Mining for novel human gut bacteriophages against Bacteroidales

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Guerin, Emma
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University College Cork
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The importance of the gut microbiome in human health and disease has become apparent in recent years. The bacterial component is well characterised in comparison to the viral fraction, that is predominantly composed of bacteria infecting viruses called bacteriophages (phages). Phages are largely understudied in the context of the human gut despite their potential as driving forces of microbiome composition and function. Changes in the composition of the microbiome have been linked to disease states such as IBD, colorectal cancer and diabetes. However, the root causes of these changes remain to be elucidated. While gradual improvements in virome metagenomic analyses have provided us with important insights, we are only just beginning to understand the complex roles of gut phages. If we are to truly understand how phages may shape our microbiome and influence our health, it will be necessary to determine the mechanisms behind phage-bacterium host interactions as well as how phages affect the human host such as through interactions with the immune system. As metagenomics provides little insight into the biological properties of phages, the isolation of novel phage-host pairs from the human gut is required in conjunction with characterisation using in vitro, ex vivo and in vivo models with naturally relevant conditions. However, this is not without challenges. To gain some scope into the human gut phageome, this thesis focuses on one of the most interesting and abundant phages identified within the “dark matter” of viral sequences, crAssphage. This phage was predicted to infect hosts of the Bacteroidales order, one of the most important bacterial groups in the healthy human gut. We performed bioinformatic analyses of crAss-like phage relatives using faecal phageome datasets originating from multiple human cohorts. This led to the de novo assembly of 244 novel crAss-like phages and their taxonomic classification at the genus-level. We demonstrated the first evidence of ex vivo crAss-like phage propagation and generated preliminary electron micrographs using faeces from a crAss-rich donor, confirming a podovirus morphology. Following this, the objective was to isolate members of the crAss-like phage family in vitro using a targeted screening approach. Both phage and host enrichments were performed from the same crAssphage-rich faeces which was aided by the use of antibiotics to selectively promote the growth of Bacteroidales with the parallel expansion of associated crAss-like phages. This led the isolation of a novel member of the crAss-like family in pure culture, ΦcrAss002, the characterisation of which provided interesting insights into gut phage-host dynamics. A similar methodology was applied with the goal of isolating further Bacteroidales associated phages, however, a more traditional screening approach was implemented. This resulted in the isolation and first detailed characterisation of a virulent Parabacteroides distasonis phage to be reported as of 2020. Given that no other virulent phages targeting this important bacterial genus are deposited on NCBI Taxonomy, the need for in vitro isolation of further phage-host pairs is emphasised. These novel gut phages show that virulent phages do not necessarily present with traditional biological properties such as plaque formation or complete lysing of host cultures. Interestingly, both phage isolates were able to co-exist with their host over time. This is consistent with the observed persistence of virulent phages in the human gut over extensive periods of time. Consequently, our view of phage-host interactions in the human gut is likely to change and expand in years to come. This will be necessary if we are to fully understand the extent to which these bacteria infecting viruses shape the microbiome and mediate homeostasis of the gut. This will be crucial if we are to manipulate them for therapeutic applications.
Bacteriophages , Human gut phageome , CrAssphage , CrAss-like phages , Human microbiome , Phage-host interactions , Human gut bacteriophages , Persistent virulent phages
Guerin, E. 2020. Mining for novel human gut bacteriophages against Bacteroidales. PhD Thesis, University College Cork.
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