Staphylococcus aureusProtein A Recognizes Platelet gC1qR/p33: a Novel Mechanism for Staphylococcal Interactions with Platelets

Abstract

The adhesion ofStaphylococcus aureusto platelets is a major determinant of virulence in the pathogenesis of endocarditis. Molecular mechanisms mediatingS. aureusinteractions with platelets, however, are incompletely understood. The present study describes the interaction betweenS. aureusprotein A and gC1qR/p33, a multifunctional, ubiquitously distributed cellular protein, initially described as a binding site for the globular heads of C1q. Suspensions of fixedS. aureusor purified protein A, chemically cross-linked to agarose support beads, were found to capture native gC1qR from whole platelets. Moreover, biotinylated protein A bound specifically to fixed, adherent, human platelets. This interaction was inhibited by unlabeled protein A, soluble recombinant gC1qR (rgC1qR), or anti-gC1qR antibody F(ab′)₂fragments. The interaction between protein A and platelet gC1qR was underscored by studies illustrating preferential recognition of the protein A-bearingS. aureusCowan I strain by gC1qR compared to recognition of the protein A-deficient Wood 46 strain, as well as inhibition ofS. aureusCowan I strain adhesion to immobilized platelets by soluble protein A. Further characterization of the protein A-gC1qR interaction by solid-phase enzyme-linked immunosorbent assay techniques measuring biotinylated gC1qR binding to immobilized protein A revealed specific binding that was inhibited by soluble protein A with a 50% inhibitory concentration of (3.3 ± 0.7) × 10⁻⁷M (mean ± standard deviation;n= 3). Rabbit immunoglobulin G (IgG) also prevented gC1qR-protein A interactions, and inactivation of protein A tyrosil residues by hyperiodination, previously reported to prevent the binding of IgG Fc, but not Fab, domains to protein A, abrogated gC1qR binding. These results suggest similar protein A structural requirements for gC1qR and IgG Fc binding. Further studies of structure and function using a truncated gC1qR mutant lacking amino acids 74 to 95 demonstrated that the protein A binding domain lies outside of the gC1qR amino-terminal alpha helix, which contains binding sites for the globular heads of C1q. In conclusion, the data implicate the platelet gC1qR as a novel cellular binding site for staphylococcal protein A and suggest an additional mechanism for bacterial cell adhesion to sites of vascular injury and thrombosis.