The Impact of Diet on the Human Gut Microbiota in the Context of Colorectal Cancer
Though strong evidence suggests the gut microbiota contributes to colorectal cancer (CRC) onset and/or progression, there is no universal signature of a gut microbe or microbiota associated with the disease. The functional capability of gut communities could be more useful in predicting CRC risk and more valuable in understanding the role of the gut microbiota in CRC than gut microbiota composition. Additionally, diet is a key contributor to the function of the gut microbiota and certain diets (e.g., protein- and fibre-rich) have been associated with CRC risk. As such, this thesis was driven by a global purpose: to characterize potential microbe-microbe and diet-microbe interactions that may influence CRC. To fulfill this purpose, taxonomic composition, metabolic function, and carriage of CRC-linked virulence determinants were characterized for CRC biopsy-derived gut microbial communities and known CRC-linked virulence determinants were characterized in individual strains. The influence of protein- and fibre-rich diets on the taxonomic composition and metabolic output of three CRC biopsy- and three healthy fecal donor-derived communities was assessed with a focus on taxa and metabolites linked to CRC. Lastly, free amino acid pools representative of major dietary protein sources were applied to individual strains of CRC-linked bacteria Fusobacterium spp. to assess potential diet-influenced differences in growth among CRC-relevant fusobacterial strains. Although colonic isolates of Fusobacterium nucleatum subsp. animalis were found to co-aggregate with strains of several CRC-relevant species, no known strain-level CRC-relevant virulence determinants were found in the CRC biopsy-derived communities, and no CRC-linked metabolites could be consistently linked to taxa between the characterized gut microbial communities. Protein- and fibre-rich diets were found to enrich for CRC- and colonic health-associated bacterial taxa and metabolites within human colonic sample-derived bacterial communities, respectively. Dietary protein-representative free amino acid pools differentially influenced the growth of individual Fusobacterium strains, including CRC-relevant strains, although the amino acid preferences and growth strategies employed by fusobacterial strains were highly heterogeneous. Ultimately, this work underlines the complexity in host-microbiota interactions and serves as a preliminary step towards holistic characterization of interactions between the human host, the resident gut microbiota, and CRC.
A substantial fraction of the world’s bacteriophages can be categorized as ‘temperate’ or ‘lytic.’ In mammalian microbiomes, temperate enrichment and lytic reduction are distinguishing features of the viral community. This contrasts aquatic and soil ecosystems where lytic phages tend to dominate. As of 2023, researchers are beginning to understand the ecological factors that give temperate phages a competitive edge in gut microbiomes. There is value in understanding why temperate phages are enriched, but understanding the consequences of this enrichment will be just as valuable, especially for human health. Little evidence exists to suggest how temperate prevalence influences microbiome composition and function. While there are strong correlations between temperate enrichment and microbiome features, few studies have had the capability to detect causal influence. To move from correlation to causation, researchers must move away from descriptive metagenomic snapshots and employ experimental manipulation of complex community models. There are several methodological limitations that forestall these studies, including but not limited to: a lack of methods for reducing lytic signals (whilst enriching temperate signals) in complex microbiome models, and a lack of methods for quantifying non-plaque-forming phages. The inability to reduce lytic signals is especially restrictive to studying temperate dynamics, as temperate signals are often subtle and easily washed out by the prominent effects of lytic predation. The driving goal of this thesis is to innovate new methods and tools for studying temperate phage dynamics in complex microbiome models. To validate a method for depleting lytic signals whilst enriching temperate signals, I rely on dilution and colony isolation: a technique as old as microbiology itself. This technique is widely assumed to remove lytic virions from host colonies, but has never been applied to a complex gut community and has never been thoroughly validated as a mechanism to exclude lytic viruses. To quantify non-plaque-forming phages, I utilize a qPCR assay paired with DNase I treatment to distinguish encapsidated vs. non-encapsidated phage genomes. This work, which uses these tools to provide glimpses of temperate phage influence, opens the door to novel experimental designs and approaches, and ultimately, a deeper understanding of temperate phage influence in mammalian microbiomes.
