29和unc-38突变引起的耐药性作为标记，为这些实验提供了快速方便的首次筛选。如果寄生虫cDNA确实挽救了抗药性表型，那么这将表明C.线虫是表达和研究念珠菌抗性等位基因的合适宿主。最终，我们可能能够在C.线虫宿主，然后可以利用这些受体的比较药理学和生理学的详细研究，并可能形成寄生虫特异性药物筛选的基础。我们的具体目标是：1）建立一个优化的表达寄生虫离子通道cDNA的方法。优雅的; 2）测定转基因蠕虫品系对驱虫药的敏感性; 3）直接记录表达寄生虫烟碱亚基的转基因品系肌肉烟碱受体的特性。该项目旨在减少我们对受感染动物和人类作为生物材料来源的依赖，并提供一种重要的研究人类寄生虫遗传学的新方法，我们将扩展到其他已确定的药物靶点。 公共卫生相关性：寄生线虫感染全球数十亿人，美国数百万人。这些生物很难研究，因为它们的生物学，因此我们对它们的遗传学知之甚少，特别是与耐药性有关的遗传学， 这可能会受到当前药物管理计划的影响。这项提案的目的是看看我们是否可以使用一个著名的实验室模型，秀丽隐杆线虫来研究寄生虫的基因，以更好地了解耐药性，并开发新的方法来帮助开发新药。

DESCRIPTION (provided by applicant): Parasitic nematodes cause many of the most prevalent Neglected Infections of Poverty and Neglected Tropical Diseases. Programs for the elimination and control of these infections rely on mass administration of anthelmintic drugs, but resistance may become a problem and there is an inadequate supply of alternatives. Current experimental methods for studying the genetics of anthelmintic resistance, and for screening potential new drugs, are inadequate, due to the inherent difficulties in working with these organisms. Functional expression of parasite genes in the free-living species, Caenorhabditis elegans, has the potential to address these problems but the complexity of the drug targets may cause difficulties. Most anthelmintics target ligand-gated ion channels, such as the ligand-gated ion channels (LGIC). The LGIC are multi-subunit proteins, encoded by a large gene family in C. elegans, and dependent on many other gene products for their correct assembly and trafficking. In addition, the size and composition of the gene family varies greatly between nematode species. We will focus on two well-characterized LGIC that are widely conserved in nematodes - the glutamate-gated chloride channel (GluCl) formed by the AVR-14B gene product and the levamisole-sensitive receptor found at the neuromuscular junction. We have previously shown that we can express AVR-14B from parasitic nematodes in C. elegans, and will now optimize the method for doing this. We will attempt to rescue mutations in the C. elegans unc-29 and unc-38 genes, which encode subunits of the levamisole receptor, via the expression of the parasite orthologues under the control of the C. elegans promoters. The use of drug resistance, caused by the unc-29 and unc-38 mutations, as a marker provides a quick and convenient first screen for these experiments. If the parasite cDNAs do rescue the drug resistance phenotype, then this will indicate that C. elegans is a suitable host for the expression and study of candidat resistance alleles. Eventually we may be able to reconstruct a complete 'parasite' nAChR in a C. elegans host, which could then be exploited for detailed study of the comparative pharmacology and physiology of these receptors and potentially form the basis of parasite-specific drug screens. Our specific objectives are; 1) To develop an optimized protocol for the expression of parasite ion channel cDNAs in C. elegans; 2) To measure the anthelmintic sensitivity of transgenic worm strains; 3) To directly record the properties of the muscle nicotinic receptors from transgenic strains expressing parasite nicotinic subunits. This project aims to reduce our reliance on infected animals and humans as a source of biological material and provide an important new way of studying the genetics of human parasites, which we would extend to other identified drug targets. PUBLIC HEALTH RELEVANCE: Parasitic nematodes infect billions of people worldwide, and millions in the United States. These organisms are difficult to study, because of their biology, and as a result we know little about their genetics, especially in relation to drug resistance, and how this might be affected by current drug administration programs. The goal of this proposal is to see if we can use a well-known laboratory model, Caenorhabditis elegans to study genes from parasites, to better understand drug resistance and to develop new methods for to help the development of new drugs.