Abstract
Macrocyclic lactones (MLs) represent the major drug class for control of parasitic infections in humans and animals. However, recently reports of treatment failures became more frequent. In addition to human and ruminant parasitic nematodes this also is the case for the horse-nematode Parascaris equorum. Nevertheless, to date the molecular basis of ML resistance is still not understood. Unspecific resistance mechanisms involving transporters such as P-glycoproteins (Pgps) are expected to contribute to ML resistance in nematodes. Here, complete sequences of two P. equorum Pgps were cloned and identified as orthologs of Caenorhabditis elegans Ppg-11 and an unnamed Caenorhabditis briggsae Pgp designated as Pgp-16 using phylogenetic analysis. Quantitative real-time PCR was used to compare expression between tissues. Significantly higher PeqPgp-11 expression was found in the gut for both genders, whereas for PeqPgp-16 the body wall was identified as predominant expression site. Furthermore, Pgps were analyzed regarding their participation in resistance development. Using SeqDoC analyses, Pgp-sequences of P. equorum populations with different ML susceptibility were compared. This approach revealed three single nucleotide polymorphisms (SNPs) causing missense mutations in the PeqPgp-11 sequence which correlated with decreased ML susceptibility. However, no resistance associated differences in mRNA expression levels were detected between embryonated eggs of these populations. In contrast, comparison of two pre-adult groups with different ivermectin (IVM) susceptibility revealed the presence of the three SNPs and in addition statistically significant PeqPgp-11 overexpression in the group of worms with reduced susceptibility. These results indicate that Pgp-11 might be involved in IVM resistance in P. equorum as it shows increased expression in an IVM exposed life-cycle stage of an IVM resistant population as well as occurrence of putatively resistance associated SNPs in populations with reduced IVM susceptibility. These SNPs are promising diagnostic candidates for detection of ML resistance with potential also for other parasitic nematode species.
Highlights
For decades macrocyclic lactones (MLs) have been used for the treatment and control of infections with endoparasitic nematodes and ectoparasitic arthropods in human and veterinary medicine [1]
Sequencing of P. equorum Pgps RT-PCR using degenerated primers resulted in 12 fragments localized in the internucleotide binding domain (IBD) of Pgps
The first P. equorum Pgp sequence is orthologous to C. elegans Pgp-11 (Fig. 1) and was designated as PeqPgp-11
Summary
For decades macrocyclic lactones (MLs) have been used for the treatment and control of infections with endoparasitic nematodes and ectoparasitic arthropods in human and veterinary medicine [1]. Even though the prevalence of ML resistant nematode populations increases, the resistance mechanisms remain largely unclear. Several possible mechanisms are discussed, including the activity of ATP-bindingcassette (ABC)-transporters, in particular P-glycoproteins (Pgps) [5]. Pgps are located in the apical cell-membrane, act as ATPdependent transporters for hydrophobic xenobiotics such as anthelmintics and decrease the concentration of the drugs at their target sites [6].
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