2025 Theses Doctoral

Genes Needed for Benomyl Resistance and Hypersensitivity in Caenorhabditis elegans

Guo, Zhenhao

Benzimidazole anthelmintics, such as benomyl, paralyze C. elegans and slow their growth by targeting the BEN-1 β-tubulin. Loss of ben-1 results in normal-appearing animals that are completely resistant to benomyl and other benzimidazoles, a result suggesting that BEN-1 acts redundantly in C. elegans. Since 74% of benzimidazole-resistant C. elegans strains isolated from the wild had ben-1 mutations (Hahnel et al. 2018), defects in other genes must also convey resistance.

To identify such genes, we screened 1,751 fully sequenced Million Mutation Project strains for benomyl resistance and additionally for increased benomyl sensitivity. Two completely resistant strains, both with ben-1 mutations, 14 partially resistant strains (animals were mobile but uncoordinated on benomyl), and five strains with increased sensitivity were identified.

We identified the causative mutation in five partially resistant strains: one was a ben-1 mutation. One was a microtubule depolymerase gene klp-7/ kinesin-13. The other three were in genes expressed in ciliated sensory neurons (the intraflagellar transport particle genes osm-1/ IFT-172, che-13/ IFT57 and dyf-3/ IFT38). We tested other mutants with defects in these cells and found that mutations of daf-19 (in the presence of a daf-12 mutation to block dauer formation), which encodes a key transcription factor for ciliogenesis, and 17 other cilia-related genes also produced partial resistance.

A similar result for 12 of the latter genes, but not daf-19 and the remaining cilia-related genes, was reported in a preprint in BioRxiv by Robert A. Brinzer et al. in 2021. This finding suggests that benomyl is transported into C. elegans via ciliated neurons as Brinzer et al. (2024) found for macrocyclic lactones. Three of the five strains with increased sensitivity, which led to paralysis at a low, normally ineffective benomyl concentration, had mutations in tbb-2/ β-tubulin (a result consistent with that of Pallotto et al. 2022).

A fourth, similarly hypersensitive strain had a mutation in tba-1/ α-tubulin. Together with effects of microtubule destabilizer KLP-7 on drug sensitivity, these results suggest that the microtubule stability may affect the strength of the drug’s effect, perhaps by changing the amount of BEN-1-containing microtubules.

We also identified a Snapin gene (snpn-1) mutant with increased sensitivity, while examining the effects of vesicle transport on drug sensitivity. Since the loss of snpn-1 results in defective synaptic vesicle distribution, it may modulate the benomyl sensitivity by disrupting the proper function of motor neurons.