Bacteriophage therapy is an idea that’s been around for a long time, but although a lot of people have heard of it, not many people really know much about it. The principle is appealing: if someone has a harmful bacterial infection, why not treat it by unleashing a virus that only infects those bacteria? Why isn’t this done more often? Bacteriophages really don’t target human cells, that part of the process isn’t the problem. And no, it’s not a cash grab by Big Pharma to ram antibiotics down everyone’s gullet, either, so strike that one off the list. Nor is is historical bias of some sort against therapies that were more popular in the old Soviet Union – I know that sounds weird, but I’ve actually heard it used as an argument for some reason, probably as an adjunct to the big-pharma one.
No, the reasons that bacteriophage therapy isn’t used much are practical ones. Just as there are a lot of bacteria species in the world, there are an awful lot of bacteriophages that target them. By sheer numbers, there are probably more bacteriophages on this planet than anything else associated with the biosphere (I hesitate to describe them, or any viruses, as “living” themselves). But we know surprisingly little about them. This paper notes that the majority of bacteriophage genes are still unassigned, for example. We don’t know what proteins they code for or what those proteins are doing. So just finding a useful phage for a given infection is one challenge, and culturing it to the point that it can be used is another.
As this new paper says, “Unfortunately, the current production processes for natural and engineered phages pose considerable biosafety concerns and are often relatively inefficient and unreliable.” The biosafety part is due to the possibility of the phage culturing process bringing on more virulence factors in the bacteria under attack – you definitely do not want to be tuning up the pathogen that you’re trying to target, for sure. Even if you do isolate some sort of phage cocktail, it turns out that storing these for dosing is not easy, either, since the phages themselves can be rather unstable and prone to degradation. There never really was a golden age of phage-centric antibacterial treatment, in other words. It was better than nothing, and could sometimes be quite useful, but could easily fail or even make things worse.
So how do we amplify the useful parts while turning down the disadvantages? The authors of that last linked paper are proposing cell-free phage production as an answer. This has been described for some model systems using E. coli extracts, but not for any wider range of pathogens. But here’s the paper’s claim:
Here, we introduce the phage production pipeline ‘‘phactory,’’ which enables on-demand and safe generation of a broad spectrum of phages in a host-independent manner, including transiently engineered and therapeutic phages against antibioticresistant bacteria. This approach has also enabled us to validate the existence of dozens of highly conserved hypothetical proteins, including non-structural proteins. It also allowed us to systematically monitor phage assembly in correlation with its expression profile using time-resolved mass spectrometry. These innovations can significantly foster our understanding of molecular phage biology, accelerate future phage engineering, and improve therapeutic phage production.
It turns out that E. coli components are a good choice, because their ribosomes seem to accept mRNA from other bacterial species pretty readily. Growing phages under these conditions, as mentioned above, lets you track the various proteins being produced by mass spec, and this seems to mimic the situation in vivo (although more slowly than the wild-type), which gives you some ideas about their functions in the phage life cycle. Furthermore, comparing these protein profiles to actual replicating phages in culture gives you some other clues: the nonstructural proteins are comparitively enhanced in the cell-free system, it seems, and these are correspondingly harder to tease out in the traditional cultures.
The cell-free conditions also let you co-express engineered phage proteins in the system for the phages to take up, and this is demonstrated by bringing in a poly-His modified capsid protein and one that contains the “small” side of the split-luciferase Hi-Bit system. This demonstrated that the phages do indeed incorporate these engineered proteins and that the ones that did so can then be purified out of the system separately. You could imagine introducing some useful modifications for therapy that way, without having to actually change the genome of the phages themselves. Indeed, when these modified ones were allowed to infect bacteria, the next generation had none of the modified proteins in them, as well they shouldn’t.
One limitation is that this technique so far depends on isolating phage DNA, which sends you right back to those traditional cultures. But the hope is that this can be done soon with entirely synthetic genomes. Another difficulty is that this is being done with phages with fairly small genomes, and moving to larger ones would both make that synthetic genome idea less practical and make the cell-free culture more tricky, because you’d need more energy sources in there for the relevant enzymes. And of course the dependence on E. coli, although gives you more range than you’d think, will still limit the variety of phages that can be produced. But there may be a way to get around that by mixing cell extracts or just adding some key enzymes themselves.
I certainly don’t expect phage therapy to take over the therapeutic landscape any time soon. But I am glad to see these sorts of techniques applied to phage selection and production, because we’ll need to update those processes to really make this work. There are so many nasty pathogens out there, and antibiotic discovery is so hard, that I hope that phage therapies can make a difference. We need all the help we can get!