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Elucidating Potential Virulence Factors in Fusobacterium Nucleatum by Kyla L.S Cochrane

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Elucidating Potential Virulence Factors in Fusobacterium Nucleatum by Kyla L.S Cochrane Elucidating potential virulence factors in Fusobacterium nucleatum by Kyla L.S Cochrane A Thesis presented to The University of Guelph In partial fulfilment of requirements for the degree of Doctor of Philosophy in Molecular and Cellular Biology Guelph, Ontario, Canada © Kyla L.S. Cochrane, February, 2016 ABSTRACT ELUCIDATING POTENTIAL VIRULENCE FACTORS IN FUSOBACTERIUM NUCLTEAUM Kyla L.S. Cochrane Advisor: University of Guelph, 2016 Dr. Emma Allen-Vercoe Fusobacterium nucleatum (Fn) is a strictly anaerobic, Gram negative bacterial species that has been implicated in multiple clinical pathologies including periodontal disease, inflammatory bowel disease and colorectal cancer (CRC). The species is unusual in its phenotypic and genotypic heterogeneity, with some strains demonstrating a more virulent phenotype than others; however, the genetic basis for these differences and the association of any particular subspecies of Fn with disease has not yet been proven. With the recent confirmation of the Fusobacterium sequence enrichment in colorectal carcinomas, the characterization of cultured Fn isolates from CRC biopsies was warranted. Fn strains were isolated from human intestinal mucosal biopsies and were phenotypically and genetically analyzed. The invasive, inflammatory potential of Fn in vitro was also assessed. There was extensive variability between the phenotypic, invasive and inflammatory potentials of 8 different CRC-derived Fn strains. Further genetic characterization showed the prevalence of a class of genes of unknown function containing MORN2-domains that were expanded in Fusobacterium spp. known to be highly invasive. Proteins containing MORN2-domains are predicted to be surface-associated and as such may play a role in enabling host cell adherence and invasion by fusobacterial species. In addition, the presence of bacteriophages in Fn strains ii were also predicted to explain the genomic heterogeneity between Fn strains especially in relation to adherence and invasion. These predictions prompted the exploration of the Fn transcriptome for the purpose of determining the proteins that govern adherence and subsequent invasion of the bacteria into host cells. Going forward, RNA-seq will be used as a tool to help unravel the mechanisms of Fusobacterium pathogenesis. Continuing research in this area will enable the development of diagnostic and therapeutic strategies for the detection and treatment of Fusobacterium-associated diseases. iii Acknowledgments Firstly, I would like to express my sincere gratitude to my advisor Dr. Emma Allen- Vercoe for not only convincing me to return to the research world but also for her continuous support, patience, motivation and immense knowledge. Most of all, I want to thank her for having the faith in me that was necessary to handle this particular project. It was not always easy but her guidance and unwavering support allowed to me to succeed. Besides my advisor, I would like to thank the rest of my advisory committee: Dr. Nina Jones, Dr. Lucy Mutharia and Dr. Katrina Gee for their insightful comments, encouragement and also for their hard questions, which incented me to widen my research from various perspectives. Additional thanks go to Dr. Abigail Mason McGuire, Dr. Ashlee Earl, Dr. Reggie Lo and Dr. Robert Holt, without their bacterial, genomic and technical expertise I would have been lost. I would also like to acknowledge the government of Ontario for providing financial support throughout my graduate career in the form of scholarships, fellowships and bursaries. Additionally, I would like to recognize the Crohn's and Colitis Canada Foundation and the Canadian Cancer Society Research Institute for providing funding to the Allen-Vercoe lab. Special thanks go out to the members of the Allen-Vercoe lab, past and present, for all of your help in the lab, coffee breaks to vent and shoulders to cry or laugh hysterically on whenever it was a bad science day. In particular I would like to thank the friends who became family: you know who you are. Completing this work would have been all the more difficult were it not for the support provided by my wonderful family, my closest friends, and of course, my best friend and partner, Michael Beswick. It is always impossible to personally thank everyone who has facilitated successful completion of a project. To those of you who I did not specifically name, I give my thanks for moving me towards my goal. I will forever be indebted to you all. iv Author’s Declaration of Works Performed I declare that this dissertation is my own account, based upon work actually carried out by me and that all sources of material, not resulting from my own investigation, including observational information, have been clearly indicated in their representative sections. For the work reported in Section 2.1.1 B, isolation of bacterial strains from colorectal cancer biopsy samples from Cohort One were processed by Dr. Emma Allen-Vercoe and Dr. Jaclyn Strauss. Additional help from both Michelle Daigneault and Dr. Emma Allen-Vercoe was provided occasionally during the isolation of bacterial strains from Cohort Two and Three as well. Sequencing analysis of all bacterial strains isolated from the colorectal cancer biopsies was performed by Sindy Babinsky at the BC Cancer Agency. In Section 2.3.1 A, the Pseudomonas MORN2 recombinant protein was cloned and purified by Dr. Bryan Berger at Lehigh University in Bethlehem, Pennsylvania. Whole genome sequencing and full annotation of the two Fn 7-1 bacteriophage, from Section 2.4.1 C, were performed by Dr. Abigail Manson McGuire and colleagues at the Broad Institute of MIT in Cambridge, Massachusetts. v Table of Contents ABSTRACT .................................................................................................................................... ii Acknowledgments.......................................................................................................................... iv Author’s Declaration of Works Performed ..................................................................................... v List of Figures ................................................................................................................................ ix List of Tables ................................................................................................................................. xi List of Abbreviations .................................................................................................................... xii Chapter 1. Literature Review and Research Objectives ................................................................. 1 1.1 The Gut Microbiota ........................................................................................................... 1 1.2 Commensals, Health and Immunity .................................................................................. 2 1.3 Inflammatory Diseases in the Gut ..................................................................................... 3 1.4 Inflammatory Bowel Disease and the associated risk of Colorectal Cancer ..................... 6 1.5 Fusobacterium nucleatum ................................................................................................. 7 1.6 Fusobacterium nucleatum and Virulence Factors ............................................................. 9 1.7 Fusobacterium nucleatum and Inflammatory Bowel Disease ......................................... 16 1.8 Fusobacterium nucleatum and Colorectal Cancer ........................................................... 18 1.9 Conclusion ....................................................................................................................... 21 1.10 Research Objectives ...................................................................................................... 22 Chapter 2. Materials and Methods ................................................................................................ 23 2.1 Isolation of bacterial species from colorectal cancer biopsies and characterization of isolated Fusobacterium nucleatum (Fn) species. ...................................................................... 23 2.1.1 Bacterial Strains ............................................................................................................ 23 2.1.2 Identification of CRC-derived strains and the characterization of Fn strains .............. 40 2.2 Fn interactions with human colonic epithelial cells ............................................................ 51 2.2.1 Assessing the ability of CRC-derived Fn isolates to invade human epithelial cells in vitro ........................................................................................................................................ 51 2.2.2 Host Immune Response to Fn and CRC-derived Strains ............................................. 63 2.2.3 Assessing the effects of human cytokines on Fn invasion in vitro .............................. 68 2.3 Determining the role of MORN2 domain-associated proteins in Fusobacterium adhesion/invasion ...................................................................................................................... 71 2.3.1 Characterization of MORN2 domains in adhesion and invasion of Fn strains ............ 71 vi 2.4 Determining the presence and role of bacteriophage in invasive Fn strains ....................... 74 2.4.1 Induction and characterization of the invasive Fn subspecies animalis 7-1 bacteriophage ɸFunu1 and ɸFunu2 ......................................................................................
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