Pharyngeal pumping and tissue-specific transgenic P-glycoprotein expression influence macrocyclic lactone susceptibility inCaenorhabditis elegans

Abstract

Macrocyclic lactone (ML) resistance has emerged in many parasitic nematodes including the pathogenic horse roundworm Parascaris univalens. The underlying mechanism of ML resistance and the drug penetration routes into the nematodes remain to be elucidated. Drug efflux by P-glycoproteins is considered a potential resistance mechanism but conclusive functional evidence is lacking. To this end, we used a motility assay modified to stimulate pharyngeal pumping (PP) by bacteria or serotonin and tissue-specific expression of Pun- PGP-9 in the free-living model nematode Caenorhabditis elegans. Here, stimulation of PP was identified as an important factor for C. elegans ML susceptibility, increasing the EC50 values of ivermectin by up to 11.1-fold and of moxidectin by 1.2-fold. In this context, intestinal Pun-PGP-9 expression elicited a protective effect against ivermectin and moxidectin only in the presence of PP stimulation, increasing the EC50 values by approximately 3- to 4-fold (ivermectin) or by < 1.3-fold (moxidectin). Conversely, epidermal Pun-PGP-9 expression protected against moxidectin regardless of PP with EC50 fold changes below 1.5 but against ivermectin with a considerable 2.9-fold EC50 increase only when the drug is not actively ingested. Our results highlight the role of active drug ingestion by nematodes for susceptibility and provide conclusive functional evidence for a contribution of P-glycoproteins to ML resistance.Author SummaryParasitic nematode infections pose a serious threat to animal health, in particular in light of the widespread anthelmintic resistance in different nematode species. In equines, the roundworm Parascaris univalens is a major pathogen of foals, exhibiting widespread resistance against macrocyclic lactones (MLs). This represents a particular challenge to animal health, but the underlying mechanisms and drug penetration routes remain mostly unknown. P-glycoprotein ABC-transporters have been linked to ML resistance in several parasitic nematodes. Here we demonstrate by tissue-specific overexpression of Pun-PGP-9 in the free-living model nematode Caenorhabditis elegans their ability to reduce the susceptibility to two commonly used MLs, ivermectin and moxidectin. At the same time, active drug ingestion by pharyngeal pumping (PP) strongly enhanced ivermectin and moderately effects moxidectin susceptibility. In more detail, the effect of intestinal or epidermal Pun-PGP-9 was dependent on active drug ingestion. These observations indicate differences in the drug penetration routes between ML derivatives and allow novel insight into the functional role of P-glycoproteins.

Ivermectin-induced Gene Expression Changes in Adult Parascaris Univalens and Caenorhabditis Elegans- a Comparative Approach to Study Anthelminthic Metabolism and Resistance in Vitro

“…In addition it has a short life-cycle of 3.5-days, and there are several powerful genetic tools available for manipulation of C. elegans (33). Several previous studies directed to decipher ML-resistance in P. univalens have employed transgenic lines of C. elegans as models (28, 34,35). To improve the likelihood of deriving conclusions from P. univalens research that use C. elegans as a model organism, it is necessary to assess its applicability, at the very least in terms of gene expression following xenobiotic exposure.…”