Benzimidazole drugs are a class of broad-spectrum anthelmintics used in the control of parasitic nematodes in humans, livestock and companion animals (Kaplan and Vidyashankar, 2012, Anderson and J., Hollingsworth, T.D., , 2014, Gilleard et al., 2021). Extensive and often inappropriate use of benzimidazole drugs has led to widespread resistance in parasitic nematodes of livestock such as Haemonchus contortus and multiple other strongylid nematode species. Recent efforts to control human parasites, most commonly using the benzimidazole derivative albendazole (ABZ), have administered billions of doses worldwide and their variable efficacy is leading to concerns regarding the emergence of resistance (Soukhathammavong et al., 2012, Diawara et al., 2013, Moser et al., 2017). An understanding of the mechanisms that affect benzimidazole drug potency has potential applications for developing drug synergists, new drugs and improving the detection of resistance.

Benzimidazole (BZ) drugs bind to β-tubulin monomers and disrupt microtubule polymerization (Robinson et al., 2004). In parasitic strongylid nematodes, benzimidazole drug resistance is associated with single nucleotide polymorphisms in the gene encoding isotype-1 β-tubulin and these mutations also confer resistance in the free-living nematode Caenorhabditis elegans (Kwa and J.G., Roos, M.H., , 1994, Kotze and Prichard, 2016, Dilks et al., 2020). However, H. contortus field isolates with similar frequencies of the β-tubulin mutations have different levels of benzimidazole drug resistance, suggesting involvement of other genes (von Samson-Himmelstjerna et al., 2009). Quantitative Trait Loci (QTL) mapping of benzimidazole drug sensitivity in C. elegans field populations identified genomic intervals in addition to the β-tubulin ben-1 locus (Zamanian et al., 2018).

A possible contributor to non-β-tubulin resistance mechanisms is xenobiotic metabolism (Rowland et al., 2013, Zanger and Schwab, 2013, Yilmaz et al., 2017). Detoxification of exogenous small molecules (xenobiotics) occurs in three phases. Phase I consists mainly of cytochrome P450 (CYP), which primarily catalyzes oxidation reactions. Phase II includes the conjugation enzymes UDP-glycosyltransferase (UGT) and glutathione S-transferases. Phase III consists of drug efflux transporters, which effectively expel the detoxified xenobiotic. CYP and UGT are large gene families with members that have distinct but overlapping substrate specificities. While xenobiotic drug metabolism leads to insecticide resistance (Daborn et al., 2002, Li and M.A., Berenbaum, M.R., , 2007, David et al., 2013), this has been relatively little studied in nematodes. We are particularly interested in the role of UGT-mediated biotransformation of benzimidazole drugs in modulating nematode responses to these drugs. In H. contortus, expression of one particular UGT gene was upregulated in two benzimidazole drug-resistant strains (Matouskova et al., 2018, Kellerova et al., 2020a). Furthermore, higher amounts of the benzimidazole derivative ABZ-glycosylated metabolites (likely mediated by UGTs) form in resistant isolates (Kellerova et al., 2020a).

Xenobiotic enzymes play an important role in metabolizing and modulating benzimidazole drug potency in laboratory studies (Laing and A., Laing, R., Ravikumar, S., Butler, V., Woods, D.J., Gilleard, J.S., , 2010, Stasiuk et al., 2019). Previous work from our group showed that exposure of C. elegans to several different benzimidazole drugs, ABZ, mebendazole, fenbendazole, thiabendazole and oxfendazole, resulted in transcriptional up-regulation phase I and phase II drug metabolizing enzymes, including a subset of UGT genes, leading to increased formation of glucose-conjugated ABZ metabolites (Stasiuk et al., 2019). Haemonchus contortus also metabolizes multiple benzimidazole drugs by glucose conjugation (Laing and A., Laing, R., Ravikumar, S., Butler, V., Woods, D.J., Gilleard, J.S., , 2010, Stasiuk et al., 2019, Kellerova et al., 2020a, Kellerova et al., 2020b). Furthermore, the UGT enzyme inhibitor chrysin reduced the production of a glucose-conjugated ABZ metabolites in C. elegans (Stasiuk et al., 2019). Mutations of the C. elegans transcription factor encoded by skn-1 and its downstream transcriptional target ugt-22 both modulate ABZ efficacy (Choe et al., 2012, Fontaine and Choe, 2018). An interesting difference between mammals and nematodes is that glucose conjugation of xenobiotic molecules is rare in mammals, glucuronic acid conjugation being more common (Meech et al., 2012a, Rowland et al., 2013, Stasiuk et al., 2019, Kellerova et al., 2020a, Kellerova et al., 2020b). Therefore, inhibition of nematode glucose conjugation could be tolerated by their mammalian hosts.

In this study we wanted to identify the key ugt genes involved in benzimidazole drug biotransformation. We surveyed the 63 C. elegans ugt genes to determine which might modulate ABZ drug potency and prioritized fifteen using available transcriptomic data. RNAi knockdown and subsequent analysis of mutants revealed that ugt-9, ugt-11 and ugt-22 produced robust ABZ hypersensitive phenotypes. We found that a cluster of genes that includes ugt-9 and ugt-11, in combination with ugt-22, act in an additive fashion. The additivity may be in part explained by total ugt gene dosage, the partially non-overlapping patterns of gene expression and/or differing enzyme specificities. This work demonstrates that multiple members of the ugt gene family play an important role in modulating benzimidazole drug sensitivity in nematodes acting in an additive and partially redundant manner and have the potential to contribute to resistance among parasitic nematodes.