The molecular localisation of Staphylococcus aureus surface protein A

Staphylococcus aureus presents an important and significant burden on global healthcare systems. The ability to of S. aureus to colonise the human population and cause a wide range of diseases is heavily dependent on the display of various proteins on its cell surface. These proteins serve multiple functions, including adhesion to host molecules, chelation of host iron, and the evasion of both innate and adaptive mammalian immune systems. Therefore, the development of new and novel strategies to combat S. aureus infection dynamics and disease progression is dependent on an advanced understanding of how these key virulence determinants are displayed and develop over time.
The aim of this project was to construct reporter fusions for four surface displayed virulence determinants fused to SNAP-tag, an O6-benzylguanine-DNA-benzyltransferase, to then localise them on the cell surface of S. aureus. The proteins were iron-regulated surface determinant A (IsdA), iron-regulated surface determinant B (IsdB), clumping factor A (ClfA), and surface protein A (SpA). Due to a lack of sensitivity of the SNAP reporter fusion, an alternative approach of immunofluorescence microscopy was also used, with which the localisation of SpA and its development over time was elucidated.

Immunolabelling for SpA localisation in this study has shown that SpA is displayed only on a subset of the bacterial population of S. aureus SH1000, a well characterised and model laboratory strain. Within this subpopulation, SpA localises over the whole of single cells. S. aureus cells actively dividing present SpA at their respective peripheries but not the newly exposed septum, displaying a horseshoe-like pattern of SpA fluorescence. In conjunction with atomic force microscopy data of the cell wall architecture of live S. aureus cells, the horseshoe pattern was believed to be the result of the surface exposure of dense, nascent cell wall material at the division septum that acts as an additional layer of peptidoglycan without Spa bound to this layer. Further investigations into the role of genes involved in cell wall associated processes identified several targets that appear vital for SpA display. Finally, the impact of high methicillin resistance S. aureus (MRSA) and antibiotic challenge on SpA localisation was determined. My project has further elucidated those mechanisms which control the display of the important virulence determinant SpA on the surface of S. aureus.