A5 Identification and characterization of metabolic pathways leading to macrocyclic lactone and multi-drug resistance in Cooperia oncophora (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: Natalie Jakobs

Project Description

State of the art:
Anthelmintic resistance is widespread in parasitic nematodes of ruminants and recently also
suspected for human ascarids. While resistance against the benzimidazoles (BZs) is
considered to be due to target-site mutations in the β-tubulin isotype-1 gene, resistance
mechanisms against macrocyclic lactones (MLs) are only poorly understood. There are several
lines of evidence that P-glycoproteins (Pgps) and cytochrome P450 enzymes (Cyp) are
involved in resistance against MLs but also against BZs.

Previous own work:
In the first funding period, experiments with the model nematode Caenorhabditis elegans on
ML and BZ resistance revealed that: I. Nearly complete inactivity of Cyps in a temperaturesensitive
variant of Cyp reductase had statistically significant but only small effects on
ivermectin (IVM) and moxidectin (MOX) susceptibility suggesting that Cyps only play a minor
role in ML resistance. II. Although BZ-inducible expression and BZ metabolism have been
shown in C. elegans, no inducible expression was shown for in vitro cultivated fourth stage
larvae (L4) after exposure to thiabendazole.

Hypotheses and work plan:
1) Anthelmintic resistance is modulated by nematode xenobiotic metabolism.
2) In multi-drug resistant (MDR) pathways some of the resistance mechanisms are shared
between different anthelmintic drugs.
3) The MLs IVM and MOX share the same mode of action but only partially share resistance
mechanisms.
The project focus will shift to MDR and metabolism of xenobiotics in general. Recently, studies
identified several candidate enzymes metabolizing xenobiotics highly overexpressed in MDR
parasites, e.g. FAD-dependent monooxygenases, glutathione S- or UDP-glycosyl
transferases. We will obtain further candidates from the transcriptomic response of a MDR C.
oncophora isolate (CoNZres) to albendazole, IVM and a subtherapeutic dose of MOX.
Candidate enzymes will be overexpressed in C. elegans gut cells using mos-1 integration and
CRISPER/Cas-9. This allows integration into the same locus to achieve similar expression
levels to allow quantitative comparisons between individual genes. Members of different
enzyme families will be integrated into different loci allowing to create combinations of
transgenes by interbreeding. Aiming to identify the mechanisms that are required to become
MOX resistant, we will select the CoNZres isolate for MOX resistance using gradually
increased treatments with subtherapeutic doses of MOX. Evolution of MOX resistance will be
monitored using larval development assays with MOX and IVM in each passage. We will
sequence the entire genomes of the parental CoNZres and the selected CoNZresMOX isolates
to identify regions under selection by MOX. Transcriptome analyses of the isolates to MOX in
vivo will in addition help to understand the differences in physiology of an IVM and an
IVM+MOX resistant isolate on the same genetic background. 

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