Deciphering the molecular determinants of cholinergic anthelmintic sensitivity in nematodes: When novel functional validation approaches highlight major differences between the model Caenorhabditis elegans and parasitic species

Abstract

Cholinergic agonists such as levamisole and pyrantel are widely used as anthelmintics to treat parasitic nematode infestations. These drugs elicit spastic paralysis by activating acetylcholine receptors (AChRs) expressed in nematode body wall muscles. In the model nematode Caenorhabditis elegans, genetic screens led to the identification of five genes encoding levamisole-sensitive-AChR (L-AChR) subunits: unc-38, unc-63, unc-29, lev-1 and lev-8. These subunits form a functional L-AChR when heterologously expressed in Xenopus laevis oocytes. Here we show that the majority of parasitic species that are sensitive to levamisole lack a gene orthologous to C. elegans lev-8. This raises important questions concerning the properties of the native receptor that constitutes the target for cholinergic anthelmintics. We demonstrate that the closely related ACR-8 subunit from phylogenetically distant animal and plant parasitic nematode species functionally substitutes for LEV-8 in the C. elegans L-AChR when expressed in Xenopus oocytes. The importance of ACR-8 in parasitic nematode sensitivity to cholinergic anthelmintics is reinforced by a ‘model hopping’ approach in which we demonstrate the ability of ACR-8 from the hematophagous parasitic nematode Haemonchus contortus to fully restore levamisole sensitivity, and to confer high sensitivity to pyrantel, when expressed in the body wall muscle of C. elegans lev-8 null mutants. The critical role of acr-8 to in vivo drug sensitivity is substantiated by the successful demonstration of RNAi gene silencing for Hco-acr-8 which reduced the sensitivity of H. contortus larvae to levamisole. Intriguingly, the pyrantel sensitivity remained unchanged thus providing new evidence for distinct modes of action of these important anthelmintics in parasitic species versus C. elegans. More broadly, this highlights the limits of C. elegans as a predictive model to decipher cholinergic agonist targets from parasitic nematode species and provides key molecular insight to inform the discovery of next generation anthelmintic compounds.

A Whole Genome Re-Sequencing Based GWA Analysis Reveals Candidate Genes Associated with Ivermectin Resistance in Haemonchus contortus

“…The L2 larvae obtained from in vitro culture of eggs were subjected to gene silencing assays. The RNAi experiments were performed on 3000-4000 L2 larvae in a final volume of 500 µL in 2 mL tubes as described previously [1,56]. First, to investigate the potential ingestion of siRNA constructs for gene silencing experiments, H. contortus L2 larvae were incubated (for 2 h at 20 • C under gentle agitation) in a culture medium containing 1 µM of a nonspecific siRNA targeting gfp (nonspecific).…”

Effects of cholesterol content on activity of P-glycoproteins and membrane physical state, and consequences for anthelmintic resistance in the nematodeHaemonchus contortus

“…The roles of the various components of lipophilic molecule transport systems (passive diffusion, active influx, and active efflux) need to be investigated further, as well as the role and production of lipids in nematodes. This knowledge may therefore make it possible to identify new targets for anthelmintics, like other targets described in recent research in order to counter multiple resistance [8,14,46,53,57,79].…”

Lupin (Lupinus sp.) seeds exert anthelmintic activity associated with their alkaloid content

“…Resolving the contribution of AChRs antagonism to the observed anthelmintic effects would require additional experiments that are beyond the scope of this study. For such experiment, the knock-down of acetylcholine receptor expression by RNA interference 39 in parasitic larvae before further exposure to lupin alkaloids could be a method of choice. However, our results indicated that the E063 enriched alkaloidic fraction was not necessarily exerting as much inhibition as the total seed extract and that significant larval migration inhibition was associated with the non-alkaloid seed extract in that case.…”

“…Because of the scarcity of fractionated alkaloidic compounds (2.7 to 12.8 mg), their anthelmintic effect on infective H. contortus larvae was measured using ALMA. This technic relies on larval auto-fluorescence generated through ultraviolet excitation 38,39 . Every alkaloidic compound was incubated for 4 hours at 20°C with 7,500 H. contortus infective larvae each.…”

“…Larvae were then passed through a 20⁏μm sieve and left for a 60 s stabilization time before migration into the spectrofluorimeter cuvette. Cumulative fluorescence resulting from larval migration into the cuvette and correlating the total number of larvae 39 was recorded during 5⁏min. Each set of experiment was performed in triplicate at a concentration of 250 µg/mL but for compound 2 (150 µg/mL).…”