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A5 Metabolism of macrocyclic lactones in helminths (Demeler/Krücken)

Research Group: Institute for Parasitology and Tropical Veterinary Medicine
Address: Freie Universität Berlin, Centre for Infection Medicine, Robert-von-Ostertag-Str. 7-13, 14163 Berlin
Supervisors: PD Dr. Jürgen Krücken
Doctoral Researcher: Esra Yilmaz

Project Description   

Anthelmintic resistance in gastrointestinal nematodes (GIN), particularly to macrocyclic lactones (ML), poses a threat to livestock productivity and animal welfare. ML resistance is often seen in GINs of sheep and increasing in GINs of cattle. The mode of action of MLs is mainly resolved, involving irreversible opening of glutamate gated chloride channels (GluCls). In contrast, the mechanisms of ML resistance are still not fully understood. Non-specific resistance mechanisms, including P-glycoproteins (Pgp) and/or detoxifying enzymes, are considered to play an important role in resistance development. Nematodes possess a large group of cytochrome P450 (CYP) monooxygenases (80 CYPs have been found in Caenorhabditis elegans) which are inducible by xenobiotics. Moxidectin metabolism by CYPs has been shown and ML-resistant Haemonchus contortus showed higher CYP activity than a susceptible isolate. Despite the detailed knowledge regarding C. elegans CYPs of parasitic nematodes have not been characterised on the molecular level yet. In contrast to the model nematode C. elegans, well-characterised nematode isolates of GIN of ruminants are still not easily available. For this project, two ML resistant Cooperia oncophora and one Ostertagia ostertagi isolates were previously purified and characterised using different in vitro assays and inhibitors against Pgps and CYPs.

The aim of the project is to identify specific CYPs involved in ML resistance in GIN. Resistance might be caused by up-regulation of particular CYPs or alleles with increased ML affinity. Since it still remains unknown, which CYP might be involved in ML resistance, C. elegans will serve as the starting organism to identify candidate genes. The PhD student will screen for CYPs able to metabolise MLs using available loss of function C. elegans strains or knock-down of CYPs by RNAi. Orthologous of these CYPs will be identified and characterised in GINs (focus on C. oncophora, O. ostertagi and H. contortus) with different resistance status. Sequences (Sanger- and pyrosequencing) and expression levels (qRT-PCR) of CYP transcripts will be compared between susceptible and ML-resistant isolates. Furthermore, introduction of alleles associated with susceptibility/resistance into experimental systems for functional analyses is envisaged by recombinant expression of CYPs from GIN in Escherichia coli or Pichia pastoris. Functional analysis of recombinant CYPs concerning their role in ML-degradation will employ quantitative characterisation of metabolites by mass spectrometry. Different MLs, CYP substrates and inhibitors will be used to characterise the pharmacological properties of CYPs. Rescue of the phenotype of a CYP-deficient C. elegans strain is planned by transformation of constructs expressing parasite CYP. Protective effects of parasite CYPs against MLs will be investigated using established development/motility assays.

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