Gut microbiome and virome response to spinal cord injury THESIS Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University By Jingjie Du Graduate Program in Microbiology The Ohio State University 2020 Thesis Committee: Matthew Sullivan, Advisor Phillip Popovich Virginia Rich Copyrighted by Jingjie Du 2020 Abstract The diversity and function of the human microbiome have been extensively studied over the last 10 years. Emerging evidence suggests that the gut microbiome plays an essential role in a wide range of human diseases, including acute, traumatic spinal cord injury (SCI). Previous work has characterized gut dysbiosis in SCI, correlating changes to clinical outcomes or other biomarkers. However, the study of gut virome in health and disease remain largely unknown, because viruses lack universal marker genes for taxonomic assignment. This work represents the first time the gut virome has been characterized in SCI and provides the first look at potential functional changes in the gut microbiome in SCI at the level of gene loss. As with our previous work, we present a unique multi-level SCI model, allowing us to compare partially preserved sympathetic enteric enervation (T10) with a total loss of sympathetic enteric enervation (T4). Our results reveal level-specific patterns of gut dysbiosis, which may inform microbe-based predictions of severity and locomotor outcome in SCI. Further, this research is the first time that the viral and functional components of the gut microbiome have been characterized in SCI areas, which represent possible therapeutic targets for improving outcomes in SCI. i Acknowledgments I would like to express my sincere gratitude to my advisor, Dr. Matthew Sullivan for his exceptional mentorship and professional support. I would also like to thank Dr. Phillip Popovich for his valuable support and guidance for this work. I would also like acknowledge my committee member Dr. Virginia Rich for her time invested in teaching me to be a good scientist. I also profoundly appreciate the personal and professional support from my collaborators, Kristina Kigerl and Ahmed Zayed, and all the Sullivan lab and Rich lab members. I really appreciate the help and support from the Microbiology graduate students and the faculty and staff at the Ohio State University’s Department of Microbiology. I greatly learned from being a teacher and scientist. More importantly, I really appreciate the unconditional love and support from my parents. Thanks for your sacrifice and support for giving me the best education and sending me to elite schools, mom and dad! I really learned a lot during the three years’ hard transition to graduate school in a different country. This work was funded by a National Institutes of Neurological Disorders and Stroke R35 award (1R35NS111582) to PGP, The Belford Center for Spinal Cord Injury (PGP), The Ray W Poppleton Research Designated endowment (PGP), a National Institutes of Health Medical Scientist Training Program T32 grant, and a Gordon and Betty Moore Foundation Investigator Award (#3790) to MBS. ii Vita 2013…………………………………………Xiangyang NO.5 Middle School, China 2013………………………………………… National College Entrance Examination (NCEE)， natural-science-oriented area ranked top 1% of test takers, Hubei, China 2013 to 2017………………………………. SYSU Scholarships of the Academic Year / National Endeavor Fellowship/ SYSU Encouragement Scholarship 2017………………………………………... B.S. Biotechnology, Sun Yat-sen University, Guangzhou, China 2017 to present …………………….………Graduate Teaching/Research Associate, Department of Microbiology, The Ohio State University Fields of Study Major Field: Microbiology iii Table of Contents Abstract ............................................................................................................................................ i Acknowledgments........................................................................................................................... ii Vita .............................................................................................................................................................. iii List of Figures .............................................................................................................................................. v Chapter 1: Introduction .......................................................................................................................... 1 Chapter 2: Spinal cord injury changes the structure and functional potential of gut bacterial and viral communities in a spinal-level dependent manner ........................................ 11 Abstract ................................................................................................................................................... 12 Background ............................................................................................................................................. 13 Results and Discussion ............................................................................................................................ 16 Conclusion ............................................................................................................................................... 28 Methods ................................................................................................................................................... 29 List of abbreviations ................................................................................................................................ 36 Declarations ............................................................................................................................................. 36 Major figures: .......................................................................................................................................... 38 Supplemental Figures .............................................................................................................................. 48 Chapter 3: Conclusions ......................................................................................................................... 61 References .................................................................................................................................................. 64 iv List of Figures Figure 1. Intestinal microbial community composition was disturbed after spinal cord injury. 38 Figure 2. Genus-level bacterial abundances are altered after SCI. .............................................. 40 Figure 3. Species-level bacterial abundances are altered after SCI. ............................................ 42 Figure 4. Predicted metabolic pathways are different between healthy and spinal cord injury animals. ......................................................................................................................................... 43 Figure 5. Phage communities are altered after SCI. .................................................................... 45 Figure 6. Viral host-prediction reveals that phage abundances vary with their hosts. ................ 47 Figure 7. Flow diagrams showing the bioinformatic workflow................................................... 48 Figure 8. Differential abundance analysis of bacteria across three treatment groups.................. 49 Figure 9. Species-level differential abundance analysis of bacteria across three treatment groups. ....................................................................................................................................................... 52 Figure 10. Predicted metabolic pathways are different between Lam controls and SCI groups. 53 Figure 11. Caudovirales phage abundances increased after spinal cord injury. .......................... 55 Figure 12. Prediction of temperate phages using different methods........................................... 57 Figure 13. Virus-host prediction. ................................................................................................. 59 Figure 14. Heat map showing the differential abundance analysis of phages grouped by infected bacterial hosts................................................................................................................................ 60 v Chapter 1: Introduction Over the last 10 years, the diversity and function of the human microbiome have been extensively studied thanks to advanced sequencing technologies and methods (16s rRNA gene sequencing, metagenomics, metatranscriptomics, and metaproteomics) and large-scale initiatives such as the Human Microbiome Project (HMP)[1], the MetaHIT (Metagenomics of the Human Intestinal Tract)[2] and the Integrative HMP(iHMP)[3]. In this chapter, we will focus on discussing the roles of gut microbes, especially bacteria and viruses, in human health and disease. Development of gut microbiota Humans are sterile at birth and infants obtain their first gut microbes from environmental microbiota during birth (as reviewed in[4]). These initial microbial sources include delivery modes (virginal birth vs cesarean birth), the first forms of sustenance (breast milk vs formula), probiotics and antibiotics use. They shape the structure of the initial microbiota[5-7], and affect infant health as well as neurocognitive development in the later life[8, 9]. The development of the normal gut microbiota may be delayed or altered by cesarean section, formula feeding, and antibiotic