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.
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.