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

Lead Research Organisation: University of Warwick
Department Name: School of Life Sciences

Abstract

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

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.

Planned Impact

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.

Publications

10 25 50
 
Description I have successfully engineered a fluorescent bacteriophage and I have discovered the mechanism via which the bacteriophage is able to clear infection in human cells environment. These data are now published (https://www.ncbi.nlm.nih.gov/pubmed/30510202, Sci Rep. 2018 Dec 3;8(1):17559. doi: 10.1038/s41598-018-35859-6. Engineered K1F bacteriophages kill intracellular Escherichia coli K1 in human epithelial cells. Møller-Olsen C, Ho SFS, Shukla RD, Feher T, Sagona AP.).
We have now all the data supporting also a neonatal meningitis model of in vitro phage therapy which are submitted and under revision.
Exploitation Route My data can be used by others in many ways: the fluorescent phage that I have constructed together with the CRISPR technology of selection that I established with collaborators, is available to other researchers. Additionally, my findings open a new field in phage biology and give hope for future phage therapy. I already have great interest from industry and many collaborations, as based on my results, bacteriophages can used efficiently also as diagnostics. The impact of my results is economic & social, since my results have a direct effect in society, economy and health sector.
Sectors Education,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

URL https://www.ncbi.nlm.nih.gov/pubmed/30510202
 
Description An RFP bacterial strain I constructed together with methodology I have developed, have been used by Biocote Ltd, in a collaboration that we have recently established, which has received attention from media (https://www.biocote.com/biocote-technology-in-action/): 2018: University footage shows bug-killing effects of silver-treated surfaces https://www.google.co.uk/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&cad=rja&uact=8&ved=0ahUKEwipnfSwnMvZAhVKKsAKHfXwCJYQFggpMAA&url=https%3A%2F%2Fwww.medicalplasticsnews.com%2Fnews%2Funiversity-footage-shows-bug-killing-effects-of-silver-treat%2F&usg=AOvVaw0wXvj0s8lx9IvSN3zVK_sd 2017: New time-lapse imaging shows superbug killed in two hours https://www.buildingbetterhealthcare.co.uk/news/article_page/New_timelapse_imaging_shows_superbug_killed_in_two_hours/136910 https://www.news-medical.net/news/20171213/Time-lapse-imaging-reveals-how-antimicrobial-treated-surfaces-kill-superbugs-in-two-hours.aspx https://www.parallelstate.com/news/time-lapse-imaging-reveals-how-antimicrobial-treated-surfaces-kill-superbugs-in-two-hours/590388
Sector Education,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Societal,Economic,Policy & public services

 
Description Detection of CF lung pathogens using engineered bacteriophages
Amount £46,000 (GBP)
Funding ID VIA045 
Organisation Cystic Fibrosis Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2017 
End 12/2022
 
Description Overcoming the opportunistic pathogen Acinetobacter baumannii in Thailand- developing bacteriophages as antimicrobial agents.
Amount £100,000 (GBP)
Funding ID ID:332371796 
Organisation British Council 
Department British Council - Newton Fund
Sector Public
Country United Kingdom
Start 04/2018 
End 04/2020
 
Description The Physics of Bacteriophage-coated antimicrobial surfaces
Amount £613,277 (GBP)
Funding ID EP/S001255/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 12/2019 
End 05/2021
 
Title Development and observation of fluorescently genetically modified bacteriophages in human cellls 
Description We developed and published methodology to detect genetically modified fluorescent bacteriophages and their effects inside infected human cells. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Year Produced 2018 
Provided To Others? Yes  
Impact This is research with significant long term impact on the understanding of phage therapy in vitro. 
URL https://www.ncbi.nlm.nih.gov/pubmed/30510202
 
Description Control of gene expression in pathogenic Enteroaggregative Escherichia coli. 
Organisation University of Birmingham
Department School of Immunity and Infection
Country United Kingdom 
Sector Academic/University 
PI Contribution We have started a collaboration with Professor Stephen Busby and his team, via having a joint PhD student who is currently learning bacteriophage methods in my lab and she will continue her PhD in the Busby lab, with my supervision on the bacteriophage aspect of the project.
Collaborator Contribution The Busby lab has expertise amongst others in site-directed mutagenesis on bacterial strains and this methodology will be contributed from the Busby lab to our joint project.
Impact This collaboration has just started, the joint PhD student with the Busby lab is currently in my lab learning phage methodology. This is a multidisciplinary collaboration, consisting of microbiology methods, synthetic biology and human cell biology.
Start Year 2019
 
Description Encapsulating bacteriophages in liposomes 
Organisation Loughborough University
Department Department of Chemical Engineering
Country United Kingdom 
Sector Academic/University 
PI Contribution Together with Dr Danish Malik, we have started a project in which we have encapsulated K1F phage into liposomes and we have tested their PH dependent killing efficiency in human cells, infected with the host pathogen. We have written a manuscript together, to be submitted soon. I have provided the phage, the host bacteria, the microscopy expertise and the human cell biology expertise.
Collaborator Contribution Dr Malik's lab has provided the phage encapsulation expertise and the relevant experiments to measure the PH dependent phage release and stability.
Impact We have nice results together and we have written a manuscript to be submitted very soon.
Start Year 2017
 
Description Engineering non-proliferative K1F phages 
Organisation University of Texas at Austin
Country United States 
Sector Academic/University 
PI Contribution I am currently constructing the non-proliferative K1F phages.
Collaborator Contribution Professor Ian J Molineux from University of Texas at Austin is helping me with the design of the constructs needed to engineer non-proliferative K1F phages.
Impact I am currently do the necessary cloning needed for this assay.
Start Year 2016
 
Description Establishment of CRISPR/Cas9 for recombinant phage selection 
Organisation Hungarian Academy of Sciences (MTA)
Department Biological Research Centre (BRC)
Country Hungary 
Sector Academic/University 
PI Contribution We are trying together to establish an effective CRISPR/Cas9 system for phage selection
Collaborator Contribution The partner (Dr Tamás Fehér), is providing his technical expertise in genetic engineering in order to establish an effective system for CRISP/Cas9 as selection method.
Impact We are currently do experiments in parallel in order to establish an efficient system of CRISPR/Cas9 for phage selection, which seems to work well and we will write a manuscript soon about it.
Start Year 2016
 
Description Immobilizing phages on surfaces and track by microscopy and modelling the interplay between phage and bacteria. 
Organisation University of Edinburgh
Department School of Physics and Astronomy
Country United Kingdom 
Sector Academic/University 
PI Contribution I have constructed a T4-GFP phage to be used for the model system that we are building. Together with Dr Aidan Brown and Fixed-Phage Ltd, we have submitted an EPSRC Industrial Fellowship grant, in which I am listed as co-Investigator. This has passed the first round and we are waiting for the final results soon.
Collaborator Contribution Dr Aidan Brown and Fixed-Phage Ltd have used the phage I constructed and have started doing some immobilization and advanced microscopy/modeling experiments to understand the interplay between phage and bacteria. This collaboration will be further enhanced upon our funding success, which will enable us to work more actively in this project.
Impact I have constructed a T4-phage GFP and the Edinburgh part has been working experimentally on that. We expect to have soon funding success and common publications.
Start Year 2017
 
Description Rapifage Ltd, Phage-based diagnostics 
Organisation DSV
Department DSV UK
Country United Kingdom 
Sector Private 
PI Contribution I presented in the Scientific Entrepreneurship module of the Innovation and the Business of Science Course (Royal Society in partnership with Imperial College), suggested for BBSRC fellows, my idea on phage-based diagnostics and I got contacted by one of the founders of DSV to start a spin-out company with them. Together with Dr Richard Amaee from DSV we have initialized Rapifage Ltd. We have received money from DSV (http://deepscienceventures.com/) and investors and this has enabled me to recruit a senior research technician for 6 months to work on this project.
Collaborator Contribution Dr Richard Amaee has done the commercial search, initialized the spin-out formally and has provided the funds from DSV and investors.
Impact We have started Rapifage Ltd together and we recruited a senior research technician who started in my lab in January 2018 to work on this project. We have now renamed the company Lucidix BioLabs.
Start Year 2017
 
Company Name Rapifage Ltd 
Description Rapifage was initiated by Dr Richard Amaee in collaboration with myself, based on the phage-based diagnostics idea that I presented in the Scientific Entrepreneurship module organized for Fellows. We aim to engineer phage-based diagnostics that can be used for a discrimination between viral-bacterial infections. 
Year Established 2017 
Impact If successful, this company will have great impact in the society, health sector and in the economy.
 
Description Warwick Knowledge Center, 'Going viral: What are bacteriophages and how can they help us?' 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Undergraduate students
Results and Impact Warwick Knowledge Center, 'Going viral: What are bacteriophages and how can they help us?'
https://warwick.ac.uk/newsandevents/knowledgecentre/science/life-sciences/bacteriophages
Year(s) Of Engagement Activity 2019
URL https://warwick.ac.uk/newsandevents/knowledgecentre/science/life-sciences/bacteriophages
 
Description 'Antibiotic Awareness week' 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Postgraduate students
Results and Impact The week commencing 18/11/2019, my lab participated in an outreach event as part of the 'Antibiotic Awareness week' organized by Professor Chris Dowson, with 4 posters from my lab exhibited in the University House, University of Warwick.
Year(s) Of Engagement Activity 2019
 
Description 'Science on the Hill' event The Rise of the Superbugs: Will your next infection kill you? 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact • The 10th of December 2019, I gave a talk to the public 'Science on the Hill' event The Rise of the Superbugs: Will your next infection kill you?, and my talk was entitled: 'What are the bacteriophages and how can they help us?'
Year(s) Of Engagement Activity 2019
URL https://warwick.ac.uk/about/publicengagement/events/scienceonthehill/pastevents/AMR
 
Description British Science Festival Event 'Super drugs for superbugs' 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact The 11/09/2019, in Zeeman Building, University of Warwick, I participated in the British Science Festival Event 'Super drugs for superbugs', as part of the panel of experts.
Year(s) Of Engagement Activity 2019
 
Description Interdisciplinary event (WALL-E), as part of the Welcome Week 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Undergraduate students
Results and Impact The 26/09/2019, in The Oculus Building, University of Warwick, I participated in an Interdisciplinary event (WALL-E), as part of the Welcome Week and funded by Warwick International Higher Education Academy (WIHEA), in the panel of experts related to this event.
Year(s) Of Engagement Activity 2019
 
Description One day visit and training of year 12 students in my lab 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact The 06/07/2016, I accommodated two year 12 students from Bishop Ullathorne Catholic School in my lab, and together we did cloning experimens which were successful.
Year(s) Of Engagement Activity 2016
 
Description Organise the "Alternative antimicrobials" workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact On Wednesday 26 October, I organised the Alternative Antimicrobial Methods Workshop, which combined the expertise of biotech companies (Fixed-Phage UK, APS Biocontrol Ltd UK, Avidbiotics USA and Centauri Therapeutics Ltd UK), and academia (Dr Mark
A Webber, University of Birmingham, Professor Martha Clokie, University of Leicester, Dr Jess Healy, UCL, Dr James Spencer, University of Bristol and Dr Antonia Sagona, University of Warwick). The speakers presented research on alternative antimicrobial strategies, including phages, bacteriocins, aptamers and novel bacterial targets. The audience consisted of academics and researchers from varied disciplines (SLS, Chemistry, Physics, Medicine and Engineering).
Year(s) Of Engagement Activity 2016
URL https://www2.warwick.ac.uk/fac/sci/lifesci/people/asagona/alternativeantimicrobials/
 
Description Participation in School of Life Sciences outreach event Getting to grips with antibiotic resistance 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact The 15/11/2016, I participated in the SLS Outreach event, University of Warwick: Getting to grips with antibiotic resistance, led by Dr David Roper and Professor Christopher Dowson, where I presented the work of my lab both with a poster and a demo microscopy event.
Year(s) Of Engagement Activity 2016
URL http://www2.warwick.ac.uk/fac/sci/lifesci/intranet/staffpg/support/comms/slsupdate/sls_update_decemb...
 
Description Participation of my Master student in an outreach event 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact The 25/01/2017, a Master student from my lab, Stanley Ho participated in an Outreach event in Physics Department, University of Warwick, organised by XMas Science Gala and he presented "Phage therapy in the battle of AMR".
Year(s) Of Engagement Activity 2017
URL http://www2.warwick.ac.uk/fac/cross_fac/xmas/impact/xmas_scientist_experience_2016/xmas_science_gala...
 
Description Participation of my lab in the New Scientist Live event 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact My lab participated in the New Scientist Live event (28th September-1st October), in London.
Year(s) Of Engagement Activity 2017
 
Description Participation via interview 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Participation via interview:
https://www.whatisbiotechnology.org/index.php/science/summary/phage-therapy/phage-therapy-uses-viruses-that-attack-bacteria-to-treat
Year(s) Of Engagement Activity 2019
URL https://www.whatisbiotechnology.org/index.php/science/summary/phage-therapy/phage-therapy-uses-virus...
 
Description Public Science Evening " Back to the Future: How Studying History Helps Medical Science" 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact The 12th of March 2019, my lab and myself participated in the Public Science Evening: " Back to the Future: How Studying History Helps Medical Science", held in atrium of School of Life Sciences, University of Warwick. I gave a talk and my lab members demonstrated different phage methods.
Year(s) Of Engagement Activity 2019
 
Description SLS outreach activity in All Saints, Leek Wootton Primary School 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact I organised an outreach activity in All Saints, Leek Wootton Primary School (08/06/2016), with the help of two PhD students who are part of Warwick School of Life Sciences Outreach. It included a presentation about bacteria, viruses and phages, then the class was asked to build phages from card, and there was also a session of microscopy, during which the class observed samples from different bacterial pathogens. Finally, the class was asked to fill in a questionnaire which was very successful, since it seems they understood what I presented. The audience were all the year 6 students of the School and their teachers.
Year(s) Of Engagement Activity 2016
URL http://www2.warwick.ac.uk/fac/sci/lifesci/intranet/staffpg/support/comms/slsupdate/sls_update_july_2...
 
Description School Visit in All Saints Leek Wootton Academy 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact I visited the school to discuss about bacteria, phages and gut microbiome with children from the Reception Class. I gave a short talk and then I had prepared different games using bacteria and phage soft toys as well as tubes to mimic the gut. I explained to the children about the gut microbiome and we played relevant games on that as well. In the end, the children could understand and tell which were the good and bad bacteria.
Year(s) Of Engagement Activity 2018
 
Description School visit in All Saints Leek Wootton Primary School 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Due to its success last year, All Saints Leek Wootton Primary School asked me to visit again this year and familiarize the students with phage therapy.
Year(s) Of Engagement Activity 2017
 
Description Supporting Women's Careers in Science 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact I am part of the Athena Swan Committee in School of Life Sciences, responsible for Outreach activities.
As part of this activity, in July 2017, I organized an Athena Swan meeting " Supporting Women's
Careers in Science" (http://www2.warwick.ac.uk/fac/sci/lifesci/athenaswan/news/careers,
http://www2.warwick.ac.uk/fac/sci/lifesci/athenaswan/#hp-03-tab), based around career pathways and
work life balance, funded by Athena Swan and School of Life Sciences, University of Warwick, which
was very successful. Due to its success, we are now planning to organize a follow-up event this
summer as well.
Year(s) Of Engagement Activity 2017
URL http://www2.warwick.ac.uk/fac/sci/lifesci/athenaswan/news/careers