Description

Parasitic nematode infections continue to have an enormous impact on human and livestock health worldwide (Hotez et al., 2014; Kaplan & Vidyashankar, 2012). A limited arsenal of anthelmintic drugs are available to combat these infections. One of the most widely used classes is benzimidazoles (BZ), and resistance against this class is widespread (Kaplan & Vidyashankar, 2012). Previous studies to understand parasitic nematode resistance using the free-living model organism Caenorhabditis elegans showed that variation in the C. elegans beta-tubulin gene ben-1, an ortholog of beta-tubulins in parasitic nematodes, confers resistance to BZ drugs (Dilks et al., 2020; Driscoll et al., 1989; Hahnel et al., 2018). The most common missense mutation resistance alleles are F167Y, E198A, and F200Y (Avramenko et al., 2019; Mohammedsalih et al., 2020). Although computational models have predicted that these amino acids are involved in the binding of BZ compounds to beta-tubulins, the binding remains to be investigated empirically at the structural level because nematode-specific beta-tubulin structures have not been created (Aguayo-Ortiz et al., 2013; Hahnel et al., 2018). To better understand the mechanisms of resistance, we sought to obtain those crystallographic structures.

To isolate BEN-1 for crystallographic studies, tags would permit the purification of this beta-tubulin from the other five beta-tubulins in C. elegans. Unfortunately, endogenous tags of BEN-1 eliminate its function (Dilks and Andersen, unpublished results). Because of this limitation, we turned to expression of three different versions of nematode beta-tubulins, C. elegans BEN-1 and two beta-tubulin isotypes from the parasitic nematode Haemonchus contortus, in Saccharomyces cerevisiae, which has a single beta-tubulin gene (Figure 1A).

We used the following procedure to replace the S. cerevisiae beta-tubulin gene with each of the nematode beta-tubulin genes (Figure 1B). These replaced beta-tubulin genes were expressed using the native S. cerevisiae promoter. First, TUB2 was deleted in diploid BY4743 cells and replaced with the Nourseothricin N-acetyl transferase (NAT) cassette selection marker (Hansen et al., 2003). Successful deletion of one copy of the TUB2 coding sequence was verified using colony PCR of a sample that grew in the presence of NAT (Figure 1C). The NAT cassette was then substituted with yeast codon-optimized C. elegans ben-1 along with the HIS3 selection marker (Burke & Gould, 1994). Successful replacement of ben-1 at the TUB2 locus was verified using colony PCR on samples that grew in histidine-deficient conditions (Figure 1D). We sporulated diploids of the ben-1 replacement strain then dissected tetrads to identify haploid cells that contained the C. elegans beta-tubulin gene ben-1. We found that dissected tetrads gave rise to only two viable haploid cells instead of four when grown on complete media (YPD) (Figure 1E). The same result was obtained after replacement of TUB2 with either of the two H. contortus beta-tubulin isotype genes (data not shown). The two surviving colonies were also unable to grow in histidine-deficient conditions, suggesting that replacement of TUB2 with nematode beta-tubulin genes is lethal. Using RT-PCR of the heterozygous diploid strain with one copy of TUB2 and one copy of ben-1 at the TUB2 locus, we found that ben-1 mRNA was expressed (Figure 1F), suggesting that the nematode beta-tubulin is deficient at protein expression, lacks proper stability, and/or cannot function with the yeast alpha-tubulin gene. Our results indicate that nematode beta-tubulin genes can not substitute for the yeast beta-tubulin gene TUB2.

Beyond the phylogenetic difference between nematode and yeast beta-tubulins, it has been observed that the dynamics of microtubules in C. elegans greatly differ from other eukaryotes (Chaaban et al., 2018), suggesting that nematode beta-tubulins might be unsuitable replacements for yeast beta-tubulins. It is possible that both alpha- and beta-tubulin might need to be replaced by their nematode-specific versions to obtain viable yeast and allow for the characterization of benzimidazole and beta-tubulin binding to better understand the mechanism of resistance.