Mechanostability of the Fibrinogen Bridge between Staphylococcal Surface Protein ClfA and Endothelial Cell Integrin α_Vβ₃.

Abstract

Binding of the <i>Staphylococcus aureus</i> surface protein clumping factor A (ClfA) to endothelial cell integrin α_Vβ₃ 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 α_Vβ₃ but how this is achieved at the molecular level is not known. Here we investigate the mechanical strength of the three-component ClfA-Fg-α_Vβ₃ interaction on living bacteria, by means of single-molecule experiments. We find that the ClfA-Fg-α_Vβ₃ ternary complex is extremely stable, being able to sustain forces (∼800 pN) that are much stronger than those of classical bonds between integrins and the Arg-Gly-Asp (RGD) tripeptide sequence (∼100 pN). Adhesion forces between single bacteria and α_Vβ₃ are strongly inhibited by an anti-α_Vβ₃ 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 α_Vβ₃ blockers. Collectively, our experiments favor a binding mechanism involving the extraordinary elasticity of Fg. In the absence of mechanical stress, RGD^572-574 sequences in the Aα chains mediate weak binding to α_Vβ₃, whereas under high mechanical stress exposure of cryptic Aα chain RGD^95-97 sequences leads to extremely strong binding to the integrin. Our results identify an unexpected and previously undescribed force-dependent binding mechanism between ClfA and α_Vβ₃ on endothelial cells, which could represent a potential target to fight staphylococcal bloodstream infections.