Quantitative benzimidazole resistance and fitness effects of parasitic nematode beta-tubulin alleles

Abstract

<jats:title>Abstract</jats:title><jats:p>Infections by parasitic nematodes inflict a huge burden on the health of humans and livestock throughout the world. Anthelmintic drugs are the first line of defense against these infections. Unfortunately, resistance to these drugs is rampant and continues to spread. To improve treatment strategies, we must understand the genetics and molecular mechanisms that underlie resistance. Studies of the fungus <jats:italic>Aspergillus nidulans</jats:italic> and the free-living nematode <jats:italic>Caenorhabditis elegans</jats:italic> discovered that a beta-tubulin gene is mutated in benzimidazole (BZ) resistant strains. In parasitic nematode populations, three canonical beta-tubulin alleles, F200Y, E198A, and F167Y, have long been correlated with resistance. Additionally, improvements in sequencing technologies have identified new alleles - E198V, E198L, E198K, E198I, and E198Stop - also correlated with BZ resistance. However, none of these alleles have been proven to cause resistance. To empirically demonstrate this point, we independently introduced the three canonical alleles as well as two of the newly identified alleles, E198V and E198L, into the BZ susceptible <jats:italic>C. elegans</jats:italic> N2 genetic background. These genome-edited strains were exposed to both albendazole and fenbendazole to quantitatively measure animal responses to BZs. We used a range of doses for each BZ compound to define response curves and found that all five of the alleles conferred resistance to BZ compounds equal to a loss of the entire beta-tubulin gene. These results prove that the parasite beta-tubulin alleles cause resistance. The E198V allele is found at low frequencies in natural parasite populations, suggesting that it could affect fitness. We performed competitive fitness assays and demonstrated that the E198V allele reduces animal health, supporting the hypothesis that this allele is less fit in field populations. Overall, we present a powerful platform to quantitatively assess anthelmintic resistance and effects of specific resistance alleles on organismal fitness in the presence or absence of the drug.</jats:p><jats:sec><jats:title>Highlights</jats:title><jats:list list-type="bullet"><jats:list-item><jats:p>All three canonical parasitic nematode beta-tubulin alleles (F167Y, E198A, F200Y) and two newly identified alleles (E198V, E198L) confer equal levels of benzimidazole resistance in a defined genetic background using single-generation, high-replication drug response assays.</jats:p></jats:list-item><jats:list-item><jats:p>Beta-tubulin variants are strongly selected in albendazole conditions in multigenerational competitive fitness assays, but these alleles confer different levels of benzimidazole resistance over time.</jats:p></jats:list-item><jats:list-item><jats:p>Only the E198V allele confers a fitness cost in control (non-benzimidazole) conditions as compared to all other tested beta-tubulin alleles, suggesting that this intermediate allele might only be found in field populations at low frequency because it causes reduced fitness.</jats:p></jats:list-item></jats:list></jats:sec><jats:sec><jats:title>Graphical Abstract</jats:title><jats:fig id="ufig1" position="float" fig-type="figure" orientation="portrait"><jats:graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="191866v1_ufig1" position="float" orientation="portrait" /></jats:fig></jats:sec>

Complementary Approaches to Understand Anthelmintic Resistance Using Free-Living and Parasitic Nematodes

“…However, an experimental cycle of discovery between C. elegans and the veterinary helminth H. contortus provides an ideal complementary approach to study resistance against a wide range of anthelmintics (Figure 1). The genetic toolbox associated with C. elegans and its close phylogenetic relationship to H. contortus allows researchers to use the tools available in C. elegans to study resistance candidates from H. contortus [20][21][22]. Here, we describe how complementary approaches have benefited studies of anthelmintic resistance mechanisms in the past and why this approach is the most productive route to discovery in the future.…”

“…The overexpression experiments only show that the parasite beta-tubulin gene is sufficient for BZ sensitivity. To show that beta-tubulin is necessary for the parasite BZ response, this gene must be mutated in a defined genetic background and shown to alter the BZ response, which has been done for several parasitic nematode beta-tubulin alleles [20][21][22]. These approaches testing gene functions between C. elegans and H. contortus have enabled rapid progress in the understanding of BZ resistance.…”

“…Previous work has demonstrated that several loss-offunction acr-23 mutations cause resistance to monepantel [29], but the wild isolates could tell us how natural nematodes become resistant to this class of anthelmintic. These naturally occurring alleles need to be tested to determine if they confer resistance to monepantel, as has been done for BZ resistance [20,21]. Use of naturally occurring alleles provides insights into the diversity that is present in nature and not only laboratory-generated alleles from genetic selection experiments.…”

“…Importantly, the C. elegans CRISPR-Cas9 system has enabled the testing of long-standing hypotheses in the anthelmintic resistance field. For example, genome-editing technology enabled the functional connection between ben-1 beta-tubulin variation and resistance in a controlled genetic background without overexpression using extrachromosomal arrays or incomplete knockdown using RNAi [20][21][22].…”

“…Confirmation that the previously described beta-tubulin mutations F167Y, E198A, and F200Y confer resistance without affecting fitness required the creation of single nucleotide variant strains and then linking these mutations to resistance and fitness effects. After editing in the laboratory reference strain N2, the F167Y, E198A, and F200Y mutations all conferred resistance without causing a loss in fitness [20]. Recently, the candidate alleles E198L and E198V were identified in the parasites Teladorsagia circumcincta and Trichostrongylus axei (Avramenko et al 2019).…”