Multiple UDP glycosyltransferases modulate benzimidazole drug sensitivity in the nematode Caenorhabditis elegans in an additive manner

Abstract

Xenobiotic biotransformation is an important modulator of anthelmintic drug potency and a potential mechanism of anthelmintic resistance. Both the free-living nematode Caenorhabditis elegans and the ruminant parasite Haemonchus contortus biotransform benzimidazole drugs by glucose conjugation, likely catalysed by UDP-glycosyltransferase (UGT) enzymes. To identify C. elegans genes involved in benzimidazole drug detoxification, we first used a comparative phylogenetic analysis of UGTs from humans, C. elegans and H. contortus, combined with available RNAseq datasets to identify which of the 63 C. elegans ugt genes are most likely to be involved in benzimidazole drug biotransformation. RNA interference knockdown of 15 prioritized C. elegans genes identified those that sensitized animals to the benzimidazole derivative albendazole (ABZ). Genetic mutations subsequently revealed that loss of ugt-9 and ugt-11 had the strongest effects. The “ugt-9 cluster” includes these genes, together with six other closely related ugts. A CRISPR-Cas-9 deletion that removed seven of the eight ugt-9 cluster genes had greater ABZ sensitivity than the single largest-effect mutation. Furthermore, a double mutant of ugt-22 (which is not a member of the ugt-9 cluster) with the ugt-9 cluster deletion further increased ABZ sensitivity. This additivity of mutant phenotypes suggest that ugt genes act in parallel, which could have several, not mutually exclusive, explanations. ugt mutations have different effects with different benzimidazole derivatives, suggesting that enzymes with different specificities could together more efficiently detoxify drugs. Expression patterns of ugt-9, ugt-11 and ugt-22 gfp reporters differ and so likely act in different tissues which may, at least in part, explain their additive effects on drug potency. Overexpression of ugt-9 alone was sufficient to confer partial ABZ resistance, indicating increasing total UGT activity protects animals. In summary, our results suggest that the multiple UGT enzymes have overlapping but not completely redundant functions in benzimidazole drug detoxification and may represent “druggable” targets to improve benzimidazole drug potency.