Experimental frontiers in the study of human-associated bacteriophages

Abstract

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.