Investigating protein-mediated staphylococcal biofilm formation

Great advances in medicine, saving lives and improving the quality of life, have been made through the use of medical devices such as catheters, artificial joints and coronary stents and many millions are used every year. However, infections associated with such devices are an important problem affecting more than 100000 patients, and with costs to the NHS of hundreds of millions pounds, per year. These device-related infections arise because bacteria can form colonies, called biofilms, on the surface of the device. Biofilms are difficult to eradicate with antibiotics and the device often has to be removed to treat the infection. It has been known for some time that polymers of sugar-like molecules, produced by the bacteria, are important for the production of biofilms, but more recently bacterial proteins have also been implicated. We have been studying proteins that are found on the surface of Staphylococcus aureus (and also MRSA) and Staphylococcus epidermidis, bacteria that cause many device-related infections. We have discovered how protein molecules on one bacterial cell might be activated to attach to the same protein but on different cells, thereby allowing the bacteria to clump together in colonies. We will use a variety of techniques to determine the structure of these protein-protein complexes. In particular, will determine the high resolution structure of the interaction surface and use this to start the search for small molecules that might block the interaction and lead, in the longer term, to new drugs or device materials that would reduce the incidence of these painful, debilitating, costly and life-threatening device-related infections.

Staphylococcus aureus and Staphylococcus epidermidis form biofilms on prosthetic devices. These infections cause significant morbidity and mortality and are difficult to treat with antibiotics; device-removal is frequently required. The biofilms are formed through both polysaccharide-mediated and, more recently discovered, protein-mediated interactions between bacterial cells. The cell-wall attached proteins SasG from S. aureus and Aap from S. epidermidis mediate biofilm formation and have many similarities. Both contain a string of G5 domains, require proteolytic processing for activity and dimerise in the presence of zinc. We provide preliminary data showing we have discovered a mechanism by which proteolysis switches-on zinc-mediated dimerisation of these proteins. We have developed a model of protein-mediated biofilm formation which is quite different to a recently published model. The overall aim of the research is to test the model to reveal the mechanism and molecular details of zinc-mediated dimerisation. The work will involve the expression and purification of domains from SasG and Aap and their structural and functional characterisation using a range of biophysical techniques and bacterial biofilm assays. Mass spectrometry will be used to identify the proteolytic cleavage site and the stoichiometry of zinc binding to protein dimers. X-ray crystallography will be used to determine the high resolution structure of the zinc-mediated G5-domain dimers which, our preliminary data suggests, are key for biofilm formation. The structures will be used to design/propose lead compounds for biofilm inhibition and these will be tested in a high-throughput fluorescence-based (FRET) assay that will be developed as part of the proposed work. Lower resolution techniques such as small angle X-ray scattering and analytical ultracentrifugation will be used to determine the shape of dimers of larger fragments of SasG and Aap. The shape of these dimers will reveal whether the proteins interact through single G5 domains at the N-terminal end of the cleaved proteins or through a multi-domain zipper, as proposed previously. Mass spectrometry and nuclear magnetic resonance spectroscopy will be used to test the proposal that the G5 domains from SasG and Aap (and other bacterial proteins) bind N-acetylglucosamine; a plausible mechanism by which protein-mediated and polysaccharide-mediated biofilm formation mechanisms could interact. The relevance of the structural and biophysical studies to biofilm formation will be tested in bacterial assays in the laboratories of our collaborators. Overall, the proposed research, if successful, will lead to a fundamental shift in the understanding of protein-mediated biofilm formation in these important nosocomial pathogens.