The human gut harbours an abundance of uncharacterised bacteriophages. While metagenomics is opening up possibilities to analyse these phages in bulk, a knowledge gap remains, because detailed studies of the phages remain problematic without the availability of cultured hosts. Genome-resolved metagenomics narrows this gap by providing the complete genome sequences of uncultured phages, allowing further in silico analyses. A major step in this direction was the discovery of crAssphage. The genome of this bacteriophage, which was assembled from previously published human faecal metagenomes and is found in the majority of individuals, allowed genomic analyses and predictions of a potential host. However, its exact role within the intestinal microbiome remains unknown.
Here, we report on our ongoing efforts to find the host of crAssphage and characterise its wider role within the human gut microbial ecosystem. First, we screened 26 individuals for crAssphage using custom primers (part of the crowd-sourced global crAssphage sampling project) and found 11 crAss-positive (42%), slightly lower than previously published rates. This high prevalence suggests that the host for crAssphage is a common gut bacterium.
The presence of eight Bacteroides Associated Carbohydrate-binding Often N-linked (BACON) domains within orf00074, encoded on the crAssphage genome, may provide a clue as to the bacterial host. BACON domains, first found in members of the Bacteroides genus, are predicted to have a role in the binding of these bacteria to mucin, but their function in crAssphage remains elusive. We hypothesized that the crAssphage BACON domains might function to adhere to a bacterial host or to mucin. Thus, we identified and sequenced orf00074 in one of the subjects and found that it contained only three BACON domains. These domains were cloned and heterologously expressed as fluorescent fusion proteins. Next, these fluorescently labelled domains will be used to illuminate the binding specificity of the domains.