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

描述（由申请人提供）：寄生线虫导致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规划依赖于驱虫药物的大规模使用，但耐药性可能成为一个问题，而且替代品供应不足。目前用于研究驱虫虫耐药性遗传学和筛选潜在新药的实验方法是不充分的，因为在处理这些生物体时存在固有的困难。在自由生活的秀丽隐杆线虫（Caenorhabditis elegans）中，寄生虫基因的功能表达有可能解决这些问题，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，如配体门控离子通道（LGIC）。LGIC是一种多亚基蛋白，由秀丽隐杆线虫的一个大基因家族编码，并依赖于许多其他基因产物来进行正确的组装和运输。此外，基因家族的大小和组成在线虫物种之间差异很大。我们将重点关注线虫中广泛保守的两种具有良好特征的LGIC——由AVR-14B基因产物形成的谷氨酸门控氯通道（GluCl）和在神经肌肉连接处发现的左旋氨松敏感受体。我们之前已经证明我们可以在秀丽隐杆线虫中表达来自寄生线虫的AVR-14B，现在我们将优化这种方法。我们将尝试在线虫启动子的控制下，通过表达寄生虫同源物来挽救编码左旋咪唑受体亚基的线虫unc-29和unc-38基因的突变。使用由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第一筛选。如果寄生虫的cdna确实挽救了耐药表型，那么这将表明秀丽隐杆线虫是表达和研究候选耐药等位基因的合适宿主。最终，我们可能能够在线虫宿主中重建一个完整的“寄生虫”nAChR，然后可以用于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)建立线虫离子通道cdna的表达优化方案；2)测定转基因虫株的驱虫敏感性；3)直接记录表达寄生虫烟碱亚基的转基因菌株肌肉烟碱受体的性质。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，并提供研究人类寄生虫遗传学的重要新方法，我们将将其扩展到其他已确定的药物靶点。

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.