Scientists have developed a model of the outer membrane of the bacteria E. coli providing a brand new tool for developing new antibiotics and other drugs in the fight against infections. Disease-causing bacteria mostly belong to two groups: Gram-positive, which include MRSA, and Gram-negative bacteria which include E. coli (Escherichia coli) and the bacteria behind meningitis and plague.
All types of disease causing bacteria are becoming resistant to antibiotics but single-celled Gram negatives are of special concern because they have an extra wall around their cells which can protect them physically from our treatments.
Publishing as a "Hot Paper" in the leading chemistry journal Angewandte Chemie International Edition and cited as a research highlight in Nature Chemical Biology, the authors describe the creation in the laboratory of the model bacterial outer membrane. Its nanoscale structure was then determined at the ISIS pulsed neutron source, Harwell UK which allowed the precise molecular composition to be resolved.
Jeremy Lakey, professor of structural biochemistry at Newcastle University who led the study explains, "Our model of the bacterial outer membrane can be used as a simulator to test how antibiotic molecules can be made to cross this critical barrier.
"A stable model is so important because the detailed structure of this wall is still not clear, largely because bacteria are very small and have a protective envelope that is only 20 nanometres thick. This model gives us unprecedented access to the structure and dynamics of the membrane."
The envelope of Gram-negative bacteria, such as E. coli, is composed of two barriers -- the so-called inner and outer cell membranes. This double membrane which is unique in biology is highly impermeable to incoming molecules and acts as a highly selective filter.
Gram-negative bacteria are highly successful organisms. In evolutionary terms, they are believed to have descended from a common ancestor of cyanobacteria, which emerged 3.6 billion years ago. E. coli bacteria live in the digestive tract of people and animals and most are harmless. However, some Gram-negatives cause illnesses such as meningitis, plague, Legionnaires disease, cholera and food poisoning.
The inner and outer membranes are very different. The inner membrane is a largely symmetric bilayer composed of simple phospholipids like most biological membranes. The outer membrane is a complex, asymmetric structure composed of lipopolysaccharides (LPS) on the outer surface and phospholipids on the inner. Calcium and magnesium ions link the core polysaccharides of adjacent LPS in outer layer to present an additional barrier of densely packed long sugar polymers.
It is the physical properties of the membrane barrier that limits the effectiveness of antimicrobials and mutations to outer membrane proteins, through which many antibiotics have to pass, also contribute to the development of antibiotic resistant strains.
In the work, funded by The Wellcome Trust, the researchers started construction of the synthetic model with a flat gold layer that chemically adheres to silicon substrate through a nickel-iron magnetic alloy. The magnetic layer interacts with neutrons of differing spin states and significantly enhances the resolution of the measurements. This technique, called magnetic contrast, was developed by the same team a few years ago. The gold is coated with a monolayer of the phospholipid (the inner membrane), which is covered in a layer of water.
The deposition of the free floating outer bilayer was more challenging as the system is not self-assembling. To create it, they needed to use laboratory methods (Langmuir-Blodgett/Langmuir Schaeffer) to compress and stack monolayers of molecules horizontally and vertically.
In this case, they needed a phospholipid layer, with hydrophilic (water liking) heads pointing down towards the periplasm, joined to an LPS layer with its heads pointing up. The two layers are bonded together by their hydrophobic (water hating) cores of acyl chains.
The structure of the model was confirmed using neutron scattering carried out at the ISIS Pulsed Neutron Source which is a particle accelerator at the Rutherford Appleton Laboratory, Oxford. This showed accurate details of the model such as the molecular asymmetry and the thickness of the internal water layer.
"Neutron scattering allows us to resolve complex structures composed of mixtures of biomolecules," says lead author and ISIS instrument scientist, Dr Luke Clifton. "By combining this with isotopic labelling, to which neutrons are very sensitive, we were able to determine where each component of the model was."
Clifton and co-workers went on to test the response of the model to antimicrobial proteins produced in our bodies, including lysozyme and lactoferrin. Interactions of these proteins with the outer membrane in vivo and in vitro are well known, allowing for direct comparisons with the synthetic model. Neutron reflectivity revealed that the experiments reproduced in vivo behaviour, replicating the disruption of the outer membrane seen in living bacteria.
Lakey adds, "The model behaves in a way that you would expect of a living bacteria which opens up exciting opportunities for researchers to test new compounds."
The next challenge for the researchers is to begin incorporating membrane proteins into the bilayer.
Source: Newcastle University