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Breaching Bacterial Cell Membranes

Primary Supervisor: Dr Sarah Horswell, School of Chemistry

Secondary supervisor: Professor Timothy Dafforn

PhD project title: Breaching Bacterial Cell Membranes

University of Registration: University of Birmingham

Project outline:

Knowledge of the structure of a cell membrane is crucial to understanding its properties. It is established that bacterial cell membranes differ in composition from eukaryotic plasma cell membranes, as well as other membranes within cells, but little is known about the reasons for this diversity. If we are to develop new antimicrobial agents, we need to understand the differences between bacterial and mammalian (or even plant) membranes in order to target bacteria selectively. For example, one class of antimicrobial agents includes small, positively charged peptides, which have an affinity for the negatively charged membranes of many bacteria, such as E. Coli. The key question is whether it is sufficient for the outer half of the membrane to bear a negative charge or whether there is a specific interaction between those negatively charged lipids and the peptides, then how the functional groups facilitate or hinder insertion of that peptide and subsequent aggregation to form pores.

We have developed a means to vary the charge density of a lipid bilayer without changing composition, by supporting the bilayer on an electrode. Several in situ probes can be used to study how the structure and elasticity vary with the charge density, which allows investigation into how both charge and specific molecule structures affect packing and properties. We have considerable experience in these techniques and propose to apply them to the questions of how charge and specific lipid functional groups confer structural properties and are involved in interactions with simple cationic peptides and other small molecules that have been shown to disrupt bacterial cell membranes or cell walls.

In this project we shall carry out structural studies on a range of lipid compositions. Our initial focus will be on cardiolipin (CL). There is a scarcity of structural information on model membranes formed from this molecule and there is a strong need to fill this gap in our knowledge. CL is found in many strains of bacteria, including E. Coli and Staph. Aureus; it is also prevalent in mitochondria and chloroplasts. CL is a phosphoglycerolipid, usually with four hydrocarbon chains. Its relatively large tail:headgroup volume ratio means it can form curved structures as well as lamellar structures, which is implicated in bacterial cell division.

We shall study the structural analogue phosphatidylglycerol (PG), which has similar functionality to CL but is a two-chained glycerophospholipid. PG is also found in bacterial membranes but is rare in mammalian membranes. We shall compare the effects of these two molecules on phosphatidylethanolamine (PE) and phosphatidylcholine (PC) layers with the effects we have already determined for the mammalian lipid phosphatidylserine (PS), construct mimics of common bacterial membranes, e.g. E. Coli, and compare the properties with membranes constructed from E. Coliextracts. We shall also investigate suitable models of Staph. Aureus and S. Pneumoniae, whose membranes are formed primarily from PG, CL and lysyl-PE (a cationic lipid). We shall then select a series of compositions and study the propensity to interact with cationic peptides, to determine whether these peptides can interact selectively with compositions representative of bacterial membranes. The next phase of the work will involve studying the mechanism of disruption of Staph. Aureus membrane mimics with coumarin derivatives. Using monolayers and bilayers as membrane mimics we aim:

  • to determine the effects of externally applied charge density and intrinsic charge density on barrier properties
  • to quantify the effect of charge density on organisation and packing
  • to investigate the effect of composition on thickness and packing
  • to determine how these properties affect interaction with cationic peptides and whether there are specific interactions that can be targeted or whether the interactions are purely electrostatic

By using our combination of techniques we can build up a detailed picture of the effect of charge, size and shape of charged phospholipid on membrane structure and properties, which will lead to an understanding of the rĂ´les of various lipids in cell membranes. Further, we will shed light on the interaction of potential antimicrobial agents with model bacterial membranes and learn which factors may lead to selectively disrupting the membranes of bacteria over those of plants or animals. If model studies are successful, they have the potential to be used to screen potential target compounds to combat infection in humans, animals or plants and so would find ultimate potential application in protecting crops, animals or reducing the number of compounds tested on animals.

BBSRC Strategic Research Priority: Understanding the Rules of Life: Structural Biology

Techniques that will be undertaken during the project:

  • Protein production through microbial expression
  • Surface pressure-area isotherm measurements and monolayer transfer
  • Capacitance and impedance measurements
  • Interfacial vibrational spectroscopy (infrared and Raman)
  • Atomic force microscopy
  • Light scattering
  • Neutron and X-ray Scattering
  • Neutron and X-ray Reflectometry, Grazing Incidence X-ray Diffraction
  • There is also potential to visit a collaborator in Germany working on molecular dynamics simulations and to learn these techniques

Contact: Dr Sarah Horswell, University of Birmingham