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The Role of Small RNA in Parasite-Host Communication During Trichinella Spiralis Infection Peter Taylor

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The Role of Small RNA in Parasite-Host Communication During Trichinella Spiralis Infection Peter Taylor The role of small RNA in parasite-host communication during Trichinella spiralis infection Peter Taylor Imperial College London Department of Life Sciences Under the Supervision of Murray Selkirk and Peter Sarkies Submitted in part fulfilment of the requirements for the degree of Doctor of Philosophy in the Department of Life Sciences in the Life Science Research programme 1 Abstract The parasitic nematode Trichinella spiralis offers a bizarre and compelling example of host manipulation. Occupying both an enteric and intracellular muscle cell niche, the worm appears to hijack the host’s innate cellular biological processes to bring about morphological shifts in the muscle cell which it inhabits, creating a more hospitable environment for the its own development. The molecular mechanisms underpinning this phenomenon are largely unknown. Recently there has been much interest in extracellular small RNAs secreted within extracellular vesicles, including in parasitic nematode infection. Here I investigate whether T. spiralis secretes small RNAs as part of its pathogenesis. I profiled small RNAs secreted by T. spiralis from both its adult (enteric) and muscle larval stages of the life cycle. My data demonstrated that T. spiralis secretes miRNA (Chapters 3 and 4). Intriguingly the majority of small RNAs secreted by muscle stage larvae are not encapsulated within extracellular vesicles, consistent with its intracellular niche and implicating a novel secretory mechanism. Small RNAs enriched within the secreted fraction include those with homology to host miRNAs known to play a crucial role in muscle development and disease, such as miR-31 (Chapter 4). To test the role of secreted miRNAs in infection I generated an inducible expression system in the muscle cell line C2C12 (Chapter 5). Induction of exogenous miRNA expression and subsequent sequencing suggests that T. spiralis miR-31 is able to target host mRNA, with genes targeted including genes linked to muscular dystrophic disease (Chapter 6). I then describe further work which should be performed to solidify and expand upon the findings of this thesis (Chapter 7). 2 Statement of Originality This statement is to declare that, to the best of my knowledge, this thesis is my own work, written entirely by me, Peter Taylor, unless otherwise referenced. All intellectual content was a product of my work, with assistance in its production duly acknowledged. Licensing The copyright of this thesis rests with the author. Unless otherwise indicated, its contents are licensed under a Creative Commons Attribution-Non-Commercial 4.0 International Licence (CC BY-NC). Under this licence, you may copy and redistribute the material in any medium or format. You may also create and distribute modified versions of the work. This is on the condition that: you credit the author and do not use it, or any derivative works, for a commercial purpose. When reusing or sharing this work, ensure you make the licence terms clear to others by naming the licence and linking to the licence text. Where a work has been adapted, you should indicate that the work has been changed and describe those changes. Please seek permission from the copyright holder for uses of this work that are not included in this licence or permitted under UK Copyright Law. 3 Acknowledgements The first people to thank are my wonderful supervisors, Murray Selkirk and Peter Sarkies. I am eternally grateful for the opportunity to learn from both of them and have been incredibly fortunate to study with two people with such extensive and complementary knowledge and skills. Both have been incredibly generous with their time and have been incredibly understanding and helpful throughout. I couldn’t have wished for better supervisors. I have had the good fortune to work with many brilliant scientists at all stages of their careers. Jana Hagen has been one of the most important people to my progress and has been equally generous with her help and sharing her knowledge. I genuinely couldn’t have even come close to finishing this project without her help and friendship throughout. I have also had extensive aid from Rita Berkachy, who also helped me find my way through this project and through my time at Imperial. Corinna Schnoeller was crucial to helping me bed in when I arrived at Imperial and was always incredibly helpful and charming. Tony Belicard was an enormous help with understanding how things worked in the Sarkies lab and showed me the ropes when it comes to RNA sequencing work. Similarly, Silvana Rosic helped my find my footing when arriving at the Sarkies lab and was always extremely kind. More recently, Subhanita Ghosh and Sheeba Singh have taken over the reins in the Sarkies lab and have been wonderful, both highly skilled scientists and genuinely lovely people. I have also had the opportunity to study alongside some wonderful PhD students who have been incredibly generous with their time and aid. I’d like to thank the incredibly talented Toni Belicard and Lisa Schneider for their help and friendship and imagine we’ll be seeing both their names for years to come in high profile journals. Particular thanks to Empar Baltazar-Perez and Adam Efrat for their excellent work on the miR-sensor system. I have also had the opportunity to work in collaboration with some incredible research groups who have all been essential to the progress of this project. First of all, I must thank the entire Southall group, with whom I have shared the lab for these past 4 years, for their kindness and assistance throughout that time. Thanks to Tony, Colin McClure, Gabriel Aughey, Alicia Estacio-Gomez, Amira Hassan and Emma Walmsley for their patience and friendship. I also must thank our more recent neighbours in Calvin Tiengwe’s group for their help and support, particularly Carla Gilabert-Carbajo for her friendship and support after long days (and sometime nights!) in the lab. Thanks to Katarina Artavanis-Tsakonas and her group for the help with the C2C12 system and for donating the cell lines and vectors for this project. Thanks also to Khuloud Al-Jamal and her group, in particular Mohammad Faruqu, for their help with the quantification of extracellular vesicles. Thanks to Bonnie Chaban for her help with electron microscopy. My thanks also to Cristina Lo Celso and her group for their guidance when designing the miRNA sensor system. Thanks to Jess Rowley for her help with flow cytometry. Thanks to Michalis Barkoulas and his group, particularly Michael Fasseas, Dimitris Katsanos, Sneha Koneru and Guled Osman for their help during my Masters project and for their friendship since. Michalis has also been brilliant as one of my progress supervisors, so thank you for that. Similarly, I must thank Stuart Haslam for his help as my other PRP supervisor. I must also thank the administrative staff at Imperial for their help and support. Particular thanks to the wonderful Kleoniki Gounaris for her time throughout the project and for her sardonic wit which always cuts through the nonsense. James Ferguson has also been of immense importance and his patience and support is greatly appreciated. Thanks also to the staff at CBS for their guidance and support. Thanks to the UKRI Biotechnology and Biological Sciences Research Council (formerly BBSRC) for their funding and support throughout the project and to Imperial College for providing me a place to study and all the support and training that has come with that. Outside of the lab I must thank my friends and family for all their support. Particular thanks to Pourya for putting up with me whilst living together and for all the stimulating conversations and encouragement. Thanks to Arash for his support and for putting me up at times along with the lovely Martina. Likewise, thanks to Eleonore and Frank and Simon and Georgina for giving me a place to stay whilst finishing up and fo all their support. Particular thanks to my parents for everything, the list is too long to include here. Final and most heartfelt thanks to Claire, who has put up with me though everything and without whom I could certainly not have made it this far. My eternal thanks and love to you. Thanks to anyone I have forgotten to include or overlooked, I’m sorry but this is already too long! 4 Table of Contents 5 Table of contents Abstract 2 Licensing 3 Acknowledgements 4 Table of Contents 5 Chapter 1 – Introduction 16 The Genus Trichinella 17 Transmission and Epidemiology 19 Trichinellosis 22 Life Cycle of Trichinella spiralis 23 Nurse Cell Formation 26 Secreted Agents 28 Small RNA 31 Small RNA in C. elegans 32 Small RNA in other nematodes 36 Micro RNA 37 Micro RNA Biogenesis 38 Micro RNA and Development 41 Micro RNA and Myogenesis 42 Extracellular Small RNA 46 Proposed Mechanisms of Secretion 49 Secreted miRNA in mammalian cell-to-cell signalling 52 6 Table of contents Small RNA and cross-species communication 53 Small RNA and Plant Parasites 53 Small RNA and the parasites of Animals 54 Nematodes and other Helminths 55 Outstanding Questions 58 Trichinella spiralis as a model for small RNA mediated 59 parasite-host communication Chapter 1 Figures Figure 1.1: Schematic of T. spiralis life cycle 25 Figure 1.2: Schematic demonstrating key points in nurse cell 27 complex formation Figure 1.3: Small RNA biogenesis and function 35 Figure 1.4: Schematic demonstrating miRNA biogenesis 40 Figure 1.5: Schematic of muscle repair and myogenesis 43 Figure 1.6: Possible mechanisms of secretion of small RNA 50 Chapter 2 – Materials and Methods 61 Recovery of Muscle Stage Larvae 62 Recovery of Adult Worms 62 Parasite culture and collection of secreted products 63 Ultracentrifugation of Secreted Material 63 RNase Digest 64 7 Table of contents RNase Protection Assay 64 Extracellular Vesicle collection and quantification 65 RNA Isolation and sequencing 65 Scatterplots 66 Seed matching and Similarity scoring 66 Heatmaps 67 Cell Culture 68 Production of Tet-inducible C2C12 cell line in Brief 68 Full Protocols for Cell Line Production 69 Production of chemically competent E.
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