CRISPR targeting reveals a reservoir of common phages associated with the human gut microbiome

The bacterial community in the human gut has crucial health roles both in metabolic functions and in protection against pathogens. Phages, which are known to significantly affect microbial community composition in many ecological niches, have the potential to impact the gut microbiota, yet thorough characterization of this relationship remains elusive. We have reconstructed the content of the CRISPR bacterial immune system in the human gut microbiomes of 124 European individuals and used it to identify a catalog of 991 phages targeted by CRISPR across all individuals. Our results show that 78% of these phages are shared among two or more individuals. Moreover, a significant fraction of phages found in our study are shown to exist in fecal samples previously derived from American and Japanese individuals, identifying a common reservoir of phages frequently associated with the human gut microbiome. We further inferred the bacterial hosts for more than 130 such phages, enabling a detailed analysis of phage–bacteria interactions across the 124 individuals by correlating patterns of phage abundance with bacterial abundance and resistance. A subset of phages demonstrated preferred association with host genomes as lysogenized prophages, with highly increased abundance in specific individuals. Overall, our results imply that phage–bacterial attack–resistance interactions occur within the human gut microbiome, possibly affecting microbiota composition and human health. Our finding of global sharing of gut phages is surprising in light of the extreme genetic diversity of phages found in other ecological niches.The human microbiome, representing the collection of all microbes that live on and within a human being, is composed of 10 times more cells than human cells (Eckburg et al. 2005; Walter and Ley 2011). These naturally occurring microbes, particularly those that reside in the human gut, are known to provide humans with crucial metabolic functions. They allow harvesting and storing energy from various dietary products, influence the development of the immune system, and protect from colonization by pathogens (Hooper et al. 2002; Dethlefsen et al. 2007). In a recent study by the MetaHIT Consortium (Metagenomics of Human Intestinal Tract) (Qin et al. 2010), DNA from fecal samples of 124 Europeans was sequenced to generate deep coverage of the human gut microbiome. Analysis of the assembled DNA fragments showed that the human gut microbial gene set is 150 times larger than the human gene complement (Qin et al. 2010).In many studied ecosystems, phages outnumber bacterial cells by a factor of 10:1, posing significant predation pressure on their hosts (Chibani-Chennoufi et al. 2004). This phage pressure has been shown to play a crucial role in the evolution, diversity, and abundance of bacteria (Avrani et al. 2011; Stern and Sorek 2011). The richness and density of gut bacterial species and populations make the human gut an ideal ecological niche for phages. Indeed, the existence of phages in the human and animal gut has been demonstrated repeatedly using transmission electron microscopy (Letarov and Kulikov 2009), and virus-like particles (VLPs) were observed in high density on the surface of human gut mucosa. Furthermore, metagenomic sampling has shown that the majority of DNA viruses in the human gut are bacteriophages (Breitbart et al. 2003). However, research on the identity and prevalence of phages infecting gut-residing microbiota and of their effects on gut bacterial populations is still in its early days.Recently, VLPs isolated from feces of four pairs of monozygotic twins and their mothers (Reyes et al. 2010), as well as six unrelated individuals (Minot et al. 2011), were sequenced over several time points. Both studies detected a largely unique and stable phage complement within each individual. These studies suggested that phages are rarely shared among individuals and that a predatory viral–microbial dynamic, as characterized in various other ecosystems, may be absent in the distal human gut.Clustered regularly interspaced short palindromic repeats (CRISPR) loci, together with their associated cas genes, have been shown to constitute a defense system against propagation of phages and plasmids (Barrangou et al. 2007; Marraffini and Sontheimer 2008; Sorek et al. 2008). CRISPR loci are composed of short repeat sequences separated by hypervariable “spacer” sequences, usually sized 24–50 bp. In the first stage of bacterial defense, bacteria incorporate fragments of phage or plasmid genomes as novel spacers. These spacers are then transcribed into small RNAs, which together with the Cas protein complex guide the way to interfere with phage replication (van der Oost et al. 2009; Horvath and Barrangou 2010; Karginov and Hannon 2010; Marraffini and Sontheimer 2010). Thus, CRISPR spacers may be viewed as a database of fragments derived from phage and plasmid genomes. Indeed, CRISPR spacers have previously been used to identify phages in several metagenomic data sets, including microbiomes harvested from acid mine (Andersson and Banfield 2008), hot spring (Snyder et al. 2010), and human oral cavity (Pride et al. 2011) environments.One of the major caveats of metagenomic analyses is that in most cases, microbes and viruses are sequenced together, thereafter making it difficult to distinguish between the two. Even when VLPs are isolated and sequenced directly, it is nearly impossible to identify the specific hosts of the isolated viruses, precluding the analysis of the relationship between virus and host. Here, we use the link between bacterial CRISPR spacers and the infecting mobile elements to study these interactions in the human gut.To this end, we have analyzed the most extensive metagenomic sequencing data of the human gut microbiome produced to date (the MetaHIT set), composed of 576.7 billion bases of sequence (Qin et al. 2010). We extracted CRISPR spacers directly from the raw sequencing reads and then used these spacers as probes to search for phage genomic segments within the assembled sequences of the metagenomes (Fig. 1). This allowed us to (1) identify and characterize a large catalog of phages and mobile elements that infect bacteria in the human gut; (2) identify, for a subset of these phages, their bacterial hosts; and (3) perform a subject-wide analysis of phage–bacteria coexistence, by correlating patterns of phage abundance with patterns of bacterial abundance and resistance. These results were then compared across data sets obtained from additional microbiome sequencing projects, revealing the existence of a reservoir of both dormant and active phages frequently associated with the human gut microbiome.Open in a separate windowFigure 1.CRISPR spacers are used as probes to fish out phage genomes. In the MetaHIT metagenomics study (Qin et al. 2010), gut microbes were harvested from feces of 124 individuals, and DNA was sequenced to generate short reads (75 bp). These reads were then assembled into contigs, which mainly represent DNA of gut-residing bacteria, but potentially also contain DNA of phages associated with these bacteria. In the present study, CRISPR spacers were detected by searching for reads that match a known CRISPR repeat on both sides of the read. The spacers detected were then used to probe assembled contigs, and phage and mobile element contigs were identified as those showing high sequence identity with a spacer (but not with the CRISPR repeat).