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).     

Alternative approach to a heavy weight problem

Obesity is reaching epidemic proportions in developed countries and represents a significant risk factor for hypertension, heart disease, diabetes, and dyslipidemia. Splicing mutations constitute at least 14% of disease-causing mutations, thus implicating polymorphisms that affect splicing as likely candidates for disease susceptibility. A recent study suggested that genes associated with obesity were significantly enriched for rare nucleotide variants. Here, we examined these variants and revealed that they are located near splice junctions and tend to affect exonic splicing regulatory sequences. We also show that the majority of the exons that harbor these SNPs are constitutively spliced, yet they exhibit weak splice sites, typical to alternatively spliced exons, and are hence suboptimal for recognition by the splicing machinery and prone to become alternatively spliced. Using ex vivo assays, we tested a few representative variants and show that they indeed affect splicing by causing a shift from a constitutive to an alternative pattern, suggesting a possible link between extreme body mass index and abnormal splicing patterns.     

Targeted Gene-and-host Progenitor Cell Therapy for Nonunion Bone Fracture Repair

Nonunion fractures present a challenge to orthopedics with no optimal solution. In-vivo DNA electroporation is a gene-delivery technique that can potentially accelerate regenerative processes. We hypothesized that in vivo electroporation of an osteogenic gene in a nonunion radius bone defect site would induce fracture repair. Nonunion fracture was created in the radii of C3H/HeN mice, into which a collagen sponge was placed. To allow for recruitment of host progenitor cells (HPCs) into the implanted sponge, the mice were housed for 10 days before electroporation. Mice were electroporated with either bone morphogenetic protein 9 (BMP-9) plasmid, Luciferase plasmid or injected with BMP-9 plasmid but not electroporated. In vivo bioluminescent imaging indicated that gene expression was localized to the defect site. Microcomputed tomography (µCT) and histological analysis of murine radii electroporated with BMP-9 demonstrated bone formation bridging the bone gap, whereas in the control groups the defect remained unbridged. Population of the implanted collagen sponge by HPCs transfected with the injected plasmid following electroporation was noted. Our data indicate that regeneration of nonunion bone defect can be attained by performing in vivo electroporation with an osteogenic gene combined with recruitment of HPCs. This gene therapy approach may pave the way for regeneration of other skeletal tissues.     

Distribution of immunoreactive melanin-concentrating hormone in the central nervous system of the rat

The distribution of melanin-concentrating hormone-like immunoreactivity (MCH-LI) in 41 microdissected brain and spinal cord regions was determined using radioimmunoassay with antibodies to salmon MCH. The highest concentration of MCH-LI was detected just ventral to the zona incerta (subzona incerta) (2923.2 fmol/mg protein). Very high concentrations of MCH-LI (greater than 1000 fmol/mg protein) were detected also in the nucleus of the diagonal band, medial forebrain bundle, posterior hypothalamic nucleus and medial mammillary nucleus. High concentrations of the peptide (between 500-1000 fmol/mg protein) were measured in 11 brain regions, including bed nucleus of stria terminalis, paraventricular nucleus, anterior hypothalamic nucleus, median eminence, parabrachial nucleus. Moderate concentrations of MCH-LI (between 250-500 fmol/mg protein) were measured in 16 brain regions, such as frontal cortex, central amygdaloid nucleus, medial septum, periventricular nucleus (preoptic) and nucleus of the solitary tract. Low concentrations of MCH-LI (less than 250 fmol/mg protein) were measured in 9 brain regions such as cortical areas, hippocampus, caudate nucleus and substantia nigra. Cervical spinal cord and neurointermediate lobe of the pituitary gland contain low concentrations of the peptide.     

Core body temperature in patients with seasonal affective disorder and normal controls in summer and winter

The rationale for phototherapy in seasonal affective disorder (SAD) was originally based on the notion that SAD patients were light deprived during the wintertime and needed more light. We previously found normal temperature profiles of untreated SAD patients during the winter, and that phototherapy significantly enhanced the amplitude of the circadian temperature profile in SAD patients during the winter (Rosenthal et al 1990). We hypothesized that summer would act similarly on the temperature rhythm of these patients. In this study we examined the temperature data from SAD patients and normal controls during the summer and compared it to the results of our previous study. We found identical profiles for SAD patients and normal controls during the summer and that summer significantly lowered the overall temperature profiles of both groups and did not alter the amplitudes. These results raise questions about the validity of the current theories of the mechanism of light therapy.