Metabolism of macrocyclic lactones and benzimidazoles in parasitic nematodes Inaugural-Dissertation to obtain the academic degree Doctor rerum naturalium (Dr. rer. nat) submitted to the Department of Biology, Chemistry and Pharmacy of Freie Universität Berlin by ESRA YILMAZ from Berlin 2019 This work was prepared from April 2015 to April 2019 under the supervision of PD Dr. Jürgen Krücken at the Institute for Parasitology and Tropical Veterinary Medicine of Freie Universität Berlin, Germany. 1st Reviewer: PD Dr. Jürgen Krücken Institute for Parasitology and Tropical Veterinary Medicine Department of Veterinary Medicine Freie Universität Berlin Germany 2nd Reviewer: Prof. Dr. Rupert Mutzel Institute of Biology Department of Biology, Chemistry and Pharmacy Freie Universität Berlin Germany Date of defense: 13th November 2019 Acknowledgements At this point, I would like to take the opportunity and reflect on the people who have supported and helped me in realizing my doctoral research. First and foremost, I would like to show my deepest gratitude to Dr. Jürgen Krücken, my research supervisor, for his never-ending technical support and always having an open ear for any concerns or questions that I had. I am thankful for his contribution to my personal growth and intellectual maturation; he has been a great and supportive teacher. I would also like to thank Prof. Georg von Samson-Himmelstjerna for his continuous support, Prof. Janina Demeler for her supervision during the first one and a half years of the project, Dr. Ralph Menzel from the Humboldt University for being my mentor and his valuable contribution to my experiments with C. elegans, and Prof. Rupert Mutzel for agreeing to review my thesis. My thanks are extended to all my current and former colleagues. In particular, I am very thankful to Sabrina Ramünke who introduced me to and helped me with experiments related to H. contortus and Dr. Bastian Gerst who taught me the basics of C. elegans. I would like to acknowledge the Research Training Group GRK 2046 for the awarding of my scholarship and for funding this project. Finally, I am very grateful to my entire family, all of whom have constantly supported me morally throughout these years. Table of contents 1 Introduction ....................................................................... 12 2 Literature review ............................................................... 14 2.1 The phylum Nematoda ............................................................ 14 2.2 Haemonchus contortus ............................................................ 15 2.2.1 Taxonomy and phylogenetic context ............................................... 15 2.2.2 General ............................................................................................. 16 2.2.3 Haemonchosis .................................................................................. 17 2.2.3.1 Pathology and clinical signs ............................................................................. 17 2.2.3.2 Identification of Haemonchus contortus and diagnosis of haemonchosis .... 18 2.2.3.3 Host immunity and other host-related determinants of disease outbreak ... 19 2.2.3.4 Climatic determinants of disease outbreak .................................................... 20 2.2.4 Control strategies ............................................................................. 21 2.2.4.1 Anthelmintic control ........................................................................................ 21 2.2.4.2 Vaccines ............................................................................................................ 22 2.2.4.3 Alternative control strategies .......................................................................... 23 2.3 Caenorhabditis elegans ........................................................... 24 2.3.1 Taxonomy and phylogenetic context ............................................... 24 2.3.2 General ............................................................................................. 25 2.3.3 Caenorhabditis elegans as a model organism in parasitology ......... 26 2.4 Anthelmintics used against nematodes .................................. 28 2.4.1 Benzimidazoles ................................................................................. 29 2.4.1.1 Discovery and anthelmintic activity ................................................................ 29 2.4.1.2 Pharmacology ................................................................................................... 29 2.4.2 Macrocyclic lactones ........................................................................ 30 2.4.2.1 Discovery and anthelmintic activity ................................................................ 30 2.4.2.2 Pharmacology ................................................................................................... 31 2.4.3 Other anthelmintics with activity against nematodes ..................... 32 2.4.3.1 Piperazine ......................................................................................................... 32 2.4.3.2 Emodepside (Cyclooctadepsipeptide) ............................................................. 33 2.4.3.3 Levamisole ........................................................................................................ 33 2.4.3.4 Monepantel (Aminoacetonitrile derivative) ................................................... 34 2.4.3.5 Tribendimidine (Symmetric diamidine) ........................................................... 34 2.4.3.6 Derquantel (Spiroindole) .................................................................................. 35 2.5 Anthelmintic resistance ........................................................... 35 2.5.1 Prevalence of macrocyclic lactone and benzimidazole resistance in Haemonchus contortus .......................................................................... 36 2.5.2 Resistance-enabling factors and management of anthelmintic resistance in sheep .................................................................................... 37 2.5.3 Proposed mechanisms for macrocyclic lactone and benzimidazole resistance .......................................................................... 39 2.5.3.1 Target-site related mechanisms ...................................................................... 39 2.5.3.2 Non-target-site related mechanisms ............................................................... 41 2.5.4 Diagnosis of benzimidazole and macrocyclic lactone resistance ..... 42 2.5.4.1 Coprological methods....................................................................................... 42 2.5.4.2. In vitro methods .............................................................................................. 43 2.5.4.3. Molecular methods ......................................................................................... 44 2.6 Cytochrome P450 monooxygenases ........................................ 45 2.6.1 Structure, function and enzyme mechanism .................................... 45 2.6.2 CYP-mediated drug resistance.......................................................... 47 2.6.2.1 CYP-mediated drug resistance in humans ....................................................... 47 2.6.2.2 CYP-mediated drug resistance in arthropods .................................................. 48 2.6.2.3 Drug resistance by CYPs in nematodes ............................................................ 48 3. Publications ...................................................................... 50 3.1 Comparison of constitutive and thiabendazole-induced expression of five cytochrome P450 genes in fourth-stage larvae of Haemonchus contortus isolates with different drug susceptibility identifies one gene with high constitutive expression in a multi-resistant isolate .......................................... 50 3.1.1 Abstract ............................................................................................ 51 3.1.2 Introduction ...................................................................................... 51 3.1.3 Materials and Methods .................................................................... 54 3.1.4 Results .............................................................................................. 59 3.1.5 Discussion ......................................................................................... 62 3.1.6 Conflict of interest ............................................................................ 66 3.1.7 Financial Support .............................................................................. 66 3.1.8 Appendix A. Supplementary data .................................................... 67 3.1.9 References ........................................................................................ 67 3.2 Orthologs of anthelmintic resistance-associated cytochrome P450 13A genes of Teladorsagia circumcincta are not thiabendazole-inducible in cultured fourth-stage larvae of Haemonchus contortus .................................................................. 72 3.2.1 Abstract ............................................................................................ 73 3.2.2 Background ....................................................................................... 73 3.2.3 Material and Methods ...................................................................... 74 3.2.4 Results .............................................................................................. 78 3.2.5 Discussion