The P-glycoprotein repertoire of the equine parasitic nematode Parascaris univalens

Abstract

P-glycoproteins (Pgp) have been proposed as contributors to the widespread macrocyclic lactone (ML) resistance in several nematode species including a major pathogen of foals, Parascaris univalens. Using new and available RNA-seq data, ten different genomic loci encoding Pgps were identified and characterized by transcriptome-guided Rt-pcRs and Sanger sequencing. phylogenetic analysis revealed an ascarid-specific Pgp lineage, Pgp-18, as well as two paralogues of Pgp-11 and Pgp-16. comparative gene expression analyses in P. univalens and Caenorhabditis elegans show that the intestine is the major site of expression but individual gene expression patterns were not conserved between the two nematodes. In P. univalens, PunPgp-9, PunPgp-11.1 and PunPgp-16.2 consistently exhibited the highest expression level in two independent transcriptome data sets. Using RNA-Seq, no significant upregulation of any Pgp was detected following in vitro incubation of adult P. univalens with ivermectin suggesting that drug-induced upregulation is not the mechanism of Pgp-mediated ML resistance. expression and functional analyses of PunPgp-2 and PunPgp-9 in Saccharomyces cerevisiae provide evidence for an interaction with ketoconazole and ivermectin, but not thiabendazole. Overall, this study established reliable reference gene models with significantly improved annotation for the P. univalens pgp repertoire and provides a foundation for a better understanding of pgp-mediated anthelmintic resistance. Parasitic nematodes are important pathogens of livestock, companion animals and humans and the emergence and spread of anthelmintic resistance has compromised veterinary helminth control. In equines, Parascaris univalens poses a major threat particularly to juvenile horses 1,2. At present, chemotherapeutic metaphylaxis and therapy remain the most effective and commonly used strategies for veterinary helminth control although widespread anthelmintic resistance to one or multiple drug classes compromise their success 3-5. The frequent and unrestricted use of macrocyclic lactones (MLs) in domestic horses over decades has driven selection of resistant parasite populations 6. In Parascaris sp., ML resistance was first reported in the early 2000s 7 and since then has developed into a global challenge. Its presence has been demonstrated in several European countries 8-13 , North America 14-16 , as well as New Zealand 17 , Australia 18 and more recently Ethiopia 19 and Saudi-Arabia 20. Similarly, the development and spread of ML resistance has been reported in many other parasitic nematodes, including Teladorsagia circumcincta 21 and Haemonchus contortus in sheep 22 , Dirofilaria immitis 23 in dogs and even in human filarial nematodes such as Onchocerca volvulus 24. Prior to genome-wide approaches, P-glycoproteins (Pgp) were already presumed to be important contributors to ML resistance in a number of parasitic nematodes 25. Nonetheless, deciphering the mechanisms of ML resistance is challenging, partly due to the multi-genic nature of the resistanc...

Pharyngeal Pumping and Tissue-Specific Transgenic P-Glycoprotein Expression Influence Macrocyclic Lactone Susceptibility in Caenorhabditis elegans

“…The objective in this study was to elucidate the general functional role of Pgp expression in specific tissues in the context of ML susceptibility. To this end, we chose to exemplarily characterise Pun -PGP-9, as it has already been shown to interact with IVM in a yeast model [ 23 ]. An important question addressed in this work was whether there are differences in the protective effect of Pgp overexpression between different ML derivatives, by testing IVM as an avermectin and MOX as a member of the milbemycins, which are summarised in Figure 4 .…”

“…Currently, functional evidence regarding the interaction between specific parasite Pgps and different MLs has mostly been inferred from indirect assays using the above-mentioned yeast model [ 23 , 24 ] or the cell line LLC-PK1 [ 24 , 29 , 30 , 44 , 45 ]. In these model systems, MLs were used as inhibitors for a secondary Pgp substrate with a discernible effect in the model systems to characterise the interaction between a transgenic parasite Pgp and different MLs.…”

“…In contrast to mammals, which only possess one (human) to three (rodents) Pgps, the nematode Pgp gene family comprises a diverse repertoire that differs between nematodes species [20], e.g., 15 Pgps in C. elegans [21] and 10 Pgps in Haemonchus contortus [22]. More recently, the Pgp repertoire of P. univalens was completely deciphered, revealing 10 different Pgp genes with particularly strong expression levels for Pun-pgp-11.1, Pun-pgp-16.2, and Pun-pgp-9 [23]. Across different species, the pgp-9 gene lineage has been repeatedly associated with ML resistance in several nematode species such as cyathostomins [19,24], Teladorsagia circumcincta [18,25], and H. contortus [26] and is regarded as one of the most relevant candidates for a contribution to ML resistance.…”

“…Across different species, the pgp-9 gene lineage has been repeatedly associated with ML resistance in several nematode species such as cyathostomins [ 19 , 24 ], Teladorsagia circumcincta [ 18 , 25 ], and H. contortus [ 26 ] and is regarded as one of the most relevant candidates for a contribution to ML resistance. While Pgps are expressed in most tissues, expression is most prominent in the intestine in different nematode species, including P. univalens and C. elegans [ 23 , 27 , 28 , 29 ]. In addition, the epidermis (= hypodermis) also exhibits moderate Pgp expression levels in P. univalens [ 27 ] and C. elegans [ 23 ], but the tissue-specific function of these genes is unclear.…”

“…While Pgps are expressed in most tissues, expression is most prominent in the intestine in different nematode species, including P. univalens and C. elegans [ 23 , 27 , 28 , 29 ]. In addition, the epidermis (= hypodermis) also exhibits moderate Pgp expression levels in P. univalens [ 27 ] and C. elegans [ 23 ], but the tissue-specific function of these genes is unclear.…”