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Probing the mechanisms of outer membrane lipid biogenesis

Principal Supervisor: Dr Tim Knowles - School of Biosciences

Co-supervisor: Ian Henderson

PhD project title: Probing the mechanisms of outer membrane lipid biogenesis

University of Registration: University of Birmingham

Project outline:

This project focuses on the biophysical characterisation of the mechanisms involved in outer membrane lipid biogenesis in Gram-negative bacteria with the overall aim of identifying novel targets for the development of the next generation of antimicrobials.

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.

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. At present little is known about this pathway, by probing its structures and function using techniques such as NMR, X-ray crystallography, and X-ray and neutron scattering, potential druggable pockets will be identified and allow the identification of 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.

References:

  1. 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.
  2. Knowles, T.J., et al., Structure and function of BamE within the outer membrane and the beta-barrel assembly machine. EMBO Rep, 2011. 12(2): p. 123-8.

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
  • Mutagenesis
  • Enzymatic activity
  • Ligand screening assays
  • Protein structure calculation and modelling
  • Cell-based assays
  • Western blots
  • SDS_PAGE
  • Genome sequencing.

Contact: Dr Tim Knowles, University of Birmingham