Independent mechanisms of benzimidazole resistance across Caenorhabditis nematodes

Abstract

Benzimidazoles, a widely used class of anthelmintic drugs, target beta-tubulin, disrupt microtubule formation, and delay nematode development. In parasitic nematodes, mutations in beta-tubulin genes are predicted to inhibit benzimidazole binding and are associated with resistance. In the free-living nematode Caenorhabditis elegans, loss-of-function mutations in the beta-tubulin gene ben-1 cause benzimidazole resistance. Although several beta-tubulin mutations serve as established markers of resistance, the prediction of the effects of novel variants in different nematode species remains challenging. Here, we identified novel beta-tubulin variants predicted to confer benzimidazole resistance across wild strains in three Caenorhabditis species: C. elegans, Caenorhabditis briggsae, and Caenorhabditis tropicalis. The three Caenorhabditis species are experimentally tractable, have characterized beta-tubulin gene complements, and defined natural niches, which allowed us to identify variants in beta-tubulin genes and test which variants are associated with resistance. We hypothesized that, if these species experienced similar selective pressures, they would evolve resistance to benzimidazoles by mutations in a beta-tubulin gene (tbb-1, tbb-2, mec-7, tbb-4, and ben-1). In the three Caenorhabditis species, we tested all strains harboring variants in the five conserved beta-tubulin genes for benzimidazole resistance. In C. elegans, we found that a heterogeneous set of variants in ben-1 were associated with resistance. By contrast, only two variants in C. briggsae ben-1 (W21stop and Q134H) were associated with resistance. C. tropicalis was distinct from the other two species, where no strains with variants in any beta-tubulin gene were resistant. We generated deletions of ben-1 in C. briggsae and C. tropicalis and confirmed that loss of ben-1 confers resistance in both species. Our findings reveal species-specific patterns of beta-tubulin-mediated benzimidazole resistance and emphasize that prediction of variants in beta-tubulin genes alone is not sufficient to predict resistance, especially across diverse nematode species. AUTHOR SUMMARYMutations in beta-tubulin genes have been associated with benzimidazole resistance across nematode species, yet predicting novel resistance variants remains challenging. Using wild strains from three Caenorhabditis species, we identified strains with variants in beta-tubulin genes and tested each strain for benzimidazole resistance. In C. elegans, a diverse set of loss-of-function variants in ben-1 were associated with resistance. Whereas in C. briggsae, only two ben-1 alleles were associated with resistance, suggesting selection acts differently in this species despite a similar niche as C. elegans. C. tropicalis had no strains with beta-tubulin variants that were resistant. Our results highlight species-specific patterns of benzimidazole resistance.