Principal Supervisor: Dr Tim Knowles, School of Biosciences
Co-supervisor: Professor Ian Henderson, Institute of Microbiology and Infection
PhD project title: Lipid transport in gram-negative bacteria.
University of Registration: University of Birmingham
This project focuses on characterising the fundamental process of outer membrane lipid transport in Gram-negative bacteria, a largely unknown process which is only now being revealed.
The emergence of bacteria that are resistant to available antibiotics represents an enormous and growing global threat requiring new targets and strategies to combat infection. Multidrug resistance is most serious for Gram-negative bacteria, with essentially few antibiotics under development or likely to be available for clinical use in the near future. The understanding of the Gram-negative bacterial outer membrane (OM) is critical to developing new antimicrobial agents.
The lipid components of this membrane, lipopolysaccharide in the outer leaflet, and phospholipid in the inner leaflet, are essential for Gram-negative survival, both by acting as an impermeability barrier to hydrophilic molecules but also by acting to slow the penetration of small hydrophobic molecules, explaining their increased resistance to hydrophobic antibiotics and detergents. For many years, how these components got to the outer membrane remained unknown, recently however the mechanism of LPS transport has been uncovered, how lipid gets there, however, still remains enigmatic.
The research in the laboratory of Dr Tim Knowles is focused on elucidating the mechanisms involved in the fundamental processes of OM biogenesis in Gram-negative bacteria and has several important objectives: (1) to provide fundamental information about how Gram-negative bacteria form and therefore by proxy mitochondria and chloroplasts. (2) To provide new opportunities to attenuate bacteria in the pursuit of anti-infective strategies. Current antibiotics predominantly target peptidoglycan synthesis and have been very effective in the past. Targeting OM biogenesis offers the potential for a whole new class of antimicrobials urgently required to stay ahead of bacterial resistance.
Focus - The MLA pathway – Retrograde lipid transport?
Gram-negative bacteria are generally more resistant than Gram-positive bacteria to antibiotics, detergents, and other toxic chemicals because of the sophisticated asymmetry of lipids in their outer membranes. Under stress conditions this asymmetry can be disrupted leading to shedding of LPS and phospholipid migration to the outer leaflet, these phospholipid bilayer patches lead to reduced barrier function and are detrimental to the cell. Recently a 6 protein pathway, the Mla pathway, has been identified that actively prevents phospholipid accumulation at the cell surface. Knocking out any component of this pathway leads to OM barrier defects and increased susceptibility to antibiotic agents in addition to also abolishing virulence in enteroinvasive E. coli and S. flexneri, making all components of the Mla pathway important virulence factors. Furthermore recent evidence from the Knowles lab
suggests that this pathway may also be able to transport lipid to the outer membrane, and therefore would be the first evidence of a mechanism for how lipids get to the outer membrane.
At present almost nothing is known about this pathway, by probing its structures and function using techniques such as NMR, X-ray crystallography and neutron reflectometry, we will gain valuable insight in to the fundamental process of lipid transport and potentially lead to the identification of druggable pockets and novel compounds that will not only abolish virulence but also impede restoration of a damaged OM and therefore increase the effectiveness of already available antibiotics.
To probe the mechanisms by which this fundamental process occurs will require a multidisciplinary approach, working in the fields of both biophysics and molecular biology and using the latest structural biology techniques including nuclear magnetic resonance, neutron and X-ray scattering, neutron reflectometry, electron microscopy and X-ray crystallography.
- Malinverni, J.C. and T.J. Silhavy, An ABC transport system that maintains lipid asymmetry in the gram-negative outer membrane. Proc Natl Acad Sci U S A, 2009. 106(19): p. 8009-14.
- Ekiert, D.C., Bhabha, G., Isom, G.L., Greenan, G., Ovchinnikov, S., Henderson, I.R., Cox, J.S., Vale, R.D. (2017) Architectures of lipid transport systems for the bacterial outer membrane. Cell. 169(2):273-285
BBSRC Strategic Research Priority: Molecules, Cells and Systems
Techniques that will be undertaken during the project:
- Nuclear Magnetic Resonance spectroscopy
- Neutron Scattering
- X-ray Scattering
- X-ray crystallography
- Electron microscopy
- Novel polymer solubilisation techniques
- Mass spectrometry
- Linear dichroism
- Circular dichroism
- Analytical ultracentrifugation
- Isothermal titration calorimetry
- Surface plasmon resonance
- Protein expression and purification
- Enzymatic activity
- Ligand screening assays
- Protein structure calculation and modelling
- Cell-based assays
- Western blots
- Genome sequencing.
Contact: Dr Tim Knowles, School of Biosciences