Extreme allelic heterogeneity at aCaenorhabditis elegansbeta-tubulin locus explains natural resistance to benzimidazoles
Authors: Hahnel Steffen R. , Zdraljevic Stefan , Rodriguez Briana C. , Zhao Yuehui , McGrath Patrick T. , Andersen Erik C. 
Published: July 2018

Benzimidazoles (BZ) are essential components of the limited chemotherapeutic arsenal available to control the global burden of parasitic nematodes. The emerging threat of BZ resistance among nearly all nematode species necessitates the development of novel strategies to identify genetic and molecular mechanisms underlying this resistance. All detection of parasitic helminth resistance to BZ is focused on the genotyping of three variant sites in the orthologs of the β-tubulin gene found to confer resistance in the free-living nematodeCaenorhabditis elegans. Because of the limitations of laboratory and field experiments in parasitic nematodes, it is difficult to look beyond these three sites, and additional BZ resistance is observed in the field. Here, we took an unbiased genome-wide mapping approach in the free-living nematode speciesC. elegansto identify the genetic underpinnings of natural resistance to the commonly used BZ, albendazole (ABZ). We found a wide range of natural variation in ABZ resistance in naturalC. eleganspopulations. In agreement with known mechanisms of BZ resistance in parasites, we find that a majority of the variation in ABZ resistance among wildC. elegansstrains is caused by variation in the β-tubulin geneben-1. This result shows empirically that resistance to ABZ naturally exists and segregates within theC. eleganspopulation, suggesting that selection in natural niches could enrich for resistant alleles. We identified 25 distinctben-1alleles that are segregating at low frequencies within theC. eleganspopulation, including many novel molecular variants. Population genetic analyses indicate thatben-1variation arose multiple times during the evolutionary history ofC. elegansand provide evidence that these alleles likely occurred recently because of local selective pressures. Additionally, we find purifying selection at all five β-tubulin genes, despite predicted loss-of-function resistants variants inben-1, indicating that BZ resistance in natural niches is a stronger selective pressure than loss of one β-tubulin gene. Furthermore, we use genome-editing to show that the most common parasitic nematode β-tubulin allele that confers BZ resistance, F200Y, confers resistance inC. elegans. Importantly, we identified a novel genomic region that is correlated with ABZ resistance in theC. eleganspopulation but independent ofben-1and the other β-tubulin loci, suggesting that there are multiple mechanisms underlying BZ resistance. Taken together, our results establish a population-level resource of nematode natural diversity as an important model for the study of mechanisms that give rise to BZ resistance.

Author summary

Nematode parasites have a tremendous impact on human health with almost two billion people infected worldwide. The control of nematode infections relies mainly on the efficacy of a limited repertoire of anthelmintic compounds, including the benzimidazoles (BZ). Already a significant problem in veterinary medicine, increasing evidence exists for the development of BZ resistance in nematodes that infect humans. Laboratory screens and field surveys identified β-tubulin genes as major determinants of BZ resistance in nematodes but detailed population-wide genetic analyses of resistance mechanisms are only just beginning. Therefore, we took advantage of the free-living model organismCaenorhabditis elegansto study the genetic basis of resistance to the commonly used BZ, albendazole (ABZ) in a natural nematode population. Performing genome-wide association mappings, we were able to identify extreme heterogeneity in the β-tubulin geneben-1as a major determinant of ABZ resistance. Moreover, our study provided new insights into the effects of missense and loss-of-function alleles at this locus, and how anthelmintic resistance could have developed within a natural nematode population.