Understanding the mechanical properties of the Staphylococcus aureus biofilm-associated protein, SasG

In nature bacteria typically exist on substrates as multicellular communities encapsulated in extracellular polysaccharide substance (EPS), commonly known as biofilms utilising a repertoire of cell surface proteins. The cell wall anchored (CWA) Staphylococcus aureus (S. aureus) surface protein G (SasG) B domain is thought to play a role in both the formation and maintenance of biofilm structure. However, in vitro evidence is inconclusive. Here we utilise a single-molecule force spectroscopy (SMFS) protein display system to first determine whether there is homophilic* bonding of SasG in the presence of Zn2+ and the molecular basis for this. We establish that the B domain of SasG does participate in Zn2+-induced dimerisation and believe this is through the use of pleomorphic coordination of Zn2+ ions (verified using a variant designed to reduce the Zn2+ coordination capacity). In addition, as biofilms form at a solid:liquid interface, cell surface proteins are directly subject to external hydrodynamic forces. The ability of the bacterial cell-wall protein structures to withstand or respond to these forces as a mechanical cue will determine the success of a bacterial colonisation. The B domain of SasG comprises E and G5 repeats which have remarkable mechanical stability, however, the origin of this unusual mechanical phenotype is unknown. Through the use of protein engineering we create a homo-polyprotein system suitable for representing the B domain of SasG in SMFS experiments. By disrupting structures and bonding patterns through residue substitution, we uncover a novel mechanical protein motif – the collagen-like region of E and G5 domains. Furthermore, we demonstrate the main force-bearing region are the 'mechanical clamps', comprising of tandem arrays of long stretches of hydrogen bonds and side chain packing interactions, as recently predicted. These results provide in vitro evidence for the Zn2+-dependent aggregation of SasG in biofilm formation and provide a novel target for therapeutics. Furthermore, insights on novel mechanical structures adds to the toolbox for the rational design of designer proteins requiring a level of mechanical strength for function.