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Anthelmintics such as the macrocyclic lactones (MLs) are of special importance for healthcare in animal husbandry. A loss of their efficacy might cause damages of health due to infections resulting in significant adverse economic effects. As the introduction of new antiparasitics cannot be expected in the medium-term, preservation of existing effective anthelmintics should be of special interest. This requires the knowledge of underlying resistance mechanisms in parasites. ML-resistance is supposed to be a result of the activity of Pgps which belong to the ATP-binding-cassette (ABC)-superfamily of transporters. Expressed in cell-membranes, Pgps mediate the ATP-dependent transport of molecules. Therefore, changes in morphology or expression might result in reduced availability of drugs at their target-site followed by a decreased drug efficacy.
The ML ivermectin (IVM) is an excellent Pgp-substrate which was demonstrated in mammalian cells. It is widely used in treatment of parasitic infections as e. g. of Parascaris equorum, affecting predominantly young equines. Adult stages of P. equorum are found in the small intestine where obstruction may lead to rupture of the intestinal wall and death. Recently, the worldwide occurrence of this nematode with its sometimes high prevalence was accompanied by cases of IVMresistance. Unfortunately, the molecular background of this process remains largely unclear and appropriate techniques for detection of ML-resistance in P. equorum are not standardised yet.
In this work, complete sequences of two putative P. equorum Pgps were generated using degenerated primers and a following RACE-PCR based on observations in other nematodes concerning the contribution of Pgps in ML-resistance. By phylogenetic analysis these were identified orthologs of Caenorhabditis elegans Pgp-11 (PeqPgp-11) and an unnamed Pgp of Caenorhabditis briggsae CBG12969 (PeqPgp-16). Amino acid sequences of both proteins contained Pgp-specific motifs which are known to be involved in drug- and ATP-binding.
Comparison of pgp-expression between tissues was conducted by quantitative real-time PCR.
Peqpgp-11 was found to be significantly higher expressed in the gut of both genders, in contrast to Peqpgp-16 which was predominantly expressed in the body wall.
In order to detect changes associated with IVM-resistance in P. equorum, Pgp-sequences of
populations with different ML-susceptibility were compared. SeqDoC-analysis revealed three single nucleotide polymorphisms (SNPs) which correlated with decreased ML-susceptibility and caused a substitution of the corresponding amino acid (Asp931Asn, Cys951Ser, Tyr978His) in PeqPgp-11. No resistance associated differences in mRNA expression levels were detected between embryonated eggs of the same populations.
Furthermore, pre-adult worms were included in investigations, where a group of worms with reduced IVM susceptibility showed statistically significant overexpression of Peqpgp-11 compared to a randomly selected group. Apart from these descriptive observations for P. equorum, the contribution of Pgps in detoxification was also investigated in the model nematode C. elegans, first, to analyse the consequences of general inhibition of Pgp-activity in a living organism and second, to assess the importance of single Pgps for the IVM-transport. In presence of the Pgp-inhibitor Verapamil, the EC50-value was decreased little but significantly by 2.5 fold compared to the C. elegans wildtype Bristol N2 strain, which was found in terms of a reduced development rate of C. elegans. However, loss-of-function strains missing a single Pgp were hallmarked by decreased EC50-values between the 1.5- and 4.3 fold. Apart from Pgp-14, loss of Pgp-11 activity showed the greatest impact on development. In summary, the results of the present work underline the hypothesis of a contribution of Pgps to IVM-resistance in nematodes. This becomes apparent in an increase of IVM-susceptibility in C. elegans strains either missing only a single Pgp or affected by the total loss of Pgpactivity, respectively, as well as in morphological and quantitative changes of the protein in dependence of the resistance level. In both cases, Pgp-11 was of particular importance, showing SNPs which are promising diagnostic candidates for detection of ML-resistance in P. equorum.
To evaluate their suitability, further populations with known phenotypic resistance levels have to be investigated. This will be facilitated by the establishment of a pyrosequencing assay for two of the three SNPs. Furthermore, heterologous expression in a model organism of the susceptible and the presumably resistant genotype of Pgp-11 might enlighten their role. This might be conducted in C. elegans, since a pgp-11 loss-of-function strain for rescue experiments is already characterised more closely regarding its IVM susceptibility.
For that reason, transgenic lines, containing PeqPgp-11 of the presumably resistant genotype and a control line were generated. First results of comparative analyses between these different lines produced encouraging results.