A Double-Edged Sword in the Quest for Phage Spread

I have written about bacteriophages (or simply phages) quite a few times before on this blog. It seems to me that phage research often doesn’t get enough attention. It is somewhat ironic that phages are often overlooked, even though they are the most abundant type of organism on our planet.

Phages come in all shapes and sizes and they are the viruses that only infect bacteria. I’ve heard someone describing the classical T4 phage as the Apollo Lunar Module in miniature. Perhaps Thomas J. Kelly, who designed the lunar lander, did indeed get some inspiration from biology (wouldn’t be the first time for NASA), or it may have been (and quite likely was) a complete coincidence, but I still like the idea. Even though phages are so abundant, our understanding of how they affect the communities and environments they live in is relatively poor. For example, over the last several years the term ‘human microbiome’ has become almost a catchphrase and the study of the microbes living in and on human body is now a very trendy topic in research. However, with all this microbiome hype, most of the studies on the human microbiome completely ignore the presence of phages in us. When I looked at how many papers on PubMed database where published on the human microbiome in 2016 the number was 6,014, but if I added the term bacteriophage to the search bar only 195 articles came up, and that’s throughout all the available years.

6598-01
See the similarities?

One of the reasons bacteriophage research is important is because of the potential developments in phage therapy. We all know that there is a big question mark about the use of antibiotics in the future. Increasing numbers of antibiotic resistant bacteria are being reported every year and we are not very good at finding and making new antibiotics at the rate they are needed. In addition, antibiotic treatment is not the most effective way of killing pathogens. Generally, each antibiotic kills a broad range of bacteria, which means both good and bad bacteria will be killed during the antibiotic treatment. As many of those good bacteria are there to protect us from other pathogens and to supply us with essential nutrients, antibiotic use often has some side effects. By contrast, phage therapy is a very specific type of treatment. Each phage can infect only a single strain of bacteria. Combining several phages allows the targeting of a very specific group of bacteria and spares any of the good bacteria you want to keep. The big issue with getting phage therapy approved has always been that they are “live” agents. By “live” I do not mean that they are living organisms, I do not think viruses are live entities, but they are live in a sense of ‘being affected by the natural selection’. And the effect of natural selection in phage-bacteria interactions is especially notable. Bacteria evolve ways to resist phage infection and phages evolve ways to evade bacterial resistance. This arms-race goes on and on and it is not something that we are very good at controlling.

I have always thought phage therapy was a good idea, and I still do. However, reading a recently published paper on phage spread to resistant bacteria, I realised that we still understand very little about both the phage biology and their impact on bacterial communities. The paper begins with an interesting observation:

If you take a culture flask, add a phage to it and add bacteria that are sensitive to infection by this phage, unsurprisingly this bacterium will not grow because the phage infects it, replicates in it and kills it. However, if to the same flask you add bacterium that should be resistant to the phage infection, the growth of the resistant bacteria is also severely hampered. This is definitely unusual; one would expect the resistant bacteria to remain resistant. Culturing the resistant bacteria just with the phage or just with the sensitive bacteria strain does not affect its growth. So, why is it, that combining sensitive and resistant bacteria and adding the phage to this mix suddenly stops the resistant bacteria from growing? Phages produce proteins called lysins. Lysins are used to lyse the infected cells at the end of the phage life cycle to release the phage progeny. The lysins have been previously shown to be able to lyse different bacterial strains. It turns out that in this case, when the phage infected the resistant cell and produced the lysins within it, the death of the infected bacterium released the lysins into the surroundings. The released lysins bound to the resistant bacteria and lysed their cell walls, killing the bacteria.

Strangely enough, when the localisation of the lysins was investigated further, it was observed that sometimes the lysins are found inside the resistant bacteria rather than on the outside. This observation hinted to the fact that some phages may be actually entering the resistant strains. Indeed, when experiments with labelled phage DNA were conducted, the DNA was also wound inside the resistant cells, suggesting that an active phage infection of resistant cells was taking place. Acquisition of sensitivity, or ASEN, is what the research team has called this phenomenon. ASEN was shown to be a consequence of the ability to acquire foreign surface proteins by the resistant bacteria. ASEN is mediated by the release of membrane vesicles from the sensitive bacteria. The vesicles can either be made by live bacteria or are generated when the bacteria are lysed. As the membranes of the sensitive bacteria contain the receptors for phage binding, the membrane vesicles released by these bacteria will also have the receptors. When these vesicles fuse with the membranes of the resistant bacteria, the resistant strain converts into a sensitive one because it now has the receptor required for binding and entry of the bacteriophage.

Phage bound to membrane vesicle (MV)
Phage bound to the membrane vesicle (MV)

This is the first description of ASEN phenomenon and, if it is widely spread, than we really need to think more carefully about its effects on bacterial communities at large. For example, using the phage therapy to treat gut infections may have unintended adverse effects by also affecting the important commensal (i.e. good-for-you) bacterial species. Interestingly, ASEN may be a double-edged sword. Production of membrane vesicles is a widespread phenomenon. In this specific study, it seems clear that membrane vesicle production is an advantage for the phage. Acquisition of phage receptors by resistant cells increases the phage host range and allows it to increase the progeny numbers. In a different situation the case might be reversed. Marine cyanobacteria also produce a lot of membrane vesicles, however, it has been suggested that the vesicles may be a way of sequestering phages and preventing them from infecting the actual host cells. As the vesicle itself contains the receptor, the phage will bind to it regardless of it not being an actual cell. Consequently, if a large proportion of phages binds to the vesicles then less of them are present to infect the bacterial cells- a win for bacteria in this case.

All in all, the dynamics of phage spread clearly still need to be better studied. Such an abundant entity as bacteriophages deserves more attention. Perhaps “The Lunar Module in Miniature Lands on Mars“ would be a good click bait headline?

Reference:

Tzipilevich, Elhanan, Michal Habusha, and Sigal Ben-Yehuda. “Acquisition of Phage Sensitivity by Bacteria through Exchange of Phage Receptors.” Cell (2016).

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