KINOMES OF SELECTED PARASITIC HELMINTHS - FUNDAMENTAL AND APPLIED IMPLICATIONS Andreas Julius Stroehlein BSc (Bingen am Rhein, Germany) MSc (Berlin, Germany) ORCID ID 0000-0001-9432-9816 Submitted in fulfilment of the requirements of the degree of Doctor of Philosophy July 2017 Melbourne Veterinary School, Faculty of Veterinary and Agricultural Sciences, The University of Melbourne Produced on archival quality paper ii SUMMARY ________________________________________________________________ Worms (helminths) are a large, paraphyletic group of organisms including free-living and parasitic representatives. Among the latter, many species of roundworms (phylum Nematoda) and flatworms (phylum Platyhelminthes) are of major socioeconomic importance worldwide, causing debilitating diseases in humans and livestock. Recent advances in molecular technologies have allowed for the analysis of genomic and transcriptomic data for a range of helminth species. In this context, studying molecular signalling pathways in these species is of particular interest and should help to gain a deeper understanding of the evolution and fundamental biology of parasitism among these species. To this end, the objective of the present thesis was to characterise and curate the protein kinase complements (kinomes) of parasitic worms based on available transcriptomic data and draft genome sequences using a bioinformatic workflow in order to increase our understanding of how kinase signalling regulates fundamental biology and also to gain new insights into the evolution of protein kinases in parasitic worms. In addition, this work also aimed to investigate protein kinases with regard to their potential as useful targets for the development of novel anthelmintic small-molecule agents. This thesis consists of a literature review, four chapters describing original research findings and a general discussion. A detailed assessment of the literature (Chapter 1) revealed that, despite a recent increase in the availability of transcriptomic and genomic data sets for parasitic worms, very little is known about the protein kinases encoded in these genomes and their associated functions. In addition, the bioinformatic tools currently used for kinase identification and classification do not permit the accurate characterisation of kinase complements of parasitic worms as they do not take into account the draft state of genomic data sets and the substantial diversity in kinases of species that are only distantly related to well-curated model organisms. Therefore, the aims of this thesis were: (i) to comprehensively identifiy, classify, curate and functionally annotate the full complements of protein kinases in the genomes of parasitic worms, by (ii) establishing an advanced bioinformatic workflow system to carry out this task; (iii) to explore fundamental aspects of kinase signalling in worms based on developmental transcriptomes and cross-species comparisons; and (iv), from an applied perspective, to identify protein kinases with potential as anthelmintic targets. An integrated bioinformatic workflow relying on a pairwise-comparative approach was established and used to define the complete kinomes of the blood flukes Schistosoma haematobium and S. mansoni (phylum Platyhelminthes; class Trematoda; Chapter 2). For such flatworms, scant kinome data and vast phylogenetic distance from any well-curated model organism represented a challenge for kinase identification and classification. Therefore, trematode-specific stochastic models were inferred to identify and classify kinases prior to pairwise curation, which proved superior to the generalised models used in kinase identification tools available at the time. The transcription profiles of the curated kinase genes were then investigated and, employing this and other functional information, kinases were assessed in detail for their potential as drug targets. Subsequently, using an in silico approach, small-molecule effectors were inferred. Next, using the well-curated kinome of the best-characterised metazoan organism, Caenorhabditis elegans (a free-living nematode) as a reference, the complete kinome of Haemonchus contortus, a parasitic nematode of ruminants, was defined (Chapter 3). Based on this curated data set, the transcriptional regulation of kinase genes across parasite development was investigated, and these data were then integrated into an improved, ranking-based drug target prediction pipeline. This study showed that, using the kinome of the free-living nematode as the reference, the curation of the H. contortus kinome was readily iii possible. However, this was not the case for species that were distantly related to C. elegans, such that a distinct approach had to be taken. Therefore, for the identification, characterisation and curation of kinase sequences of distant taxa, such as those in in the class Enoplea, pairwise analyses were undertaken between closely related species within each of the genera Trichinella and Trichuris (Chapters 4 and 5). Kinomes of four species of enoplean, with unique biology and evolution, were investigated and compared. These analyses showed that enopleans have remarkably compact kinomes compared with other worms, and complemented with advanced three-dimensional modelling, revealed a novel enoplean-specific protein kinase. In conclusion, the present thesis has contributed significantly to gaining a deep understanding of the protein kinomes in socioeconomically important parasitic worms (Nematoda and Trematoda), and has provided a bioinformatic framework for the exploration of kinomes of the plethora of parasitic worms (Chapter 6). Although established for worm kinomes, the workflow system developed will have broad applicability to almost any group of eukaryotic organisms. Importantly, the findings presented in this thesis provide a practical resource for future functional investigations of signalling pathways in parasitic worms, which has considerable fundamental implications for studying worm biology, physiology and evolution. Given that protein kinases are recognised as attractive targets for small molecule drugs, the results should also have significant applied implications for future anti-parasitic drug discovery, repurposing and development. ________________________________________________________________ iv DECLARATION ________________________________________________________________ The work described in the thesis was performed in the Faculty of Veterinary and Agricultural Sciences of the University of Melbourne between April 2014 and July 2017. The scientific work was performed solely by the author with the exception of the assistance which has been specifically acknowledged. The thesis is less than 100,000 words in length, exclusive of tables, figures, references and appendices. No part of this thesis has been submitted for any other degree or diploma. ............................................................... Andreas Julius Stroehlein July 2017 v ACKNOWLEDGEMENTS ________________________________________________________________ I would like to express my deepest gratitude to my supervisor Robin Gasser for the opportunity to do my PhD in his group. I am very thankful for his continuous encouragement and support and for providing me with so many opportunities to become a ‘real’ parasitologist from the day I arrived in Melbourne and throughout my entire PhD. I have learned so much from him regarding parasites, research, teaching and life. I hope in the future I will be able to pass on some of his wisdom to others and become the teacher and mentor he has been for me. I am indebted to my PhD co-supervisor Neil Young. He is a great scientist and has mentored and supported me every day during my PhD candidature. His invaluable input and continuous support have helped shape my project and have allowed me to thrive as a scientist. His suggestions and our fruitful discussions have made me a better critical thinker and researcher. Thank you so much, Neil. I am looking forward to working with you in the future. I would like to thank Alexander Maier for running “Concepts in Parasitology” (CiP) organised by the Australian Society for Parasitology and everyone else who contributed to the success of this course. I was fortunate enough to participate in CiP in December 2015 and this experience has had a tremendous impact on me and my professional career as a parasitologist and scientist. I have formed many ties with great scientists in the Society and have built lasting friendships with many Australian and international researchers that are equally as passionate about parasites as I am. I would like to thank all scientists that have contributed to my work or supported me otherwise during my PhD candidature: Aaron Jex (Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia), Paul Sternberg (California Institute of Technology, Pasadena, CA, USA), Patrick Tan (Genome Institute of Singapore, Duke-NUS Graduate Medical School, Republic of Singapore), Peter Boag (Monash University, Melbourne, Australia), Andreas Hofmann (Griffith University, Brisbane, Australia), Pasi Korhonen and Abdul Jabbar (University of Melbourne, Australia), Bill Chang (Yourgene Biosciences, Taiwan), Giuseppe La Rosa and Edoardo Pozio (Istituto Superiore di Sanità, Rome, Italy) and Peter Nejsum (Aarhus University, Denmark). I would like to especially thank my friends in Melbourne that have become so important in my life during the last three years: Luke, Nina, Glenn, Julian, thank you for making Melbourne