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

苯并咪唑(BZ)类驱虫药是可用于控制全球寄生线虫负担的有限化疗药物的重要组成部分。兽医已经是一个巨大的问题，人们迫切担心人类批准的BZ衍生物会产生抗药性，这会对发展中国家流行地区的受威胁人口造成致命后果。目前对BZ耐药性的分子机制的了解仅限于药物靶点β-微管蛋白的变化。序列分析表明，有三种主要的基因类型与BZ抗性相关。这些与BZ抗性相关的单核苷酸变异(SNV)最初是在线虫Ben-1中发现的，并已成功地作为分子标记用于对家畜和人类的寄生线虫种群进行基因分型。然而，寄生线虫种群中BZ敏感性的降低不能完全用β-微管蛋白基因中的这三个SNV或一般情况下的β-微管蛋白基因中的SNV来解释。因此，我们迫切需要鉴定和鉴定BZ抗性基因，以提高寄生线虫治疗的有效性。正如其他驱虫药所证明的那样，我们假设对BZ的耐药性更可能是一个复杂的多基因性状，它涉及除了β-微管蛋白之外的额外基因。为了确定BZ抗性的这些额外机制，需要对线虫的自然变异进行更详细的观察。由于寄生线虫的种群遗传学研究非常有限，对自由生活的线虫线虫的定量遗传学研究已成为一种有前途的替代方案。利用线虫野生种群中存在的自然遗传变异，该项目的目的是更全面地了解导致线虫对BZ产生抗性的保守分子途径。为此，我将对250个线虫野生株进行全基因组关联(GWA)研究，以确定与BZ抗性有关的数量性状基因座(QTL)。已识别的QTL将通过计算精细定位和创建近等基因系来识别单个候选基因来缩小范围。CRISPR/Cas9基因组编辑将最终完成对有希望的候选基因的验证。此外，我将研究线虫野生株Ben-1基因座的基因组变异，以确定该基因座的其他变异。所有已识别的变体都将通过计算蛋白质结构模型来表征BZ与β-微管蛋白的结合效率。BZ结合效率降低的BEN-1等位基因对BZ反应的贡献将被进一步鉴定。综上所述，我们的方法侧重于鉴定新的BZ抗性基因和BEN-1的新变种，它们有可能成为筛选抗病寄生线虫种群的新的抗性标记。

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.