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Structure-function relationships in bacterial cell walls

Principal Supervisor: Dr Sarah L. Horswell - School of Chemistry Luke Alderwick - School of Biosciences

Co-supervisor: Liam R. Cox

PhD project title: Structure-function relationships in bacterial cell walls

University of Registration: Birmingham

Project outline: 

Mycobacterium tuberculosis, the causative agent of TB, has a unique cell envelope which differs substantially from the cell wall of both Gram-negative and Gram-positive bacteria. This results in very low permeability, thereby contributing to resistance against common antibiotics. The main structural element consists of a cross-linked network of peptidoglycan (PG) in which some of the muramic acid residues are substituted with a complex polysaccharide, arabinogalacta (AG), attached to which are long chain mycolic acids.The proposed project will involve the investigation of structures formed by these mycolic acids and mycolates, which will be reconstituted from bacterial cell walls, with a view to understanding how the molecules assemble together and form a barrier. If a monolayer of mycolic acids or mycolates is compressed, the conformation of the molecules changes and different constituents of the wall respond in different ways. These changes can be investigated with a Langmuir trough, where a monolayer of the molecules on the air|water (or air|buffer) interface can be compressed by changing the area available to the molecules. Alternatively, an electric field could be employed to induce a slight compression in a supported monolayer (electrostriction). Monolayers can be studied at the air|liquid interface or transferred onto a solid support, such as glass, quartz or gold, using Langmuir-Blodgett and/or Langmuir-Schaeffer techniques. In this project, a combination of polarisation modulation infrared reflection absorption spectroscopy (PM-IRRAS), shell-isolated nanoparticle-enhanced Raman Scattering (SHINERS) and atomic force microscopy (AFM) will be used to study films transferred onto solid supports. PM-IRRAS will provide information on conformation of the molecules and their orientation within the monolayer, from which it will be possible to determine how the chains of the molecules are folded. AFM will be used to investigate phase behaviour and to determine mechanical strength of the monolayers by recording force distance curves. There is also scope for investigating permeability of monolayers supported on electrodes using electrochemical techniques such as differential capacitance and electron transfer probes. Neutron reflectivity will be used to probe monolayer thickness and solvent ingress into monolayers supported on the air|liquid interface and solid|liquid interface. By combining these techniques, we aim to build a picture of how the bacterial walls are formed and how their structure influences their properties and function. This will, in turn, lead to the possibility of discovering ways to breach the walls and of screening potential drugs.

Molecules reconstituted from bacteria will be investigated, along with molecules produced by bacteria engineered to produce defects in the walls. We will determine the effect of these defects on the structure and permeability of the films. There is also scope for chemical synthesis of analogues of these molecules, labelled in particular positions, that we shall use to investigate the conformation of molecules and how they fold in forming the monolayers. Future work will include the building of more complex architectures containing other components of the cell wall (AG, then PG), which will lead to the possibility of constructing and investigating an artificial mycobacterial cell wall. Selected

References:

  1. Biophysical J. 2004, 86(3), 1763-1776. (Electric Field-Driven Transformations of a Supported Model Biological Membrane: an Electrochemical and Neutron Reflectivity Study.)
  2. Biophysical J. 2005, 89(1), 592-604. (Electrochemical and PM-IRRAS studies of the effect of cholesterol on the structure of a DMPC bilayer supported at a Au(111) electrode surface. Part 1 Properties of the acyl chains.)
  3. Faraday Disc. 2010, 145, 357-379. (Structure and Dynamics of Phospholipid Bilayer Films under Electrochemical Control.)
  4. Langmuir 2013, 29(5), 1695-1708. (Effect of Headgroup on the Physicochemical Properties of Phospholipid Bilayers in Electric Fields: Size Matters)
  5. Biochem Soc Trans. 2008, 36( 4),555-65. (Tuberculosis: a balanced diet of lipids and carbohydrates.)
  6. Proc. Nat. Acad. Sci. 2012, 109(28),11354-9. (Structural basis of inhibition of Mycobacterium tuberculosis DprE1 by benzothiazinone inhibitors.)
  7. Cold Spring Harbor Perspect. Med. 2015, 5(8), a021113. (The Mycobacterial Cell Wall-Peptidoglycan and Arabinogalactan.)

BBSRC Strategic Research Priority: Food Security

Techniques that will be undertaken during the project:

  • Molecular microbiology
  • Langmuir isotherms, Langmuir-Blodgett and Langmuir-Schaeffer deposition
  • Polarisation Modulation Infrared Reflection Absorption Spectroscopy
  • Atomic Force Microscopy
  • Neutron Reflectometry (air|liquid and solid|liquid interfaces), X-ray Reflectometry and Grazing Incidence X-ray Diffraction
  • Capacitance measurements
  • Shell-Isolated Nanoparticle-Enhanced Raman Scattering
  • Chemical synthetic strategy and techniques

Contact: Dr Sarah Horswell & Dr Luke Alderwick, University of Birmingham