**3. Phages in the gut**

As the dominant members of the gut virome, phages have been the focus of studies on the role of the gut virome in health and disease. Phages are ubiquitous viruses that are the most abundant biological entity in the world and can be found anywhere that bacteria can be found. Studying phages in the gut presents a number of difficulties. The first of which is that phages lack a universal marker, such as the 16 s rRNA gene in bacteria. Second, since phages depend on their bacterial hosts for reproduction and only 39% of bacteria in the gut can be cultured [39], many phages that are associated with the other 61% cannot be cultivated. This means that modern phage research largely depends on costly and labor-intensive viral metagenomics, which also presents challenges due to the immense genetic diversity of phages, the lack of a robust virus metagenomic classification, and still nascent use of bioinformatics to evaluate data set generated from viral metagenomic analysis. Much phage research has revolved around the practice of phage therapy, which has been used for over 100 years in some Eastern European Countries to treat single strain bacterial infections. The emergence of antibiotic resistance has led to phages gaining recent attention for their potential as an alternative to antibiotics [40]. In phage therapy, patients are administered solutions of individual phage strains, or multiple strains (i.e. phage cocktail), which are selected through *in vitro* screening for their specificity to the single bacterial agent causing the infection and for their effectiveness in eliminating that one bacterial species. Much of the interest in phage therapy rather than antibiotics is based on the specificity of phages to target a narrow host range, allowing for the targeted elimination of a bacterial pathogen while leaving commensal bacterial members of the microbiome intact, and the ability of phages to self-propagate upon infection of their bacterial host.

In general, there are two types of phages: lytic and temperate. Lytic phages reproduce via the lytic cycle and temperate phages use the lysogenic cycle (**Figure 1**). Conventional phage therapy uses lytic phages because in the lytic

lifecycle, phages infect a bacterial host, hijack the host machinery for replication of viral progeny, and eventually lyse the host cell and the release of novel phage progeny. In the lysogenic lifecycle, a temperate phage infects a bacterial host and integrates its viral DNA into the bacterial chromosome as a prophage. This process does not always end in cell lysis, instead the prophage can reproduce by propagating with the bacterial chromosome during replication. Harmful environmental stimuli in the gut, such as oxidative stress [41], antibiotics [42], or other unfavorable conditions for the bacterial host [43], can result in the induction of the prophage into the lytic cycle, thereby resulting in the lysis of the bacterial host and release of novel phage progeny. However, Lysogenic (temperate) phages are generally not used in phage therapy because lysogeny is a mechanism for bacteria to exchange DNA so lysogenic phages carry the potential for propagating genes for pathogenesis.

While lytic phages are largely seen as parasitic to their bacterial hosts, temperate phages and their host bacteria have a much more complicated relationship. Temperate phages are important drivers of bacterial evolution [44], in part through their role in horizontal gene transfer between bacterial hosts. Temperate phages are common in the gut and studies have found that a large proportion of bacteria in the microbiome have temperate phages incorporated into their genomes as prophages [21, 45]. For the bacterial host, carrying prophages has several fitness benefits. Prophages encode genes for metabolism, antibacterial resistance, and toxin production (for example, shiga toxin production) [9, 46], thereby conveying functional genes for survival to their bacterial hosts upon integration with the bacterial chromosome. Prophages also protect their hosts from infection by lytic phages through superinfection exclusion [47]. Phage-mediated horizontal gene transfer between bacterial hosts increases rates of genetic recombination and diversification of phage-encoded genes in the gut [48].

Composition, structure, and function of the gut virome contributes to health in a number of ways [49], as reviewed by Mukhopadhya and colleagues [50]. The coevolution between phages and their bacterial hosts is a well-established mechanism for driving the development of microbial communities across environments [44]. This is also the case in the gut environment where phages are thought to modulate the microbiota and, in turn, affect human health. A longitudinal study of gut microbiome and virome composition in healthy infants found that expansion of gut bacterial species was accompanied by contractions and shifts of gut phage populations, suggesting that phage predation of targeted bacteria may help drive the development of a healthy infant gut microbiome [51]. Conversely, in the setting of dysbiosis, changes in the gut phage population have been shown to precede the onset of type 1 diabetes in children [38]. Phages are also thought to form a protective barrier in the mucosa of the gastrointestinal epithelium, thereby providing the host tissue with non-host-derived defense against pro-inflammatory gut bacteria [52]. Experimental evidence suggests that they do this by using their Ig-like domains expressed on the viral capsid to attach to the glycan molecules of the host's mucin glycoproteins. Growing evidence now implicates a role for phages of the mucosa in states of dysbiosis, which have been characterized by an increased richness and abundance of the mucosal temperate phage population [9, 14, 34, 35, 53]. These changes in the phage community is opposite that of the bacterial community in which decreased richness and diversity characterize dysbiosis.

The virome also influences health through direct interaction with the human immune system by triggering both pro- and anti-inflammatory action [4, 10–14]. Phages are capable of activating TLR9-mediated IFNγ, a pro-inflammatory pathway that exacerbates intestinal colitis [14]. Conversely, phages can also ameliorate inflammation through TLR3- and TLR4-mediated interferon-β activation [11]. Several studies have found elevated abundance of phages in the mucosal surfaces of patients with IBD [36, 53]. Other studies have found an expansion of phages from the order *Caudovirales* in the setting of inflammatory bowel disease [34, 54, 55]. Norman and colleagues speculate that phages may contribute to, or be a biomarker for, inflammation and dysbiosis in the gut. Collectively, these studies indicate that phages have an important role in gastrointestinal disorder and potentially, in the corrective response to dysbiosis.
