Bacteriophages of the Human Gut: The “Known Unknown” of the Microbiome

The Human Gut Virome Is Highly Diverse, Stable, and Individual Specific

Viruses and mobile genetic elements are molecular parasites or symbionts that coevolve with nearly all forms of cellular life. The route of virus replication and protein expression is determined by the viral genome type. Comparison of these routes led to the classification of viruses into seven "Baltimore classes" (BCs) that define the major features of virus reproduction. However, recent phylogenomic studies identified multiple evolutionary connections among viruses within each of the BCs as well as between different classes. Due to the modular organization of virus genomes, these relationships defy simple representation as lines of descent but rather form complex networks. Phylogenetic analyses of virus hallmark genes combined with analyses of gene-sharing networks show that replication modules of five BCs (three classes of RNA viruses and two classes of reverse-transcribing viruses) evolved from a common ancestor that encoded an RNA-directed RNA polymerase or a reverse transcriptase. Bona fide viruses evolved from this ancestor on multiple, independent occasions via the recruitment of distinct cellular proteins as capsid subunits and other structural components of virions. The single-stranded DNA (ssDNA) viruses are a polyphyletic class, with different groups evolving by recombination between rolling-circle-replicating plasmids, which contributed the replication protein, and positive-sense RNA viruses, which contributed the capsid protein. The double-stranded DNA (dsDNA) viruses are distributed among several large monophyletic groups and arose via the combination of distinct structural modules with equally diverse replication modules. Phylogenomic analyses reveal the finer structure of evolutionary connections among RNA viruses and reverse-transcribing viruses, ssDNA viruses, and large subsets of dsDNA viruses. Taken together, these analyses allow us to outline the global organization of the virus world. Here, we describe the key aspects of this organization and propose a comprehensive hierarchical taxonomy of viruses.

https://mmbr.asm.org/content/mmbr/84/2/e00061-19.full-text.pdf

Global Organization and Proposed Megataxonomy of the Virus World

Discharge of treated sewage leads to release of antibiotic resistant bacteria, resistance genes and antibiotic residues to the environment. However, it is unclear whether increased abundance of antibiotic resistance genes in sewage and sewage-impacted environments is due to on-site selection pressure by residual antibiotics, or is simply a result of fecal contamination with resistant bacteria. Here we analyze relative resistance gene abundance and accompanying extent of fecal pollution in publicly available metagenomic data, using crAssphage sequences as a marker of human fecal contamination (crAssphage is a bacteriophage that is exceptionally abundant in, and specific to, human feces). We find that the presence of resistance genes can largely be explained by fecal pollution, with no clear signs of selection in the environment, with the exception of environments polluted by very high levels of antibiotics from manufacturing, where selection is evident. Our results demonstrate the necessity to take into account fecal pollution levels to avoid making erroneous assumptions regarding environmental selection of antibiotic resistance.

/pdf/fecal-pollution-can-explain-antibiotic-resistance-gene-4a35auouxv.pdf

Fecal pollution can explain antibiotic resistance gene abundances in anthropogenically impacted environments

Whole-Virome Analysis Sheds Light on Viral Dark Matter in Inflammatory Bowel Disease

CrAssphages are an extensive and ubiquitous family of tailed bacteriophages, predicted to infect bacteria of the order Bacteroidales. Despite being found in ~50% of individuals and representing up to 90% of human gut viromes, members of this viral family have never been isolated in culture and remain understudied. Here, we report the isolation of a CrAssphage (ΦCrAss001) from human faecal material. This bacteriophage infects the human gut symbiont Bacteroides intestinalis, confirming previous in silico predictions of the likely host. DNA sequencing demonstrates that the bacteriophage genome is circular, 102 kb in size, and has unusual structural traits. In addition, electron microscopy confirms that ΦcrAss001 has a podovirus-like morphology. Despite the absence of obvious lysogeny genes, ΦcrAss001 replicates in a way that does not disrupt proliferation of the host bacterium, and is able to maintain itself in continuous host culture during several weeks. Bacteriophages of the crAssphage family have not yet been isolated, despite being highly abundant in the human gut. Here, Shkoporov et al. isolate in pure culture one of these viruses and show that it infects the human gut symbiont Bacteroides intestinalis.

/pdf/phcrass001-represents-the-most-abundant-bacteriophage-family-3ggt54apfd.pdf

ΦCrAss001 represents the most abundant bacteriophage family in the human gut and infects Bacteroides intestinalis.

Recent years have been marked by the growing interest towards virulent and temperate bacteriophage populations inhabiting the human lower gastrointestinal tract - the gut phageome. A number of studies demonstrated high levels of specificity and temporal stability of individual gut phageomes, as well as their specific alterations in disease cohorts, in parallel with changes in the bacteriome. It has been speculated that phages might have an active role in shaping the taxonomic composition and functional properties of the human gut bacteriome. An overwhelming majority of gut bacteriophages, however, remain uncultured, unclassified, and their specific hosts and infection strategies are still unknown. They are often referred to as "the viral dark matter". A possible breakthrough in understanding of the phageome can only become possible when a significant proportion of the "the viral dark matter" is identified and linked to bacterial hosts. Here, we describe a method that enables rapid discovery and host-linking of novel bacteriophages in the gut via a combination of serial enrichment cultures and shotgun metagenomics of viral DNA. Using this approach dozens of novel and previously known bacteriophages were detected, including the ones infecting difficult-to-culture anaerobic bacteria. The majority of phages failed to produce lysis and propagate on host cultures in traditional assays. The newly identified phages include representatives of Siphoviridae, Myoviridae, Podoviridae, and crAss-like viruses, infecting diverse bacterial taxa of Bacteroidetes, Firmicutes, Actinobacteria, Verrucomicrobia and Proteobacteria phyla. The proposed new method has a potential for high-throughput screening applications for mass discovery of new phages in different environments.

/pdf/probing-the-dark-matter-of-the-human-gut-phageome-culture-52x5wptf13.pdf

Probing the "Dark Matter" of the Human Gut Phageome: Culture Assisted Metagenomics Enables Rapid Discovery and Host-Linking for Novel Bacteriophages

ΦCrAss001, isolated from human faecal material, is the first member of the extensive crAssphage family to be grown in pure culture. The bacteriophage infects the human gut symbiont Bacteroides intestinalis, confirming in silico predictions of the likely host. Genome analysis demonstrated that the phage DNA is 102 kb in size, has an unusual genome organisation and does not possess any obvious genes for lysogeny. In addition, electron microscopy confirms that φcrAss001 has a podovirus-like morphology. Despite the absence of lysogeny genes, φcrAss001 replicates in a way that does not disrupt proliferation of the host bacterium and is able to maintain itself in continuous host culture.

https://www.biorxiv.org/content/biorxiv/early/2018/06/26/354837.full.pdf

C. Brian Fitzgerald

Papers

ΦCrAss001 represents the most abundant bacteriophage family in the human gut and infects Bacteroides intestinalis.

Probing the "Dark Matter" of the Human Gut Phageome: Culture Assisted Metagenomics Enables Rapid Discovery and Host-Linking for Novel Bacteriophages

ΦCrAss001, a member of the most abundant bacteriophage family in the human gut, infects Bacteroides