Mechanostability of the Fibrinogen Bridge between Staphylococcal Surface Protein ClfA and Endothelial Cell Integrin αVβ3

Binding of the Staphylococcus aureus surface protein clumping factor A (ClfA) to endothelial cell integrin alpha(V)beta(3) plays a crucial role during sepsis, by causing endothelial cell apoptosis and loss of barrier integrity. ClfA uses the blood plasma protein fibrinogen (Fg) to bind to alpha(V)beta(3) but how this is achieved at the molecular level is not known. Here we investigate the mechanical strength of the three-component ClfA-Fg-alpha(V)beta(3) interaction on living bacteria, by means of single-molecule experiments. We find that the ClfA-Fg-alpha(V)beta(3) ternary complex is extremely stable, being able to sustain forces (similar to 800 pN) that are much stronger than those of classical bonds between integrins and the Arg-Gly-Asp (RGD) tripeptide sequence (similar to 100 pN). Adhesion forces between single bacteria and alpha(V)beta(3) are strongly inhibited by an anti-alpha(V)beta(3) antibody, the RGD peptide, and the cyclic RGD peptide cilengitide, showing that formation of the complex involves RGD-dependent binding sites and can be efficiently inhibited by alpha(V)beta(3) blockers. Collectively, our experiments favor a binding mechanism involving the extraordinary elasticity of Fg. In the absence of mechanical stress, RGD572-574 sequences in the Aa chains mediate weak binding to alpha(V)beta(3), whereas under high mechanical stress exposure of cryptic Aa chain RGD95-97 sequences leads to extremely strong binding to the integrin. Our results identify an unexpected and previously undescribed force-dependent binding mechanism between ClfA and alpha(V)beta(3) on endothelial cells, which could represent a potential target to fight staphylococcal bloodstream infections.