Exploring the impact of gastrointestinal parasitic helminths on the human microbiome using advanced biomolecular and bioinformatics technologies Timothy P. Jenkins Department of Veterinary Medicine University of Cambridge BBSRC-DTP This dissertation is submitted for the degree of Doctor of Philosophy (PhD) in Molecular Parasitology Homerton College June 2019 DECLARATION This dissertation is the result of my own work and includes nothing which is the outcome of work done in collaboration except as declared in the Preface and specified in the text. It is not substantially the same as any that I have submitted, or, is being concurrently submitted for a degree or diploma or other qualification at the University of Cambridge or any other University or similar institution except as declared in the Preface and specified in the text. I further state that no substantial part of my dissertation has already been submitted, or, is being concurrently submitted for any such degree, diploma or other qualification at the University of Cambridge or any other University or similar institution except as declared in the Preface and specified in the text It does not exceed the prescribed word limit for the relevant Degree Committee. ………………………………………. Timothy P. Jenkins May 2019 ii Exploring the impact of gastrointestinal parasitic helminths on the human microbiome using advanced biomolecular and bioinformatics technologies By Timothy P. Jenkins SUMMARY Our understanding of the biology of human gastrointestinal (GI) parasitic helminths is greater than ever before. However, so far, the research has focused on gene expression profiling, immune- and protein-protein interactions in host-parasite systems. The importance of parasite- microbiota interactions has, so far, been largely overlooked. The microbiome is key to host health and it has been demonstrated that the balance between the gut microbiota and the host is crucial for health maintenance and that a disturbance of this balance can result in a range of diseases. Hence, given that GI nematodes and the gut microbiota share the same ecological niche within the human host, it is plausible that GI helminths and the host microbiota interact, and that this could significantly impact on the health and homeostasis of the parasite-infected hosts. Fortunately, the availability and affordability of next generation sequencing now enables us to investigate such host-parasite-microbiota interactions in depth and at high throughput. Therefore, the aims of this thesis were to explore the impact of such helminth infections in various systems, ranging from natural multi-species infections in a developing country to highly controlled and experimental infections involving a single species of parasitic helminth. This would allow the identification of microbiome changes that are consistent across different settings, as well as help detect alterations that are specific to a certain host-helminth system. Thus, the main aims of the thesis were: (i) to investigate the consequences of natural multi- or mono-species infections by helminth parasites on the composition of the human gut microbiota (Chapters 2 and 3), (ii) to elucidate the longitudinal impact of experimentally controlled mono- species helminth infections on the human gut microbiota (Chapter 4), (iii) and to examine whether an extra-intestinal (EI) helminth infection has an impact on the host microbiome in a murine model of human schistosomiasis (Chapter 5). Overall, I found that GI and EI helminths have a substantial impact on the host gut iii microbiota, both on individual taxa and on a community level. Many of the observed changes appeared to be specific to the host-helminth system that was being investigated, yet some consistencies emerged. Firstly, low level, long term, and single species infections that were not accompanied by pathology appeared to increase the gut microbial diversity within their host and promote a stable and healthy gut microbial composition (Chapters 3 and 4). Contrarily, acute heavy burden infections, associated with pathology, appeared to have the opposite effect, i.e. reducing the overall diversity of the host’s gut microbiome and promoting the proliferation of opportunistic pathogens (Chapters 2 and 5). This suggests that parasitic helminth infections could detrimentally impact the hosts they infect besides the direct pathology they induce, but also adds further weight to the idea of a therapeutic use of helminths in the context of helminth therapy. Indeed, the beneficial effect helminths can have on the host gut microbiota, together with the mounting evidence towards an intrinsic link between autoimmune diseases and the microbiome, might present a mechanism through which helminths could exert a therapeutic effect on patients suffering from such conditions. In, conclusion the present thesis has contributed significantly by providing entirely new insights into the impact of natural and experimental parasitic helminth infections on the human gut microbiome (Chapter 6). The findings provide a sound basis for future fundamental investigations of, for example, the relationship of helminth species, abundance, and host to microbiome changes in the context of infection. However, the results also act as a stepping stone for studies exploring the translational potential of helminth-microbiota interactions, such as the role that helminth induced microbiome modulations play in infection pathology, or whether such changes play a key role in the therapeutic potential of helminth therapy. iv ACKNOWLEDGEMENTS "It is the long history of humankind that those who learned to collaborate and improvise most effectively have prevailed." – Charles Darwin Foremost, I would like to express my sincere gratitude to my advisor Dr Cinzia Cantacessi for her continuous support, patience, motivation, enthusiasm, and immense knowledge throughout my PhD. Her guidance helped me improve in countless ways and provided me with the key skills I require to become an independent researcher. I cannot imagine receiving comparable opportunities to improve and progress both on an academic and personal level elsewhere. For that I will be forever grateful. I would also like to take this opportunity to thank Dr Terrence Miller, since it was he, who first introduced me to the field of parasitology and the practicalities of research. His contagious enthusiasm and endless encouragement were pivotal in my decision to pursue a PhD. At such an early career stage, such confidence in my skills and abilities was the greatest motivation I could ask for. Many thanks to my colleagues Dr Laura Peachey, Dr Alba Cortes-Carbonell, Dr Riccardo Scotti, and Xiaopei Su for their helpful discussions, valuable advice, the sleepless nights we were working together before deadlines, and for all the fun we have had over the past four years. My deepest and sincerest thanks to my friends in Cambridge, all of whom, transformed my time here into arguably the best experience of my life. Especially, Chris Guillermo, Kanwar, Sohaib, and Thom, ensured fun was always at hand. Also, a big thank you to all of my friends that have stuck with me for even longer. Martin, Cosimo, Lotte, Kaja, and Trenton, you have always been there for me and helped keep me sane. I am also infinitely grateful to Tina and Uwe, who have kept me alive over the years, even on my most daring adventures. During my PhD programme I was fortunate to receive help and support from many well-regarded scientists from around the world. Sincerest thanks to Prof Rupika S. Rajakaruna, v Dr Piyumali K. Perera, and their colleagues (Department of Zoology, University of Peradeniya, Peradeniya, Sri Lanka) for collecting the faecal samples from the study populations of Sri Lankans naturally infected with mixed helminth infections, identifying the parasite content, and extracting the microbial DNA (Chapter 2). Thanks to Dr Zeno Bisoffi, Chiara Piubelli, Francesca Perandin, and Dora Buonfrate (Centre for Tropical Diseases, Sacro Cuore-Don Calabria Hospital, Negrar, Verona, Italy) for the sampling, parasite identification, microbial DNA, and metabolite extraction of elderly people infected with Strongyloides stercoralis (Chapter 3). I would also like to thank Prof Jules Griffin and Cecilia Castro (Department of Biochemistry, University of Cambridge, Cambridge, UK) for their help with the metabolite screening of the above mentioned samples (Chapter 3). Great thanks also to Prof Cris Constantinescu and Radu Tanasescu (Division of Clinical Neurology, School of Clinical Sciences, University of Nottingham, Queen's Medical Centre, Nottingham, UK) for conducting the helminth therapy trial and to Prof David Pritchard and Dr Gary Telford (Department of Pharmacy, University of Nottingham, Nottingham, UK) for collecting the faecal samples from the trial participants (Chapter 4). Finally, I want to thank Dr Gabriel Rinaldi (Wellcome Sanger Institute, Wellcome Genome Campus, UK), Prof Paul Brindley, Dr Michael H. Hsieh (Department of Microbiology, Immunology & Tropical Medicine, and Research Center for Neglected Diseases of Poverty, School of Medicine & Health Sciences, George Washington University, USA), and Dr Micahel Nadim J. Ajami (Department of Molecular Virology and Microbiology, Baylor College of Medicine, USA) for conducting the murine Schistosoma mansoni infections and providing the raw sequencing reads for this study (Chapter 5). Many thanks also to Prof Lutz Krause (The University of Queensland Diamantina Institute, Translational Research Institute, Australia)