Mechanistic studies of Fusobacterium genetic and defense systems
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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.