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.
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This work was supported by the Deutsche Forschungsgemeinschaft (DFG) (grant number 111144555). Additional funding was provided by the DFG (grant number 251133687/GRK 2046) and the Karl-Enigk Foundation (grant number S0229/1100191/19). Open access funding provided by Projekt DEAL. Deutsche Forschungsgemeinschaft Grant Number: 111144555. Deutsche Forschungsgemeinschaft Grant Number: 251133687/GRK 2046.
Supplementary information is available for this paper at https://doi.org/10.1038/s4159 8-020-70529-6.
BankIt2307849 PunPgp-2: MT001899, BankIt2307849 PunPgp-3: MT001900, BankIt2307849 PunPgp-9: MT001901, BankIt2307849 PunPgp-10: MT001902, BankIt2307849 PunPgp-11.2: MT001904, BankIt2307849 PunPgp-12: MT001905, BankIt2307849 PunPgp-16.2: MT001907, BankIt2307849 PunPgp-18A: MT001908, BankIt2307849 PunPgp-18B: MT001909. The updated annotation (gff3) of P-glycoproteins has been shared with WormBase ParaSite and will be available on the next WormBase Parasite release.
Gerhard, Alexander P.; Krücken, Jürgen; Heitlinger, Emanuel; Janssen, I. Jana I.; Basiaga, Marta; Kornaś, Sławomir; Beier, Céline; Nielsen, Martin K.; Davis, Richard E.; Wang, Jianbin; and von Samson-Himmelstjerna, Georg, "The P-Glycoprotein Repertoire of the Equine Parasitic Nematode Parascaris univalens" (2020). Maxwell H. Gluck Equine Research Center Faculty Publications. 43.