Extreme allelic heterogeneity at aCaenorhabditis elegansbeta-tubulin locus explains natural resistance to benzimidazoles

Abstract

32Benzimidazoles (BZ) are essential components of the limited chemotherapeutic arsenal available to 33 control the global burden of parasitic nematodes. The emerging threat of BZ resistance among nearly all 34 nematode species necessitates the development of novel strategies to identify genetic and molecular 35 mechanisms underlying this resistance. All detection of parasitic helminth resistance to BZ is focused on 36 the genotyping of three variant sites in the orthologs of the β-tubulin gene found to confer resistance in the 37 free-living nematode Caenorhabditis elegans. Because of the limitations of laboratory and field 38 experiments in parasitic nematodes, it is difficult to look beyond these three sites, and additional BZ 39 resistance is observed in the field. Here, we took an unbiased genome-wide mapping approach in the 40 free-living nematode species C. elegans to identify the genetic underpinnings of natural resistance to the 41 commonly used BZ, albendazole (ABZ). We found a wide range of natural variation in ABZ resistance in 42 natural C. elegans populations. In agreement with known mechanisms of BZ resistance in parasites, we 43 find that a majority of the variation in ABZ resistance among wild C. elegans strains is caused by variation 44 in the β-tubulin gene ben-1. This result shows empirically that resistance to ABZ naturally exists and 45 segregates within the C. elegans population, suggesting that selection in natural niches could enrich for 46 resistant alleles. We identified 25 distinct ben-1 alleles that are segregating at low frequencies within the 47 C. elegans population, including many novel molecular variants. Population genetic analyses indicate that 48ben-1 variation arose multiple times during the evolutionary history of C. elegans and provide evidence 49 that these alleles likely occurred recently because of local selective pressures. Additionally, we find 50 purifying selection at all five β-tubulin genes, despite predicted loss-of-function resistants variants in ben-1, 51indicating that BZ resistance in natural niches is a stronger selective pressure than loss of one β-tubulin 52 gene. Furthermore, we use genome-editing to show that the most common parasitic nematode β-tubulin 53 allele that confers BZ resistance, F200Y, confers resistance in C. elegans. Importantly, we identified a 54 novel genomic region that is correlated with ABZ resistance in the C. elegans population but independent 55 of ben-1 and the other β-tubulin loci, suggesting that there are multiple mechanisms underlying BZ 56 resistance. Taken together, our results establish a population-level resource of nematode natural diversity 57 as an important model for the study of mechanisms that give rise to BZ resistance. 58 . CC-BY 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint (which . http://dx.doi.org/10.1101/372623 doi: bioRxiv preprint first p...

Quantitative benzimidazole resistance and fitness effects of parasitic nematode beta-tubulin alleles

“…To show that beta-tubulin is necessary for BZ response, only the beta-tubulin gene must be mutated in a defined genetic background and shown to cause BZ resistance. Recently, the F167Y and F200Y alleles were shown to confer BZ resistance in C. elegans in a controlled genetic background (Hahnel et al, 2018;Kitchen et al, 2019).…”

“…Measurements of the exact levels of resistance and any growth effects associated with the alleles are nearly impossible in field populations, so controlled laboratory experiments are required to test these hypotheses. In C. elegans, the F200Y and a loss-of-function ben-1 allele conferred strong BZ resistance but no growth consequences compared to a wild-type control strain in conditions that lacked BZ (Hahnel et al, 2018), suggesting that the maintenance of resistance is not a detriment to fitness.…”

“…The existing strains harboring the ben-1 deletion allele (ean64) or the F200Y alleles (ean100, ean101) (Hahnel et al, 2018) were used as controls for all experiments in this study. The barcoded wild-type control strain PTM229 dpy-10(kah81) was used in competitive fitness assays (Zhao et al, 2018).…”

“…The barcoded wild-type control strain PTM229 dpy-10(kah81) was used in competitive fitness assays (Zhao et al, 2018). All strains in this study (Supplemental Table 1) were generated in the N2 genetic background with modifications introduced using the CRISPR-Cas9 genome-editing system as described previously (Hahnel et al, 2018).…”

“…High-throughput fitness assays were performed as previously described (Evans and Andersen, 2020;Zdraljevic et al, 2017 (Brady et al, 2019;Hahnel et al, 2018;Zdraljevic et al, 2017). Animal optical density integrated by the animal length (EXT) was measured with the COPAS BIOSORT for each individual.…”