The microbiome has long been an alluring target to study and recent advancements in microbial detection and omics-technologies has further revolutionized our view of how human diseases are impacted by the microbiome. A member of the human microbiome that has garnered such attention is Fusobacterium nucleatum, a Gram-negative, anaerobic bacterium, that normally inhabits the human oral cavity. Interestingly, F. nucleatum is highly invasive into surrounding cells and tissues of the periodontal pocket (below the gymline) and capable of disseminating throughout the entire body. Because of this, F. nucleatum is associated with a wide variety of diseases, most recently and strikingly, colorectal cancer. Despite the pathogenic potential of F. nucleatum, there is limited knowledge about the molecular mechanisms contributing to the invasive nature and virulence of this oral bacterium.
This gap in knowledge can be attributed to the absence of genetic tools and resources to investigate and study host-pathogen interactions of Fusobacterium. Progress in dissecting the role of Fusobacterium in disease has been hindered by a lack of fully sequenced and annotated genomes, and the absence of genetic systems to generate target virulence gene deletions to validate mechanisms contributing to host-pathogen interactions. Breakthroughs discussed in this work focus on developing and expanding the genetic toolkits and resources available for studying F. nucleatum interactions in relation to human health and disease.
As part of this work, herein, I introduce FusoPortal, an online database of fully sequenced and annotated Fusobacterium genomes, that enabled the bioinformatic annotation and correction of large protein encoding reading frames, that were previously misannotated. This database features a custom basic local alignment search tool (BLAST) server that establishes this resource as a powerful tool for identifying potential virulence factors that contribute to Fusobacterium pathogenesis. Most notably, FusoPortal facilitated my discovery of DNA uptake machinery involved in natural competence and transformation in F. nucleatum. This work is the first to characterize natural competence in a Fusobacterium species, and also enables the expansion of Fusobacterium genetics utilizing the newly found competence mechanism. The findings within this dissertation encompass a paradigm shift in efficient and robust tools to study F. nucleatum biology and pathogenesis. By creating tools for identifying key genes, proteins, and mechanisms involved in Fusobacterium induced or accelerated diseases, there is the potential to accelerate the development of novel therapeutics and vaccines against the emerging 'oncomicrobe' Fusobacterium nucleatum. / Doctor of Philosophy / The trillions of microbes living on or in the human body, collectively called the microbiome, has long been a captivating target to study and understand its role in human health and disease. Recent advances in technology have revolutionized our view of the individual components of the human microbiome, which has led to a renaissance in understanding how specific bacterial species could be used to modulate human health and fight a myriad of diseases. A member of this microbiome that has garnered such attention is Fusobacterium nucleatum, a bacterium that lives in oxygen free pockets along the gumline in the human mouth. A striking feature of F. nucleatum is its ability to invade surrounding tissue, driving bacterial spread throughout the entire body. This bacterium is associated with a wide variety of diseases, most importantly colon cancer.
Although F. nucleatum is implicated in these diseases, we still know very little about the mechanisms used by Fusobacterium to promote disease. This roadblock in studying F. nucleatum can largely be attributed to the lack of molecular tools and resources to investigate and study the interactions between the bacteria and its human host. Therefore, research discussed in this work revolved around developing and resources available for studying F. nucleatum interactions in relation to human health and disease.
One such resource developed was FusoPortal, an online website with fully sequenced and annotated genomes. This resource was critical in the bioinformatic annotation and correction of large proteins that were previously misannotated. This website features a tool that allows one to search this complete set of genes for a specific sequence establishing this resource as an important tool for identifying key genes and mechanisms that could influence F. nucleatum ability to infect and cause disease. Most notably, FusoPortal provided the means to discover a bacterial system that can import DNA and integrate it into the bacterial genome, a process called natural competence. This work is the first to characterize natural competence in a Fusobacterium species, and has allowed me to utilize the newly found natural competence mechanism to enhance Fusobacterium genetics. In summary, the findings within this dissertation brings about a new horizon for studying F. nucleatum biology, thereby, providing the framework for creating future therapeutic strategies to treat diseases including colorectal cancer.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/106638 |
| Date | 21 May 2020 |
| Creators | Sanders, Blake Edward |
| Contributors | Biochemistry, Slade, Daniel J., Caswell, Clayton C., Tu, Zhijian, Helm, Richard F. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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