Studying phage-host coevolution in the lab

Raise your hand if you've used the terms "coevolution" or "arms race" to describe how phages and their hosts interact. Chances are, if you've written a grant, a paper, an abstract or a scholarship application about phages, you've done it. But how is this actually measured in the lab, and what can it tell us?

The problem:

Collectively, we don't know much about phage-host interactions on a molecular level, but we're already using phages to treat antibiotic-resistant infections and making plans to use phages to manipulate the microbiome. This is a problem that can (and should) be fixed.

For most phages:

• We don't know which parts of their hosts are important for phage infection (e.g. bacterial surface receptors are unknown)
• We don't know how bacteria evolve phage resistance (what do they change about themselves?), nor do we know much about the properties of phage-resistant bacteria (are they more or less pathogenic? More or less antibiotic-susceptible?)
• We don't know how phages respond to these newly-evolved phage-resistant bacteria

The paper:

Stephen Wandro and colleagues have elegantly shown how they've come closer to answering these questions for a strain of Enterococcus faecium and a phage that targets this pathogen.

First of its kind?

This isn't the first time this kind of experiment has been done on a phage-host pair (E. coli and Pseudomonas fluorescens have been well-studied in this manner), but this group has done it using an opportunistic gut pathogen (isolated from human stool). This is especially interesting because we don't know much about phage-host interactions in the gut yet.

A few things up front:

• Everything was done in vitro, and the authors studied one bacterial strain and one phage.
• The bacterial host: Enterococcus faecium (isolated from human stool, obtained from BEI Resources)
• The phage: a Myovirus called EfV12-phi1 (isolated from sewage; obtained from the Felix d'Herelle Center)

The experiment:

• Grow phage and bacteria together. Twice a day for about a week (~53 generations total), transfer some of the mixture to a flask of fresh growth medium. Follow bacterial growth dynamics / phage susceptibility over time. At the same time, track phage and bacterial sequences to watch which genes change.
• The controls: 1) Do the same, except transfer just the phages to naïve bacteria each time (follow what happens when only the phage is allowed to evolve). 2) Do the same with bacteria grown on their own (follow changes in the bacteria that have nothing to do with the phage).
• Their hypothesis: that they'd see changes in phage tail fibers and bacterial surface receptors over time.

(Some of) what they found:

Coevolution dynamics:

• When the host was allowed to evolve alongside the phage, one of two things happened: phage-resistant cells emerged, then were overcome by the evolving phage, then new phage-resistant cells re-emerged, OR phage-resistant mutant cells emerged and then held strong, with no further population crashes
• When phages were introduced onto naïve hosts each time, the phage population was soon able to consistently clear the culture

Host genome changes:

• Mutations in the host consistently accumulated in two genes: yqwD2 (capsular polysaccharide production) and rpoC (RNA polymerase)

Phage genome changes:

• Regardless of whether the bacteria were allowed to evolve or not, three phage genes were consistently altered: a capsid gene and two hypothetical genes.
• There was one phage mutation that consistently occurred ONLY when the host was allowed to co-evolve: this was a duplication of a portion of the tail fiber gene.

In conclusion:

Not only did this group show coevolutionary dynamics of a new phage-host pair representing an understudied, clinically-relevant pathogen, but they showed exactly which genes change over time during this coevolution. Importantly, these dynamics were predictable.

As a bonus, they highlighted an unexpected mechanism by which the phage is able to alter its tail fiber gene, and their results suggest that this might happen in response to the host altering its extracellular sugars (capsule).

So many new questions!

Does this mean the phage receptor is part of the host's capsule? Does the host become more or less pathogenic (or more or less antibiotic resistant) when it evolves changes in its capsule genes? What happens if you combine this phage with a phage that DOESN'T provoke changes in the capsule, but instead leads to changes in something else... is the cell able to manage? What happens if you combine this phage with an antibiotic that targets RNA polymerase? Do the phage-induced changes in rpoC alter the cell's susceptibility to the drug?

Let's do more experiments like this one!

Doing studies like this one with other phage-host pairs will give us the power to more accurately predict phage-bacterial interaction outcomes. Armed with this kind of insight, we'll be better able to make decisions about which phages to use on which pathogens, and whether or not to combine them with antibiotics, other phages, both, or neither.

Thoughts?

Phage researchers and those in the phage therapy industry: are you doing these kinds of experiments on your favourite phages? Why or why not? Email us at capsid@phage.directory or tweet @phagedirectory using the hashtag #coevolution if you'd like to weigh in.

Thanks for reading! – Jessica <>={

Main Source:

Wandro et al. 2019. Predictable Molecular Adaptation of Coevolving Enterococcus faecium and Lytic Phage EfV12-phi1. Frontiers in Microbiology.