Engineering synthetic phages against pathogenic E. coli as an innovative tool for phage therapy

Summary

A major challenge to patient safety is the hospital infections caused by Gram-negative bacteria that are resistant to antibiotics. A well-defined bacterial strain of this kind is Escherichia coli (E. coli) O18:K1:H7, which is responsible for secondary infections in burn patients, neonatal meningitis and sepsis, and acute cystitis. One of the possible solutions to this problem is the use of bacteriophages as antimicrobial agents. Bacteriophages are viruses that infect and kill bacteria. They show high specificity to their bacterial target, while having minimal side effects on the host, so they can potentially be used to treat bacterial infections in humans. However, there are still concerns for phage therapy, over the potential for immune response, rapid toxin release by the phages and difficulty of dose determination in clinical situations. Additionally, little is known about the cell biology underlying phage therapy, due to the challenges in the field, so that has been an obstacle in the rapid progress of phage therapy.
The key aim of the research proposal is to engineer a model system as a tool for phage therapy consisting of 3 parts: a synthetic phage able to target a well-known pathogen, the pathogen (E.coli O18:K1:H7), and mammalian cells to test the phage-bacterium interplay mimicking the conditions of the human body. This system, with proper validation, can be used further for studies, establishing a promising proof of concept for safe phage therapy, which can treat conditions such as infection in burn wound patients, neonatal meningitis or acute cystitis, caused by the target pathogen. A combination of molecular biology, synthetic biology and microscopy methods will be enable me to achieve the objectives. The research will be undertaken in the School of Life Sciences, University of Warwick, in the lab of Professor Alfonso Jaramillo. The University of Warwick has been ranked as the 7th top institutions within the UK according to the Research Excellence Framework 2014. The School of Life Sciences was rated as world-leading (80% of its outputs were rated as world leading or internationally excellent).

Technical Summary
A solution to the emerging problem of antibiotic resistance in many bacterial pathogens is the use of bacteriophages as antimicrobial agents. By the means of molecular biology, synthetic biology and microscopy, I propose to engineer a model system as a tool for phage therapy consisting of synthetic T7 phage able to target Escherichia coli (E. coli) O18:K1:H7, a defined bacterial strain responsible for various human diseases, in a mammalian cell environment. The synthetic T7 phage will be non-lytic, so that the release of endotoxin is minimal and it can therefore be used therapeutically. The synthetic phage will be engineered to encode endosialidase, an enzyme that will allow the phage to recognize and degrade the K1 capsule. The synthetic phage will be engineered to be detectable by microscopy, by fusing an EGFP protein to the major capsid protein of T7 phage. The synthetic phage will be evaluated for its killing efficiency and endotoxin release upon infection of E.coli K1 cells. The model system will be tested in mammalian cells, infected with E. coli O18:K1:H7, using a K-12/K1 hybrid strain (EV36) constructed in the lab. The final goal is to find an efficient and simple way to provide a tool for phage therapy against pathogenic E. coli K1. The synthetic phage should efficiently kill the pathogen (E. coli K1) without harming the mammalian cell environment. The mammalian cells infected by pathogenic E. coli should recover (be healthy with no bacteria) after addition of the synthetic phage. This system, upon proper validation, can be used further for in vivo mouse studies, establishing a promising proof of concept for safe phage therapy.

Impact Summary

More than 20,000 European citizens die every year from untreatable bacterial infections that are resistant to conventional antibiotics. The proposal aims to develop new technologies and foundational advances in Synthetic Biology, by creating phage therapy approaches using recombinant phages made in the lab. From this proposal, the UK economy will benefit, since due to the antibiotic resistance problem, the phage therapy will provide a direct and financially affordable approach for tackling a serious problem. The research will also impact directly on human and animal health and wellbeing through providing an alternative to antibiotics for treating bacterial infections.

The engineering of synthetic phages will have a significant impact in biological manufacturing and the development of novel therapeutics. The modular phage engineering will facilitate future engineering projects aimed at the creation of organism-specific antibiotics and the delivery of non-invasive therapeutics to specific cells.

The engineering-driven focus on healthcare applications aimed at developing new tools for personalized medicine will engage young citizens towards careers in synthetic biology.

This research will have an impact in the industrial biotechnology and bioenergy: the methodology proposed will be useful to engineer biomolecules, phages and bacteria, with the desirable characteristics for each approach.

Phages can also be used in contexts other than phage therapy, such as agriculture, environment and decontamination. Synthetic phages could be used against bacterial pathogens of fresh fruits and plants for food. Phages could also be used in bacterial diseases of wild trees, where small-molecule antibiotics are not an option.

The proposal will increase the public awareness of phage therapy, which will contribute to the engagement of citizens in science.

The impact in biotechnology and medical research industries is very high, as the key aim of the proposal is to produce a new approach for antimicrobials that could be patented and generalised to other systems.

The model system of the application could foster the creation of new start-up companies.

The current proposal is an example of personalized therapeutics for the medical-oriented industry, which will impact on the way medicine, is practiced. It focuses on creating molecular therapies specific to the individual and the pathogen, more efficient therapy development, efficient delivery of personalized drugs, the development of non-invasive treatments for disease, and the production of organism-specific antimicrobials. Overall, the potential impacts on health are significant.