Transmission of crAsspahge in the microbiome

Big questions in the microbiome field surround the topic of microbiome acquisition. Where do we get our first microbes from? What determines the microbes that colonize our guts form birth, and how do they change over time? What short and long term impacts do these microbes have on the immune system, allergies or diseases? What impact do delivery mode and breastfeeding have on the infant microbiome?

A key finding from the work was that mothers and infants often share identical or nearly identical crAssphage sequences, suggesting direct vertical transmission. Also, I love heatmaps.

As you might expect, a major source for microbes colonizing the infant gut is immediate family members, and the mother is thought to be the major source. Thanks to foundational studies by Bäckhed, Feretti, Yassour and others (references below), we now know that infants often acquire the primary bacterial strain from the mother’s microbiome. These microbes can have beneficial capabilities for the infant, such as the ability to digest human milk oligosaccharides, a key source of nutrients in breast milk.

The microbiome isn’t just bacteria – phages (along with fungi and archaea to a smaller extent) play key roles. Phages are viruses that predate on bacteria, depleting certain populations and exchanging genes among the bacteria they infect. Interestingly, phages were previously shown to display different inheritance patterns than bacteria, remaining individual-specific between family members and even twins (Reyes et al. 2010). CrAss-like phages are the most prevalent and abundant group of phages colonizing the human gut, and our lab was interested in the inheritance patterns of these phages.

We examined publicly available shotgun gut metagenomic datasets from two studies (Yassour et al. 2018, Bäckhed et al. 2015), containing 134 mother-infant pairs sampled extensively through the first year of life. In contrast to what has been observed for other members of the gut virome, we observed many putative transmission events of a crAss-like phage from mother to infant. The key takeaways from our research are summarized below. You can also refer my poster from the Cold Spring Harbor Microbiome meeting for the figures supporting these points. We hope to have a new preprint (and hopefully a publication) on this research out soon!

  1. CrAssphage is not detected in infant microbiomes at birth, increases in prevalence with age, but doesn’t reach the level of adults by 12 months of age.
  2. Mothers and infants share nearly identical crAssphage genomes in 40% of cases, suggesting vertical transmission.
  3. Infants have reduced crAssphage strain diversity and typically acquire the mother’s dominant strain upon transmission.
  4. Strain diversity is mostly the result of neutral genetic variation, but infants have more nonsynonymous multiallelic sites than mothers.
  5. Strain diversity varies across the genome, and tail fiber genes are enriched for strain diversity with nonsynonymous variants.
  6. These findings extend to crAss-like phages. Vaginally born infants are more likely to have crAss-lke phages than those born via C-section.

References
1. Reyes, A. et al. Viruses in the faecal microbiota of monozygotic twins and their mothers. Nature 466, 334–338 (2010).
2. Yassour, M. et al. Strain-Level Analysis of Mother-to-Child Bacterial Transmission during the First Few Months of Life. Cell Host & Microbe 24, 146-154.e4 (2018).
3. Bäckhed, F. et al. Dynamics and Stabilization of the Human Gut Microbiome during the First Year of Life. Cell Host & Microbe 17, 690–703 (2015).
4. Ferretti, P. et al. Mother-to-Infant Microbial Transmission from Different Body Sites Shapes the Developing Infant Gut Microbiome. Cell Host & Microbe 24, 133-145.e5 (2018).

What is crAssphage?

CrAssphage is a like mystery novel full of surprises. First described in 2014 by Dutilh et al., crAssphage acquired it’s (rather unfortunate, given that it colonizes the human intestine) name from the “Cross-Assembly” bioinformatics method used to characterize it. CrAssphage interests me because it’s prevalent in up to 70% of human gut microbiomes, and can make up the majority of viral sequencing reads in a metagenomics experiment. This makes it the most successful single entity colonizing human microbiomes. However, no health impacts have been demonstrated from having crAssphage in your gut – several studies (Edwards et al. 2019) have turned up negative.

Electron micrograph of a representative crAssphage, from Shkoporov et al. (2018). This phage is a member of the Podoviridae family and infects Bacteroides Intestinalis.

CrAssphage was always suspected to predate on species of the Bacteroides genus based on evidence from abundance correlation and CRISPR spacers. However, the phage proved difficult to isolate and culture. It wasn’t until recently that a crAssphage was confirmed to infect Bacteroides intestinalis (Shakoporov et al. 2018). They also got a great TEM image of the phage! With crAssphage successfully cultured in the lab, scientists have begun to answer fundamental questions about its biology. The phage appears to have a narrow host range, infecting a single B. intestinalis strain and not others or other species. The life cycle of the phage was puzzling:

“We can conclude that the virus probably causes a successful lytic infection with a size of progeny per capita higher than 2.5 in a subset of infected cells (giving rise to a false overall burst size of ~2.5), and also enters an alternative interaction (pseudolysogeny, dormant, carrier state, etc.) with some or all of the remaining cells. Overall, this allows both bacteriophage and host to co-exist in a stable interaction over prolonged passages. The nature of this interaction warrants further investigation.” (Shakoporov et al. 2018)

Further investigation showed that crAssphage is one member of an extensive family of “crAss-like” phages colonizing the human gut. Guerin et al. (2018) proposed a classification system for these phages, which contains 4 subfamilies (Alpha, Beta, Delta and Gamma) and 10 clusters. The first described “prototypical crAssphage” belongs to the Alpha subfamily, cluster 1. It struck me how diverse these phages are – different families are less than 20% identical at the protein level! When all crAss-like phages are considered, it’s estimated that up to 100% of individuals cary at least one crAss-like phage, and most people cary more than one.

Given the high prevalence of crAss-like phages and their specificity for the human gut, they do have an interesting use as a tracking device for human sewage. DNA from crAss-like phages can be used to track waste contamination into water, for example (Stachler et al. 2018). In a similar vein, our lab has used crAss-like phages to better understand how microbes are transmitted from mothers to newborn infants. The small genome sizes (around 100kb) and high prevalence/abundance make these phages good tools for doing strain-resolved metagenomics. Trust me, you’d much rather do genomic assembly and variant calling on a 100kb phage genome than a 3Mb bacterial genome!

Research into crAss-like phages is just beginning, and I’m excited to see what’s uncovered in the future. What are the hosts of the various phage clusters? How do these phages influence gut bacterial communities? Do crAss-like phages exclude other closely related phages from colonizing their niches, leading to the low strain diversity we observe? Can crAss-like phgaes be used to engineer bacteria in the microbiome, delivering precise genetic payloads? This final question in the most interesting to me, given that crAss-like phages seem relatively benign to humans, yet incredibly capable of infecting our microbes.

References
1.Dutilh, B. E. et al. A highly abundant bacteriophage discovered in the unknown sequences of human faecal metagenomes. Nature Communications 5, 4498 (2014).
2.Edwards, R. A. et al. Global phylogeography and ancient evolution of the widespread human gut virus crAssphage. Nature Microbiology 1 (2019). doi:10.1038/s41564-019-0494-6
3.Guerin, E. et al. Biology and Taxonomy of crAss-like Bacteriophages, the Most Abundant Virus in the Human Gut. Cell Host & Microbe 0, (2018).
4.Shkoporov, A. N. et al. ΦCrAss001 represents the most abundant bacteriophage family in the human gut and infects Bacteroides intestinalis. Nature Communications 9, 4781 (2018).
5.Stachler, E., Akyon, B., de Carvalho, N. A., Ference, C. & Bibby, K. Correlation of crAssphage qPCR Markers with Culturable and Molecular Indicators of Human Fecal Pollution in an Impacted Urban Watershed. Environ. Sci. Technol. 52, 7505–7512 (2018).