Identification of genes underlying nematode benzimidazole resistance using Caenorhabditis elegans statistical and population genetics

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Anthelmintic drugs of the benzimidazole (BZ) class are essential components of the limited chemotherapeutic arsenal available to control the global burden of parasitic nematodes. Already a huge problem in veterinary medicine, an urgent fear for the development of resistance in human-approved BZ derivatives exists, which has fatal consequences for threatened populations in endemic areas of the developing world. The current knowledge of molecular mechanisms underlying BZ resistance is restricted to variation in the drug target, beta-tubulin. Sequence analyses revealed three major genotypes correlated with resistance to BZ. These BZ resistance-related single nucleotide variants (SNVs) were initially discovered in the C. elegans ortholog ben-1 and have been successfully used as molecular markers to genotype parasitic nematode populations in livestock and humans. However, reduced BZ sensitivity in parasitic nematode populations cannot be fully explained by these three SNVs in the beta-tubulin gene alone or in beta-tubulin genes in general. Therefore, we have an urgent need to identify and characterize BZ resistance genes to increase the efficacy of parasitic nematode treatments.As already demonstrated for other anthelmintic drugs, we hypothesize that resistance to BZ is more likely a complex, polygenic trait, which involves additional loci beyond beta-tubulin. To identify these additional mechanisms of BZ resistance, a more detailed view on natural variation in nematodes is required. Because population genetics on parasitic nematodes is highly limited, quantitative genetic studies on the free-living nematode Caenorhabditis elegans have become a promising alternative. Taking advantage of the natural genetic variation present within wild populations of C. elegans, the aim of this project is to achieve a more complete understanding of the conserved molecular pathways that cause BZ resistance in nematodes. To this end, I will perform genome-wide association (GWA) studies on 250 C. elegans wild strains to identify quantitative trait loci (QTL) contributing to BZ resistance. Identified QTL will be narrowed by computational fine mapping and the creation of near-isogenic lines to identify single candidate genes. Validation of promising candidate genes will be finally done by CRISPR/Cas9 genome editing. Furthermore, I will investigate the genomic variation at the ben-1 locus in C. elegans wild strains to identify additional variants in this locus. All identified variants will be characterized in silico for the binding efficiency of BZ to beta-tubulin by computational modeling of protein structures. Alleles of ben-1 with reduced BZ binding efficiency will by further characterized for their contribution to BZ response. In summary, our approach focuses on the identification of new BZ resistance genes and new variants of ben-1 that have the potential to serve as novel resistance markers for the screening of resistant parasitic nematode populations.