Staphylococcus aureus is a commensal organism capable of causing a diverse array of acute and chronic infections. Biofilm formation plays an important role in pathogenesis for many chronic infections by promoting antibiotic tolerance and immune evasion. Both protein and eDNA have previously been identified as major components of the biofilm matrix. Many recent studies have shown that DNA-binding proteins contribute to biofilm structure and stability in other organisms, and that these proteins represent potential targets for therapeutic development. However, little has been done to investigate the role of eDNA-binding proteins in S. aureus biofilms. To address this, we conducted a screen to identify potential eDNA-binding proteins in the S. aureus biofilm matrix. Secreted and cell surface-associated proteins displaying DNA-binding activity were detected using Southwestern blotting and were identified by mass spectrometry. From this screen, we selected Eap, IsaB, and SaeP as candidates for further study.
To gain insight into the function of these proteins, in vitro biofilm formation of single and double mutants was assessed in several strain backgrounds. While no major change in overall biomass was observed in these mutants, we found that loss of both eap and isaB resulted a loss of surface-associated eDNA from the biofilm matrix. Furthermore, we observed that overexpression of SaeP resulted in a significant increase in both overall biofilm formation and an increase in surface-associated eDNA. This phenotype was still observed in both sae and nuc mutants at high levels of SaeP expression, suggesting that SaeP-based biofilm enhancement was not completely dependent on Sae-based regulation of the staphylococcal nuclease. The high pIs of these proteins suggest that the association with eDNA is primarily mediated through ionic interactions. These findings suggest that Eap, IsaB, and SaeP function as non-specific eDNA-binding proteins that are important for the maintenance of eDNA in the biofilm matrix. We suggest these proteins represent a new class of functionally redundant matrix proteins capable of significantly impacting biofilm matrix composition.