Fusobacterium are Gram-negative anaerobic bacteria that colonize a variety of eukaryotes including cattle and humans. In humans, Fusobacterium coordinates the central architecture of the oral biofilm by expressing an abundance of outer membrane adhesins that mediate bridging between early and late colonizing bacteria. While Fusobacterium are mostly considered commensal microorganisms, they can also become an opportunistic pathogen that spreads throughout the human body and promote the development of oral and extra-oral infections and diseases including colorectal cancer. Importantly for this work, many Fusobacterium species and strains are recalcitrant to genetic manipulation, the majority of which has led to hindrance in the study of their biology, molecular mechanisms, and pathogenesis.
The genetic intractability of Fusobacterium is an obstacle for the development of future treatments for diseases associated with these anaerobic bacteria. Therefore, the creation of tools to enhance genome editing in target species is crucial to understand the molecular mechanisms driving Fusobacterium infections. This dissertation exploits innate and adaptive defense systems present in Fusobacterium for their use as molecular tools for genome editing.
Accordingly, we first investigated restriction-modification systems with a focus on the role of DNA methyltransferases and endonucleases in host defense and genetic recalcitrance in several strains of Fusobacterium through bioinformatic and biochemical approaches. Altogether, over 15 DNA methyltransferases were characterized. Most notably, we identified and characterized two type II DNA methyltransferases that are capable of methylating plasmid DNA by treating with purified enzymes in-vitro and coexpression approaches in Escherichia coli strains, enabling an statiscally improved transformation efficiency via electroporation in F. nucleatum.
Also contained in this dissertation is the first detailed description of CRISPR-Cas adaptive immunity systems present in Fusobacterium strains. Most of the discovered CRISPR-Cas systems in Fusobacterium belong to Class 1 systems. Nonetheless we identified Type II-A and Type VI-C Class 2 systems. The discovery of Cas9 and Cas13c effectors respectively from these systems will be crucial in the development of a new generation of genome-editing tools in Fusobacterium.
The studies included in this dissertation provide the framework for overcoming Fusobacterium genetic recalcitrance by the implementation of host mimicking techniques. By utilizing restriction-modification system enzymes and the adaptive immunity CRISPR-Cas systems, we will gain a better understanding of how Fusobacterium modulates infections and diseases, and ultimately explore the potential of novel therapeutic treatments. / Doctor of Philosophy / The oral cavity has one of the most diverse and largest microbial populations, where microorganisms are capable of colonizing hard surfaces of the teeth and the soft tissues of the oral mucosa. A fundamental member of the oral microbiome is Fusobacterium, a Gram-negative bacterium which coordinates the oral biofilm formation by interacting with other microorganisms. In recent studies, Fusobacterium has been associated with oral and extra-oral infections and diseases including periodontitis, preterm birth, Lemiere syndrome, inflammatory bowel disease and colorectal cancer. Importantly, many Fusobacterium species and strains are challenging to study due to their inability to uptake exogenous DNA and lack of genetic tools, which has hindered the study of their biology, molecular mechanisms and pathogenesis.
The challenges in the genetic manipulation of Fusobacterium present a significant obstacle for the development of future treatments for diseases associated with these bacteria. Therefore, the creation of tools to expand bacterial transformation of exogenous DNA and genome editing to more than just one Fusobacterium species is crucial to understand how Fusobacterium is causing these infections. This dissertation explores the presence and utilization of defense systems, which defend bacteria from phage attack, as an alternative to improve Fusobacterium genetics.
Accordingly, we first studied a set of over 15 enzymes that recognize a specific DNA pattern and add a methyl group (DNA methyltransferases) to specific nucleotides in several strains of Fusobacterium. We discovered that two of these enzymes improve Fusobacterium's ability of importing and genomically incorporating exogenous DNA after an electric discharge permeabilizes the bacterial membrane. Furthermore, for the first time we have described the composition of CRISPR-Cas bacterial defense systems, that detect invading DNA from viruses and provide protection to Fusobacterium strains. These systems have previously been successfully used as genetic tools to achieve genome editing. Thus, their further characterization is warranted to create novel molecular tools in Fusobacterium. Altogether, these discoveries will lead to a better comprehension of Fusobacterium biology in infections and diseases, while exploring novel therapeutic strategies.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/110368 |
| Date | 07 December 2020 |
| Creators | Umana Torres, Ariana |
| Contributors | Biochemistry, Slade, Daniel Joseph, Allen, Irving C., Zhu, Jinsong, Allen, Kylie Dawn |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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