Structural and biological insights into the most abundant phages of the human gut
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Date
2025
Authors
Tobin, Ciara A.
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Publisher
University College Cork
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Abstract
The importance of the gut microbiome in both health and disease states has emerged over recent years. However, the focus has mostly been on the bacterial portion of the human gut. As obligate predators of bacteria, bacteriophages (phages) have the potential to shape their microbial environment, through interactions with their bacterial hosts. The relationship between phages and their bacterial hosts is complex, each deriving various defence and counter-defence strategies in a bid to survive and persist.
In order to gain further insights into phages of the human gut (the gut phageome), this thesis explores two key areas of research. The first examines current literature on biological and methodological factors that can result in gut phageome variation in individuals. Biological factors include age, diet and geography, as well as disease states such as irritable bowel syndrome, irritable bowel disease, colorectal cancer, and infectious diseases such as severe acute respiratory syndrome coronavirus 2 (SARS-COV-2), the causative agent of coronavirus disease 2019 (COVID-19). This led to the compositional analysis of both the virome and bacteriome from the gut and oral cavity of 43 COVID-19 patients, during the acute and convalescence stages of infection. Overall, the gut and oral virome and bacteriome of acute patients was found to be similar to those in recovery.
The second aim of this thesis was to explore structural and biological aspects of the most abundant phages of the human gut, Crassvirales phages. These phages are remarkable for their ability to establish a long-term equilibrium with their bacterial hosts, without noticeable detrimental effects to the latter. CRISPR interference was used to target a number of genes predicted to encode for structural proteins and enzymes involved in various stages of the phage lifecycle in one Crassvirales phage, DAC15. Targeting of the terminase large subunit (TerL) resulted in an enrichment of the number of empty phage capsids, visualised by transmission electron microscopy (TEM), while still maintaining a large proportion of viable phages. Minimal reductions in efficiency of plaquing (EOP) when targeting additional essential and non-essential genes led to the analysis of the DAC15 genome for potential defence strategies. A combined bioinformatic and structural homology approach identified five putative anti-CRISPR proteins (Acrs). This approach was extended to Crassvirales phages CrAss001, CrAss002 and p-crAssphage, identifying 16 more potential Acrs across the three genomes. This is the first instance of putative Acr genes being identified in Crassvirales phages.
A combination of structural, biochemical and bioinformatic approaches were used to elucidate the structure and function of the receptor binding proteins (RBP) of Crassvirales phage CrAss001. CrAss001 was determined to have three tail spike proteins (TSP), that make up inner and outer TSP assemblies. The structure of the inner TSP, gp22_23, was resolved to 3.4 Å and shown to act as a capsule depolymerase, producing zones of clearing on overlays of the bacterial host, Bacteroides intestinalis, that predominantly expresses capsule polysaccharide (CPS) PVR9. A second TSP that makes up part of the outer assembly, gp25, was resolved to 2.6 Å and was shown to exhibit esterase activity. The structures of the remaining two outer assembly proteins, gp24 and gp26, were predicted by AlphaFold. Gp24 is thought to be a linker protein for TSP’s gp25 and gp26. This work provides the first mechanistic insights into the RBPs of a Crassvirales phage.
The final chapter of this thesis looks at the capsid structure of Crassvirales phage DAC15. This represents the second Crassvirales capsid to be resolved in full. The capsid was resolved to 2.95 Å and has a triangulation number of nine. Each asymmetric subunit consists of nine copies of the major capsid protein (MCP), nine copies of the auxiliary protein, two copies of the head fibre dimer (HFD) and one copy of the head fibre trimer (HFT). The MCP has the canonical HK97 fold and both the MCP and auxiliary protein are structural matches to the MCP and auxiliary protein of CrAss001. Extensive non-covalent interactions were identified between MCP subunits and between MCP and auxiliary proteins that are thought to lend stability to the capsid. While the HFD and HFT are structurally similar to their counterparts in CrAss001, there are some differences. The C-terminal domains of the HFD protrude upwards from the capsid, while the domains in the HFD of
CrAss001 point away from each other. The HFT of DAC15 has a 26-residue insert that allows it to extend several nanometres further from the capsid than the HFT of CrAss001.
Overall, the work in this thesis advances our understanding of several aspects of the gut phageome, primarily through structural and biological work focused on Crassvirales phages. This builds on over a decade of work elucidating the various aspects of Crassvirales phage biology in the human gut.
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Keywords
Crassvirales , Gut virome , Gut microbiome , Phage , Structural virology , Tailspikes , SARS-COV-2 , CRISPR interference , Anti-CRISPR proteins , Capsid
Citation
Tobin, C. A. 2025. Structural and biological insights into the most abundant phages of the human gut. PhD Thesis, University College Cork.