Phage therapy is the therapeutic use of bacteriophages to treat bacterial infections. Bacteriophages, or phages, are viruses that infect bacteria. They are highly specific, often infecting only a single strain of bacteria. Because of this specificity, phage therapy has potential advantages over traditional antibiotic treatment, which can kill beneficial bacteria along with the harmful ones. Phage therapy is seen as a solution to the problem of antibiotic resistance, which is becoming prevalent worldwide. At the current trend, in 26 years, the number of deaths attributed to antibiotic resistance are projected to become comparable with the number of deaths caused by cancer.
This project represents our HTGAA large-scale group research effort, where every participant has the opportunity to contribute to state-of-the-art research using advanced techniques. The potential impact of this work is significant, with the potential to make a real difference in people's lives. For instance, consider the Patterson story, which highlights the transformative power of phage therapy. This Group Final Project is not just about academic advancement, but about making a tangible difference in the global fight against antibiotic resistance.
A famous example: Tom Patterson and Steffanie Strathdee’s story
As evidenced in the Patterson story, a significant challenge in phage therapy is the ability of bacteria to rapidly develop resistance to the phages. In Patterson's case, the initial phage cocktail became ineffective after a few days. Consequently, another cocktail was developed and administered. Once again, it became ineffective after a short period. It was not until the third cocktail was introduced that the patient was finally cured. This highlights the need for continual monitoring and adaptation in phage therapy, reflecting the dynamic nature of bacterial resistance.
You can find a detailed introduction into bacteria, phages and phage therapy in our HTGAA Bootcamp Part 1
Despite the great advantages of phage therapy over conventional antibiotics, bacteriophages have a major limitation: Bacteria can develop ways to defend themselves against the phages and become resistant. But, in contrast to the static nature of antibiotics, phages have the power to evolve too. For billions of years, there has been an arms race between phages and bacteria. But now, with the development of new tools in synthetic biology such as protein engineering and the synthesis of new genomes harboring advantageous mutations, we can try to give the phages a head start. We attempt this by engineering their DNA or RNA, so they are prepared in case they encounter bacteria developing a resistance. This is the overall goal of the group final project. Specifically, we want to engineer the bacteriophage MS2 to be more prepared and more efficient in killing its host bacteria Escherichia coli (E. coli).
MS2 bacteriophages infect E. coli bacteria with a high specificity. It is a very small virus consisting of coat proteins, a maturation protein and genetic material. They infect bacteria by attaching to the F-pilin protein on the host cell membrane and entering the cell. Once inside, the viral RNA acts as a messenger for phage protein production. All proteins for virion assembly are translated and the virus RNA is replicated. Next, new viruses form by assembling the coat and maturation proteins and encapsulating the virus RNA. Finally, a lysis protein expressed from the viral RNA triggers bacterial lysis by causing cell wall breakdown, whereby the new phages are released into the environment to infect new bacteria.
To make phages stronger against their host and to prepare them against host resistance, we can introduce mutations into their genes to slightly change the structure and/or function of the encoded proteins. Four genes are present on the MS2 RNA: 1. the maturation protein (A), 2. the coat protein (coat), 3. the lysis protein (L) and 4. the replicase (rep). For this course, we focus on the L gene encoding the lysis protein.
Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5446614/
The exact function of the L protein is unknown, however, it is thought to form oligomers that can then integrate into the cell membrane to form pores, which ultimately lyse and kill the bacterial cell. This lysis protein is crucial for the phage to complete its life cycle and release new phages to infect more bacteria. Because of its importance, E. coli can try to intervene in the lysis protein production to stop the spread of the phages. For the translation and processing of the viral proteins, the phage heavily relies on the bacterial protein machinery. One example for such a protein is DnaJ, a chaperone responsible for proper protein folding, which has been shown to be important for MS2 lysis protein processing. E. coli can mutate this chaperone, preventing the lysis protein from interacting with DnaJ. This in turn causes the lysis protein to lose its function and stops MS2 from infecting more bacteria.