Staphylococcus aureus vWF-binding protein triggers a strong interaction between clumping factor A and host vWF

Abstract

The Staphylococcus aureus cell wall-anchored adhesin ClfA binds to the very large blood circulating protein, von Willebrand factor (vWF) via vWF-binding protein (vWbp), a secreted protein that does not bind the cell wall covalently. Here we perform force spectroscopy studies on living bacteria to unravel the molecular mechanism of this interaction. We discover that the presence of all three binding partners leads to very high binding forces (2000 pN), largely outperforming other known ternary complexes involving adhesins. Strikingly, our experiments indicate that a direct interaction involving features of the dock, lock and latch mechanism must occur between ClfA and vWF to sustain the extreme tensile strength of the ternary complex. Our results support a previously undescribed mechanism whereby vWbp activates a direct, ultra-strong interaction between ClfA and vWF. This intriguing interaction represents a potential target for therapeutic interventions, including synthetic peptides inhibiting the ultra-strong interactions between ClfA and its ligands.

Fig. 1. Staphylococcus aureus adhesion to von Willebrand factor (vWF).

a The modular architecture of mature vWF. vWF-binding protein (vWbp) binds to the A1 domain. b Cartoon depicting S. aureus ClfA and vWbp in a hypothetical complex with vWF under the shear stress of arterial blood flow. Tangled and coiled globular vWF is stretched out by the arterial flow. vWbp mediates ClfA-dependent binding of S. aureus to vWF via its A1 domain.

Fig. 2. vWbp augments ClfA-dependent binding of S. aureus to vWF and the ternary complex is very strong.

a S. aureus cells expressing ClfA at high levels and treated with recombinant vWbp probed with vWF-modified AFM tips. Data for a representative cell are shown. For more cells, see Supplementary Fig. 1. On the left are histograms of rupture forces with insets showing the respective adhesion maps (500 × 500 nm, 32 × 32 or 16 × 16 pixels, gray scale = 0–3 nN, each dot represents a binding event) and a cartoon in the top graph illustrates the experimental setup. Green ovals represent ClfA, golden spheres vWbp, and blue lines vWF. On the right are shown histograms of the rupture lengths with insets showing three representative retraction profiles. b Data for ClfA⁺ S. aureus cells (not treated with vWbp) probed with vWF-functionalized tips treated with vWbp. c Data for vWbp-untreated ClfA⁺ cells probed with vWF-modified tips. d Data for vWbp-treated ClfA⁻ cells probed with vWF-modified tips.

Fig. 3. Force-loading rate dependence for the vWF-vWbp-ClfA ternary interaction follows Bell–Evans dynamics.

Left: dynamic force spectroscopy plot of rupture force vs loading rate (n = 2992 curves from 3 cells, for individual data from 6 cells see Supplementary Fig. 2). The dotted line shows the extrapolated Bell–Evans fit through the most probable (mean) rupture forces and loading rates for five log-equispaced loading rate bins shown as solid circles. Error bars are the standard deviations. Right: corresponding histograms of the plot shown on the left.

Fig. 4. The vWbp-vWF and vWbp-ClfA binary interactions are much weaker than the ternary complex.

a vWbp-modified surfaces probed with vWF-modified tips. Representative data—one surface-tip combination—are shown. Left panel: Histograms of rupture forces (recorded on a 10 µm × 10 µm area, 16 × 16 pixels) and a cartoon illustrating the experimental setup. Right panel. Histograms of the rupture lengths and insets of three representative curves. b S. aureus ClfA⁺ cells probed with vWbp-modified AFM tips. A pause after contact step of 500 ms (contact time) was included to enhance binding frequency. Data shown are for one cell-tip combination. Left panel: Histogram plot of rupture forces. Insets show the respective adhesion map (500 × 500 nm, 32 × 32, gray scale = 0–1.5 nN, each dot represents an adhesion event) and a cartoon to illustrate the experimental setup. c Data for a ClfA⁻ cell probed with a vWbp-modified tip, with 500 ms contact time.

Fig. 5. The vWF-A1 domain is insufficient for a highly stable vWF-vWbp-ClfA interaction.

vWbp-treated ClfA⁺ S. aureus cells probed with vWF-A1-modified tips. Left panel: Histogram plot of rupture forces with insets showing the respective adhesion map (500 × 500 nm, 16 × 16 pixels, gray scale = 0–1.5 nN, each dot represents a binding event) and a cartoon of the experimental approach. Right panel: Histogram plot of the rupture lengths with insets showing three representative curves. Data shown come from one representative cell.

Fig. 6. A dock, lock, and latch (DLL) interaction might be the key for a highly stable vWF-vWbp-ClfA complex.

a ClfA⁺ S. aureus cells were first treated for 15 min with a peptide with a random sequence and then with recombinant vWbp before being probed with vWF-modified AFM tips. Data for a representative cell are shown. On the left are histograms of rupture forces with insets showing the respective adhesion maps (500 × 500 nm, 16 × 16 pixels, gray scale = 0–3 nN, each dot represents a binding event) and a cartoon in the top graph illustrates the experimental setup. The random peptide is illustrated by blue curled lines. On the right are shown histograms of the rupture lengths with insets showing three representative curves. b Data for ClfA⁺ S. aureus cells treated for 15 min with a peptide with the Fg γ-chain C-terminal sequence that binds to ClfA (black curled lines). vWbp was then added for 15 mins and the cells probed with vWF-modified tips. c ClfA_PY cells were treated with recombinant vWbp and then probed with vWF-modified AFM tips.