Principal Supervisor: Dr Antonia Sagona, School of Life Sciences
Co-supervisor: Professor David Roper, School of Life Sciences
PhD project title: Investigation of the localization of Filamentous temperature sensitive protein Z (FtsZ) in pathogenic E.coli as a biomarker for antibacterial agents.
University of Registration: University of Warwick
The problem of antimicrobial resistance is very severe worldwide and new approaches are required to tackle it. Part of the problem in relation to human health is the presence of highly resistant bacteria to small molecule antibiotics in community and hospital settings, leading to delayed treatment outcomes, reinfection or even death1. In this project we wish to develop and explore a model system for host-pathogen interaction in the context of bacterial pathogenesis and its utility in the study and discovery on novel antibacterial agents.
We will use a non-pathogenic form of E. coli O18:K1:H7 (strain EV36) in conjunction with a human cerebral microvascular endothelial cell line (hCMEC/D3) 2 to establish an in-vitro neonatal meningitis model. E. coli O18:K1:H7 is responsible for secondary infections in burn patients 3, neonatal meningitis and sepsis and acute cystitis 4. Our approach will be to monitor the growth of rod shaped E. coli in this human cell line infection model focussing on the cell division protein FtsZ as a marker for bacterial cell division and growth. FtsZ: Filamentous temperature sensitive protein Z, is the bacterial homolog of tubulin 5 and is absolutely required for cell division in E. coli and other bacterial species. A variety of known antibacterial compounds are known to cause aberrant cell division that correlate with the mis-localization of FtsZ in the bacterial cell, leading to defective cell division that can result in clearage of the infection.
In this project, we will use confocal microscopy and advanced electron microscopy techniques, to study the change in structure and localization of the FtsZ protein in the infection model. Importantly we have already established a “tool kit” of reagents, cell lines and background knowledge to support this project. We will first develop a cell culture model of the blood-brain barrier consisting of human cerebral microvascular endothelial cell line (hCMEC/D3) 2 before infecting these cells with E.coli O18:K1:H7 (using the non-pathogenic hybrid K1/K12 strain EV36) which we have already made fluorescent, by transforming a RFP plasmid into its genome. This will enable the visualisation of the bacterial cells and their interaction with the human cell line by live and confocal microscopy. We will further observe localization and structure of FtsZ with confocal and advanced electron microscopy using antibody staining of fixed cells to probe the fine structure of the cells as well as FtsZ, once infected with E. coli O18:K1:H7. It has been shown previously that certain bacteriophages and their related peptides are able to block E.coli cytokinesis via the inhibition of FtsZ assembly 6. As a further step, we will investigate the potential effect of K1F phage 7, which can specifically target the K1 polysaccharide capsule of pathogenic E. coli K1, in E.coli K1 cell division and the relevant assembly of FtsZ involved in this process. To this end, we will apply K1F phage using the same cell model and we will observe the structure and localization of FtsZ upon phage addition with the methodology described as above. Our final goal is to investigate whether FtsZ localization and structure can be used as an antibacterial biomarker for potential agents including bacteriophages.
Aims/objectives: The aim of this project is to understand by following the structure and localization of FtsZ inside human brain cells and under different conditions, whether this could be used as an antibacterial biomarker.
The specific objectives are:
- Establish a cell culture model of the blood-brain barrier, consisting of human cerebral microvascular endothelial cell lines.
- Infect the cells with fluorescent E.coli O18:K1:H7 and observe by microscopy the progress of infection, using suitable software.
- Stain with suitable antibodies for FtsZ under these conditions.
- Repeat the staining and analyse the localization upon inhibition of FtsZ, using different inhibitors of cell growth as controls.
- Apply bacteriophage K1F into the culture and further test Ftsz localization and structure.
A combination of live, confocal and electron microscopy, molecular biology, structural biology, biochemistry, microbiology and human cell culture will be implemented towards the objectives.
- Peleg, A. Y. & Hooper, D. C. The New England journal of medicine 362, 1804-1813 (2010).
- He, Y. et al. Stroke. August ; 45(8): 2514–2526. (2014).
- Busch, N. A. et al. Infect Immun 68, 3349-3351 (2000).
- Johnson, J. R., Kuskowski, M. A., Smith, K., O'Bryan, T. T. & Tatini, S. The Journal of infectious diseases 191,1040-1049 (2005).
- Eswara, P.J. et al. Annu Rev Microbiol Sep 8;71:393-411 (2017).
- Haeusser, D.P. et al. PLoS Genet. Mar 20;10(3):e1004217 (2014).
- Scholl, D. & Merril, C. J Bacteriol 187, 8499-8503, (2005).
BBSRC Strategic Research Priority: Molecules, Cells and Systems
Techniques that will be undertaken during the project:
Human cell culture
Contact: Dr Antonia Sagona, School of Life Sciences