Zinc-dependent mechanical properties of Staphylococcus aureus biofilm-forming surface protein SasG

Staphylococcus aureus surface protein SasG promotes cell–cell adhesion during the accumulation phase of biofilm formation, but the molecular basis of this interaction remains poorly understood. Here, we unravel the mechanical properties of SasG on the surface of living bacteria, that is, in its native cellular environment. Nanoscale multiparametric imaging of living bacteria reveals that Zn²⁺ strongly increases cell wall rigidity and activates the adhesive function of SasG. Single-cell force measurements show that SasG mediates cell–cell adhesion via specific Zn²⁺-dependent homophilic bonds between β-sheet–rich G5–E domains on neighboring cells. The force required to unfold individual domains is remarkably strong, up to ∼500 pN, thus explaining how SasG can withstand physiological shear forces. We also observe that SasG forms homophilic bonds with the structurally related accumulation-associated protein of Staphylococcus epidermidis, suggesting the possibility of multispecies biofilms during host colonization and infection. Collectively, our findings support a model in which zinc plays a dual role in activating cell–cell adhesion: adsorption of zinc ions to the bacterial cell surface increases cell wall cohesion and favors the projection of elongated SasG proteins away from the cell surface, thereby enabling zinc-dependent homophilic bonds between opposing cells. This work demonstrates an unexpected relationship between mechanics and adhesion in a staphylococcal surface protein, which may represent a general mechanism among bacterial pathogens for activating cell association.The bacterial pathogen Staphylococcus aureus causes a wide range of infections in humans, which are often associated with the ability of the bacteria to form biofilms on indwelling medical devices such as central venous catheters and prosthetic joints (1–4). Biofilm formation involves initial adhesion of the bacteria to surfaces, followed by cell–cell adhesion (aggregation) to form microcolonies and a mature biofilm, and finally dispersal by the detachment of cell aggregates from the biofilm (5). Currently, little is known about the molecular interactions driving biofilm formation by S. aureus due to the paucity of appropriate high-resolution probing techniques. Such knowledge may contribute to the development of novel compounds for therapy.Adhesion and biofilm formation by S. aureus involve a variety of cell wall components. Whereas adhesion to host proteins is mediated by cell-wall–anchored (CWA) proteins (6, 7), intercellular adhesion was until recently thought to be promoted by the expression of the polysaccharide intercellular adhesin (PIA), also known as the poly-N-acetyl-glucosamine (PNAG) (8, 9). This positively charged polymer is able to bind the negatively charged bacterial surfaces. PIA, encoded by genes in the ica operon, represents the most well-understood biofilm-mediating pathway in staphylococci (10, 11). However, many strains do not produce PIA and rely on CWA proteins to promote intercellular adhesion in an ica-independent manner (6, 7).A prototype of biofilm-forming CWA protein is SasG (12–15), which mediates cell–cell adhesion through its “B” multidomain region (5, 7). B repeat sequences contain “G5” domains (∼78 residues) in a tandem array, separated by 50-residue sequences known as the “E” regions (Fig. 1A) (14, 15). SasG forms β-sheet–rich protein fibrils that protrude from the cell surface, which can be visualized by electron microscopy (12). The proposed mechanism for SasG-mediated cell association is based on homophilic protein–protein interactions. SasG is covalently attached to the cell wall and undergoes limited cleavage within the B region to remove the N-terminal “A” region. The cleaved and exposed SasG B domains on neighboring cells interact with each other in a Zn²⁺-dependent manner, leading to cell–cell adhesion (13). The G5–E domains of the related accumulation-associated protein (Aap) of Staphylococcus epidermidis are also responsible for the Zn²⁺-dependent biofilm formation (15). However, recent work also suggests that Aap could bind a ligand protein, the small basic protein (Sbp), which accumulates on the cell surface and within the biofilm matrix (16). Therefore, whereas SasG and Aap are believed to mediate intercellular adhesion via zinc-dependent homophilic bonds between opposing proteins, it is unclear whether this is the only mechanism at play. Also, the mode of action of zinc is controversial. Whereas SasG dimerizes in vitro in a zinc-dependent manner, a direct link between homodimerization and biofilm formation has not yet been established. Rather, it has been suggested that zinc could mediate binding to anionic cell surface components like teichoic acids (14). Direct biophysical analysis of SasG proteins on the surface of living cells would help to clarify these important issues.Open in a separate windowFig. 1.Role of SasG in cell–cell adhesion. (A) Schematic representation of the SasG structure emphasizing the A domain, not engaged in cell–cell adhesion, and the B repeat sequence containing G5 domains (78 residues) in a tandem array, separated by the E regions (50 residues). (B–E) Optical microscopy images of S. aureus cells expressing full-length SasG [SasG₈⁽⁺⁾ cells] after resuspension in TBS buffer (B) or in TBS buffer containing 1 mM of ZnCl₂ (C), after addition of 1 mM EDTA (D), and further addition of 1 mM ZnCl₂ (E). (F and G) Control experiment using S. aureus expressing no SasG [SasG⁽⁻⁾ cells] in TBS buffer (F) or in TBS containing 1 mM of ZnCl₂ (G).Recent advances in atomic force microscopy (AFM) techniques have enabled researchers to gain insight into the biophysical properties and molecular interactions of microbial cells (17, 18), including S. aureus (19–22). A variety of AFM-based force spectroscopy methods have been developed, in which the force acting on the AFM probe is measured with piconewton (10⁻¹² N) sensitivity as the probe is pushed toward the sample, then retracted from it (17). In the past few years, a new force spectroscopy-based imaging mode, multiparametric imaging, has offered the possibility to image the surface structure of living cells, while mapping their mechanical and adhesive properties at unprecedented spatiotemporal resolution (23–28). Unlike in conventional imaging, the method involves recording arrays of force curves across the cell surface, at improved speed, positional accuracy, and force sensitivity (26). As the curves are recorded at high frequency, correlated images of the structure, adhesion, and mechanics of the cells can be obtained at the speed of conventional imaging. This technology has been used to image single filamentous bacteriophages extruding from living bacteria (25) and to map adhesive nanodomains on fungal pathogens (28). Furthermore, recent progress in single-cell force spectroscopy (SCFS) (18, 29, 30) has made it possible to understand the forces driving cell adhesion and biofilm formation. Here, a living cell is attached to the AFM probe, thereby enabling researchers to measure the interaction forces between the cell and a target surface (18). Applying these newly developed modalities to staphylococci is a challenging problem, which would provide novel insights into the molecular bases of biofilm formation and biofilm-associated infections.Here, we combine multiparametric imaging and SCFS to investigate the mechanical strength of SasG on living bacterial cells, thus in its fully functional environment. We use a S. aureus strain carrying a plasmid expressing SasG with eight consecutive G5–E repeats [hereafter S. aureus SasG₈⁽⁺⁾ cells]. We show that intercellular adhesion involves the Zn²⁺-dependent–specific association of G5–E repeats on opposing cells and that the elongated structure and mechanical strength of SasG make it ideally suited for that purpose. In addition, our results show that Zn²⁺ plays a dual role that is more complex than anticipated before: adsorption of Zn²⁺ to cell wall components increases the cohesion of the cell surface, thereby favoring the projection of highly elongated SasG proteins beyond other surface components and making them fully functional for Zn²⁺-dependent homophilic interactions.