Engineering Bacteroidales and Their Phages Towards Biomedical Interventions in the Gut Microbiome

The human microbiome plays an integral role in shaping health and disease. The bacteria that inhabit the gut directly influence the immune system, metabolism, susceptibility to infectious disease, and more. This impact makes the microbiome of particular interest in the development of live therapeutics through the manipulation of targeted bacteria and their bacteriophages. In this dissertation, I will address areas of need regarding the manipulation of the microbiome. Engineering bacteria for live therapeutics comes with inherent risk, as they may persist past the therapeutic window or escape into a new environment. Advances in biocontainment are essential for the future of these promising avenues of treatment. Here, we evaluate the potential of two inducible toxin systems for use in a biocontainment system and show controlled knockdown of engineered strains in a mouse gut. We also evaluate horizontal gene transfer to bypass a multilayered CRISPR based system and show robust containment of transgenes in vivo. Next, we identify and characterize novel Bacteroidales lysogenic phages active in the gut. Lysogenic phages represent an understudied area of the microbiome despite their importance in horizontal gene transfer and potential as genetic delivery systems. We demonstrate that novel identified phages are present in the human microbiome and that one of these phages is capable of transduction both in vitro and in vivo. Finally, we address the rise in antibiotic-resistant pathogenic Escherichia coli by engineering commensal members of the gut microbiome to secrete heterologous antimicrobials. Secreted colicin by Phocaeicola vulgatus prevented colonization of patient-isolated uropathogenic E. coli in a prophylactic mouse model. Together, this work contributes to the body of knowledge on interactions in the microbiome and towards biomedical applications of engineered commensals as living therapeutics.