Modulation of intestinal homeostasis and inflammation by Prevotella intestinalis (nov. sp.) Von der Fakultät für Lebenswissenschaften der Technischen Universität Carolo-Wilhelmina zu Braunschweig zur Erlangung des Grades einer Doktorin der Naturwissenschaften (Dr. rer. nat.) genehmigte D i s s e r t a t i o n von Aida Iljazovic aus Rijeka, Kroatien 1. Referentin: Prof. Dr. Petra Dersch 2. Referent: Prof. Dr. Stefan Dübel eingereicht am: 25.07.2018 mündliche Prüfung (Disputation) am: 26.10.2018 Druckjahr 2019 Vorveröffentlichungen der Dissertation Teilergebnisse aus dieser Arbeit wurden mit Genehmigung der Fakultät für Lebenswissenschaften, vertreten durch die Mentorin der Arbeit, in folgenden Beiträgen vorab veröffentlicht: Tagungsbeiträge Aida Iljazovic, Eric J.C. Galvez, Till-Robin Lesker, Sophie Thiemann and Till Strowig: Microbial Interactions of Prevotella spp. within the Intestinal Ecosystem. Presentation. 9th Seeon Conference on „Microbiota, Probiota and Host“, 24-26 June 2016, Kloster Seeon, Germany. Aida Iljazovic, Eric J.C. Galvez, Till-Robin Lesker, Sophie Thiemann and Till Strowig: Microbial Interactions of Prevotella spp. within the Intestinal Ecosystem. Poster. Symposium Microbiota and Mucosal Immunity: Rules of Engagement in Health and Disease, 28-30 July 2016. Toronto, Canada. Per Marco, senza il cui amore e supporto la fine di questa dissertazione sarebbe stata impensabile. Acknowledgments Doing a PhD research often appears a solitary undertaking. However, it is unthinkable to maintain the degree of dedication and focus necessary for its completion without the help and support of many people. I take this opportunity to extend my sincere gratitude and appreciation to all those who made this PhD thesis possible. Foremost I would like to thank my supervisor, Dr. Till Strowig, who decided to believe in me and offered me an opportunity to learn and work in this exciting field of research. I am extremely grateful for having a supervisor who shared with me the same motivation and enthusiasm for my work, and who was always available to offer his help, advice, end encouragement. Without his continuous support and patient guidance throughout my time as a PhD student, completion of this thesis would be unimaginable. I would like to thank the members of my thesis committee, Prof. Dr. Ingo Schmitz and Prof. Dr. Marc Erhardt for their time and insightful suggestions to improve my project. I am also thankful to Prof. Dr. Petra Dersch and Prof. Dr. Stefan Dübel for agreeing to be on my examination committee, as well as Prof. Dr. Anett Schallmey for chairing my disputation. Performing this work was possible only due to the support of numerous people. I would like to thank the staff of the animal unit and the Genome analytics core facility at the Helmholtz Institute for Infection Research, Dr. Marina Pils, Dr. Meina Neumann-Schaal, Dr. Kerstin Schmidt-Hohagen, and Dr. Sabine Gronow for their help, advice and technical support. I am thankful to DFG, German Research Foundation, for providing the funding, which allowed me to undertake this research, and to the HZI Graduate School for the financial support and for the several career development opportunities. Completing this work would have been all the more difficult without the support provided by many past and current members of the MIKI group. Special thank you goes to Urmi and Achim for willingness to always give a helping hand in the lab and Eric for joining forces on several aspects of this project. I am indebted to them for their help. Besides good advice and collaborations, MIKI group has been a source of friendships. I am especially grateful to Adrian, Urmi, Eric and Sophie for all the enjoyable and memorable moments we shared together. Thank you for sharing all the ups and downs during my PhD. Because of you, I only remember the good times. My gratitude goes to all my friends that I had the pleasure to meet in Braunschweig. They provided a much needed form of escape from my work and helped me keep things in perspective. The impact they had on my life is invaluable. iv A special thank you goes to Prof. Dr. Marina Šantić whose enthusiasm and love for science was contagious and has inspired me to do research. I am extremely grateful for the positive and lasting impact she had on my life. I can only hope one day to pay it forward. I am grateful to my family for their love and support for every choice I made. Especially, I am grateful to my brother Amir who unknowingly always challenged me to be better and work harder, and for being a role model in my life. Finally, I express my deepest appreciation and gratitude to Marco, who has been by my side throughout this PhD, celebrating every little achievement as his own as well as giving me the strength and encouragement during the difficult times. Thank you for your continuous support, patience and understanding and for being my best friend as well as my family away from home. v Summary Intestinal homeostasis is maintained by the dynamic interplay between the gut microbiota and the host immune system. Alterations in the composition and function of the gut microbiota have been associated with a wide range of human disease including inflammatory bowel disease and rheumatoid arthritis. Diverse microbial signatures within the intestinal microbiota have been associated with increased susceptibility to intestinal inflammation, but whether these candidate microbes actively modulate host phenotypes is frequently not known. Metabolites produced through microbiota activity have recently been linked as mediators to distinct intestinal and systemically immune-related disorders. The role of members of the Prevotella genus within the intestinal microbiota and their effects on the host is not completely understood and somewhat conflicting interpretations have been reported. Whereas association with plant-rich diet and improved glucose metabolism suggested Prevotella spp. are beneficial for the host, their increased relative abundances in microbial ecosystems at multiple body sites have been associated with diverse diseases. Yet, whether Prevotella spp. actively contribute to the development of these diseases is not known. The detailed investigation of the immunomodulatory properties of Prevotella spp. has been prohibited by the poor characterization of Prevotella species and high strain diversity, as well as the lacking availability of diverse intestinal Prevotella isolates. In the present work, we isolated three novel intestinal Prevotella species from mice prone to intestinal inflammation. Characterization of the impact of Prevotella colonization on the interplay between host and the microbiota during intestinal homeostasis and inflammation was performed using P. intestinalis, which among the three novel species shared the highest similarity to the predominant human gut Prevotella species - P. copri. We identified that colonization by this novel member of the Prevotella genus significantly decreased the production of the bacterial fermentation product SCFAs and the immunomodulatory cytokine IL-18, which was associated with an increase in the severity of intestinal inflammation. Our findings suggested that Prevotella-mediated intestinal injury may be influenced via different pathways, yet the ability to ameliorate Prevotella-induced disease severity by supplementation of IL-18 suggested that remodeling of the microbial metabolome and specifically SCFA production may be the dominating pathomechanism. Finally, the consequences of modulation of SCFA production in the intestine by Prevotella spp. may have far-reaching consequences for the host, as SCFA have immunomodulatory effects in distant sites such as the liver, bones or the brain. vi Zusammenfassung Die intestinale Homöostase wird durch dynamische Wechselwirkungen der Darmmikrobiota und dem Immunsystem aufrecherhalten. Verämderungen bei der Zusammensetzung und Funktion der Mikrobiota wurden mit diversen Krankheitsbildern im Menschen assoziiert, wie entzündliche Darmerkrankungen und rheumatoider Arthritis. Auch wenn diverse mikrobielle Signaturen mit einer erhöhten Anfälligkeit für Darmentzündungen assoziiert werden konnten, bleiben die ursächlichen Mikroben, welche die auftretenden Phenotypen aktiv modulieren, unbekannt. Kürzlich konnten von der Mikrobiota produzierte Metabolite als Mediatoren für Darmentzündungen und systemische Immunerkrankungen identifiziert werden. Die Rolle von Bakterien aus dem Prevotella Genus und ihre Effekte auf den Wirt im Kontext von Darmentzündungen führte zu gegensätzliche Interpretationen. Zum einen wurde eine erhöhte Anzahl an Prevotella spp. mit einer pflanzenreichen Ernährung und einem verbesserten Glucose-Metabolismus assoziiert, zum anderen wurde Prevotella spp. in mikrobiellen Gemeinschaften mit diversen Krankheiten in Verbindung gebracht. Ob Prevotella spp. aktiv bei der Entstehung dieser Krankheiten beiträgt ist nicht bekannt. Eine detaillierte Untersuchung der immunmodulatorischen Eigenschaften von Prevotella spp. wurde bisher durch mangelnde Charakterisierungen der Prevotella-Spezies, die große Vielfalt der bekannten Stränge und die fehlende Verfügbarkeit verschiedener Prevotella-Isolate des Darmes erschwert. In dieser Arbeit wurden drei neue intestinale Prevotella Spezies aus Mäusen isoliert, die anfällig für intestinale Entzündungen sind. Die Rolle von Prevotella beim Wechselspiel zwischen Wirt und der Mikrobiota bei intestinaler Homöostase und Entzündung wurde mit dem Isolat P.intestinalis untersucht, welches von den neu isolierten Spezies