Principal Supervisor: Dr Antonia Sagona - SLS
Co-supervisor: Dr Freya Harrison - SLS
PhD project title: Synthetic engineering of in vitro and ex vivo models of phage therapy for E.coli K1 lung infections
University of Registration: School of Life Sciences
The interest in phage therapy has grown increasingly over the past decade, due to the emerging problem of antibiotic resistance in many bacterial pathogens. A major challenge to patient safety is the hospital infections due to gram-negative bacteria resistant to antibiotics. A well-defined strain of this kind is E. coli O18:K1:H7, responsible for neonatal meningitis and sepsis and acute cystitis (1), as well as pneumonia in elderly or more susceptible patients with prior history of urinary tract infections (2,3). The most specific characteristic of this strain is the K1 capsular polysaccharide (K1 antigen), an α-2-8-linked homopolymer of sialic acid (NeuNAc), which is essential for the virulence of invasive E. coli. It provides the bacterium with natural antiphagocytic barrier but also structural similarities between K1, and human tissue components indicate that immune tolerance may also be a factor of capsular E. coli pathogenesis (4).
One of the possible solutions to this problem is the use of bacteriophages as antimicrobial agents. However, there are still concerns for phage therapy, over the potential for immune response, rapid toxin release by the lytic action of phages and difficulty of dose determination in clinical situations. Additionally, there is not much knowledge in the cell biology lying behind phage therapy, due to the challenges in the field, and that is an obstacle in the rapid progress of phage therapy.
In order to overcome these hurdles and establish sound experimental data on phage therapy, we propose to synthetically engineer an in vitro model system and an ex vivo lung system as tools for phage therapy against E. coli O18:K1:H7 lung infections. The in vitro system will consist of 3 parts: a synthetic (recombinant) fluorescent non-proliferative phage (so as to decrease the endotoxin release caused by phage lysis in human cells) able to target the pathogen inside mammalian cell environment by means of confocal and live microscopy, the pathogen (E. coli K1) and lung epithelial mammalian cells to test the phage-bacterium interplay mimicking the conditions of the human body. Accordingly, the ex vivo model will consist of pig lung tissue which will be infected with E. coli K1 and treated with the corresponding phage. For the specific research proposal, the phage that will be used is K1F. This phage was first isolated in sewage (5) and can target the K1 polysaccharide capsule of pathogenic E.coli. Like several other K1 capsule-specific phages, K1F encodes an endo-neuraminidase (endosialidase) that is part of the tail structure.
The specific objectives are the following:
- Engineer a synthetic fluorescent K1F phage, with the addition of GFP to the capsid of the phage via homologous recombination and CRISPR/Cas9 selection.
- Engineer the phage to be non-proliferative, by deleting the RNA polymerase and express it in trans (trans complementation) in a lab controlled manner, in order to decrease the endotoxin release cause by the phage’s lysis.
- Establish an in vitro model system of phage therapy against E.coli K1 lung infections, consisting of a human lung epithelial cells infected with E.coli K1 and treated with fluorescent non-proliferative K1F phage.
- Establish an ex vivo model system of phage therapy against E.coli K1 lung infections.
- Compare the suitability of the two systems based on the killing efficiency of the phage and the recovery of the cells/tissue, for exploring different aspects of phage biology. A combination of synthetic biology, microbiology and microscopy methods will be implemented towards the objectives.
- Johnson JR et al., 2005. Antimicrobial-resistant and extraintestinal pathogenic Escherichia coli in retail foods. The Journal of infectious diseases 191:1040-9
- Sligl WI et al., 2015. Nosocomial Gram-negative bacteremia in intensive care: epidemiology, antimicrobial susceptibilities, and outcomes. International Journal of Infectious Diseases 37:129-34.
- Berk SL et al., 1982. Escherichia coli pneumonia in the elderly with reference to the role of E. coli K1 capsular polysaccharide antigen. American Journal of Medicine 72(6):899-902.
- Silver RP et al.,. The K1 capsular polysaccharide of Escherichia coli. Reviews of infectious diseases 10 Suppl 2:S282-6
- Scholl D et al.,. 2005. The genome of bacteriophage K1F, a T7-like phage that has acquired the ability to replicate on K1 strains of Escherichia coli. J Bacteriol 187:8499-503
BBSRC Strategic Research Priority: Molecules, cells and systems
Techniques that will be undertaken during the project:
- Synthetic biology techniques
- Cell biology techniques (confocal and live microscopy)
- Biochemistry techniques
- Microbiology techniques
Contact: Dr Antonia Sagona, University of Warwick