Mining the Microbiome to Modulate Host Defense Against Clostridioides difficile Infections 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 Nathiana Smit aus Haarlem, Niederlande 1. Referent: Prof. Dr. Dieter Jahn 2. Referent: Prof. Dr. Till Strowig eingereicht am: 18-12-2019 mündliche Prüfung (Disputation) am: 01-09-2020 Druckjahr 2020 II Vorveröffentlichungen der Dissertation Teilergebnisse aus dieser Arbeit wurden mit Genehmigung der Fakultät für Lebenswissenschaften, vertreten durch den Mentor der Arbeit, in folgenden Beiträgen vorab veröffentlicht: Publikationen Lagkouvardos, I., Lesker, T.R., Hitch, T.C.A., Gálvez, E.J.C., Smit, N., Neuhaus, K., Wang, J., Bained, J.F., Abt, B., Stecher, B., Overmann, J., Strowig, T., Clavel, T.: Sequence and cultivation study of Muribaculaceae reveals novel species, host preference, and functional potential of this yet undescribed family. Microbiome 7: 28 (2019). doi:10.1186/s40168-019-0637-2 Ünal C.M., Berges, M., Smit, N., Schiene-Fischer, C., Priebe C., Strowig, T., Jahn, D., Steinert, M.: PrsA2 (CD630_35000) of Clostridioides difficile is an active parvulin-type PPIase and a virulence modulator. Frontiers of Microbiology 9: 2913 (2018). doi: 10.3389/fmicb.2018.02913 Tagungsbeiträge Smit, N., Lesker, TR., Kirstein, S., Neumann-Schaal, M., Stecher, B., Clavel, T., Strowig, T.: Exploring Oligo-MM12 mice to study precision microbiome reconstitution against Clostridium difficile infections. (Presentation). Vortragstagung der Deutschen Gesellschaft für Hygiene und Mikrobiologie, Bochum, Deutschland (2018). Posterbeiträge Smit, N., Lesker, TR., Kirstein, S., Neumann-Schaal, M., Stecher, B., Clavel, T., Strowig, T.: Exploring Oligo-MM mice to study precision microbiome reconstitution against Clostridioides difficile infections. (Poster) 12. Vortragstagung der Clostpath 11, Leiden, Netherlands (2019). III IV Acknowledgements The previous four years I have been working on my PhD research. However, I could not have completed it without the help and support of a lot of people, friends and family. In this section I would like to show my gratitude and appreciation to all of those who made it possible for me to finish this thesis. To start, I would like to thank my supervisor, Prof. Dr. Till Strowig, for giving me the opportunity to conduct my research and to learn and work in the exciting field of microbiology and microbiota research. I am grateful for his help and support throughout my PhD studies, and his guidance that made the completion of this thesis possible. Additionally I would like to thank Prof. Dr. Dieter Jahn for agreeing to be my mentor and both members of my thesis committee, Prof. Dr. Petra Dersch and Dr. Matthias Lochner, for their time, valuable insights, suggestions to continue my work and to improve my research project. I also want to thank Prof. Dr. Stefan Dübel for agreeing to chair my disputation. My work would not have been possible without the support of numerous colleagues and collaborations. Most importantly I would like to thank the staff of the animal facility and the genome analytics core facility of the HZI, and additionally the help and advice of Dr. Meina Neumann-Schaal and Dr. Kerstin Schmidt-Hohagen concerning bile acids. Another thanks goes to the colleagues within the CDIFF consortium. I also would like to acknowledge my past and current colleagues from the MIKI group, not only for support and help during the work hours, but also next to work, for all the fun moments and enjoyable events we shared during the past years. However, a special thank you goes to Till Robin Lesker, who helped me analyze a lot of different data. I also want to thank Sophie Thiemann, who was my former supervisor during my Master thesis and the reason I got interested in microbiota research in the first place and why I started my PhD here at the HZI. In the time I was living in Braunschweig, I had the pleasure to meet and become friends with numerous people. I have learned so much new things next to learning about the microbiota, infections and C. difficile. During these years, I learned how to do scuba diving, climbing, bouldering and salsa dancing. I specifically want to name Tanya, Lisa, Simon, Charlotte and Richard for making my life great! V I also don’t want to forget to thank the friends I still have in the Netherlands (and Belgium): Evelien, Hilde, Amy, Serena and Jayron. Thank you for not forgetting about me, you are the best! I want to thank my family for their love and support during my life, including the time I spend performing my PhD. A thank you to my parents: my mom, Remco and my dad, who always believed I could do it, even in times I did not believe it myself and Remco for helping and instructing me in fields I was unfamiliar with. I also want to thank my grandparents for always being there for me and for always supporting me. Another thank you goes to Yoran and Glenn, my two “little” brothers for being in my life. And a special thank you goes to Ronald and Pauline who were always willing to drive back and forth for me! Last but definitely not least: a very special thank you and a lot of appreciation and gratitude goes to Jan-Maarten, who listened to me, supported me, believed in me, encouraged me and was at my side during the hardest parts of this thesis. You gave me the strength to keep going during the difficult times. Thank you for being there for me and believing in me. VI ~To the stars who listened and to the dreams that were answered~ Sarah J. Maas VII VIII Summary The intestinal microbiota consists of numerous and diverse microbes forming a dynamic ecosystem in close association with the host. One of the many functions of the microbiota is the colonization resistance against intestinal pathogens. Alterations in the composition of the microbiota, for example caused by antibiotics, may cause the loss of colonization resistance, making the host vulnerable towards infections, e.g. with Clostridioides difficile. C. difficile is a spore-forming anaerobic bacterium which can cause severe diarrhea and life-threatening complications in infected individuals. In many developed countries, C. difficile infection is now a major cause of antibiotic-related nosocomial infections. The disease affects mostly elderly individuals. Antibiotics are the standard treatment, however, up to 30% of the patients are suffering from recurrent C. difficile infections. Transfer of fecal matter from a healthy individual to the patient (fecal transplant) is able to cure these infections. Recent studies have demonstrated that the conversion of primary to secondary bile acids by specific commensal bacteria is important for this colonization resistance, but further evidence suggested that the restoration of the secondary bile acid production may not be sufficient for complete protection against this pathogen. In the present study, the effect of the microbiota on C. difficile infection has been explored. Therefore, a C. difficile mouse model has been established initially by exploring the effect of different ages and microbiota compositions on the severity of C. difficile infections. Second, the effect of specific mutations in the genome of C. difficile on the ability to colonize mice and to produce spores has been examined. Third, the inhibitory effect of specifically isolated commensal bacteria of the microbiota of resistant mouse models on C. difficile infections has been explored in depth in order to create a consortium of bacteria that protects the host against C. difficile. Our findings confirm that the age of the mice, comparable as in humans, is important for the severity of the disease of mice. They also confirm that secondary bile acids are indeed important to protect the host against C. difficile infections. However, in our model, adding a secondary bile acid producer to the microbiota is not sufficient for complete protection of the mice, but that together with other commensal bacteria, secondary bile acid producing bacteria are able to provide complete resistance against C. difficile induced pathogenesis. IX Zusammenfassung Die intestinale Mikrobiota bezeichnet ein komplexes Ökosystem aus zahlreichen und diversen Mikroorganismen, welches in enger Verbindung mit dem Wirt steht. Eine Hauptfunktion der Mikrobiota stellt die Besiedelungsresistenz gegen intestinale Pathogene dar. Veränderungen in der Zusammensetzung, zum Beispiel durch den Einsatz von Antibiotika, kann zum Verlust der Besiedelungsresistenz führen und den Wirt anfällig für Infektionen, mit z.B. Clostridioides difficile machen. C. difficile ist ein sporenbildendes, anaerobes Bakterium, welches besonders in immunsupprimierten und älteren Individuen schwere Durchfälle, bis hin zu lebensbedrohlichen Komplikationen führen kann. In Industrieländern ist C. difficile der häufigste Erreger nosokomialer und Antibiotika- assoziierter Durchfallerkrankungen. Nach Standardtherapie mit Antibiotika leiden bis zu 30% der Patienten an wiederkehrenden C. difficile Infektionen. Eine wirkungsvolle Alternativbehandlung stellen Stuhltransfers dar, bei der Bakterien gesunder Person auf den Patienten übertragen werden. Studien haben belegt, dass die Umwandlung von primären zu sekundären Gallensäuren durch spezifische Bakterien ein wichtiger Faktor für die Besiedelungsresistenz gegen C. difficile darstellt, dieser allein aber nicht ausreichend ist, um einen kompletten Schutz