Characterization of the Interaction Between the Staphylococcus Aureus Clumping Factor (ClfA) and Fibrinogen

Abstract

The ability of Staphylococcus aureus to adhere to adsorbed fibrinogen and fibrin is believed to be an important step in the initiation of bioinaterial and wound-associated infections. In this study, we show that the binding site in fibrinogen for the recently identified S. aureus fibrinogen-binding protein clumping factor (ClfA) is within the C-terminus of the fibrinogen y chain. S. aureus Newman cells expressing ClfA adhered to microtitre wells coated with recombinant fibrinogen purified from BHK cells, but did not adhere to wells coated with a purified recombinant fibrinogen variant where the 4 C-terminal residues of the y chain were replaced by 20 unrelated residues. In addition, a synthetic peptide corresponding to the 17 C-terminal amino acids of the fibrinogen y chain effectively inhibited adherence of ClfA-expressing cells to fibrinogen. In western ligand blots, a recombinant truncated ClfA protein called Clf33 (residues 221 -550) recognized intact recombinant fibrinogen y chains, but failed to recognize recombinant fibrinogen y chains where the 4 C-terminal amino acids were altered by deletion or substitution. Previous studies have shown that the C-terminal domain of fibrinogen y chains contains a binding site for the integrin . We now show that Clf33 inhibits ADPinduced, fibrinogen-dependent platelet aggregation in a concentration-dependent manner and inhibits adhesion of platelets to immobilized fibrinogen under fluid shear stress, indicating that the binding sites for the platelet integrin and the staphylococcal adhesin overlap. The interaction between Clf33 and fibrinogen was further characterized using the BIAcore biosensor. When soluble Clf33 was allowed to bind to immobilized fibrinogen, a Kd of 0.51 t-0.19 pM was experimentally determined using equilibrium binding data. It was also shown that the synthetic C-terminal y-chain peptide effectively inhibited this interaction.

Extracellular Fibrinogen-binding Protein, Efb, fromStaphylococcus aureus Blocks Platelet Aggregation Due to Its Binding to the α-Chain

“…The D fragment includes residues 111-197 of the A␣-chain, 134 -461 of the B␤-chain, and 88 -406 of the ␥-chain (20, 21). Clf did not recognize the D fragment, as expected since it is the C-terminal region of the ␥-chain that is recognized by Clf (22), and this part is not included in the D fragment. The recombinant Fbe from S. epidermidis, which has been shown to bind to the B␤-chain (18), did not recognize the D fragment, indicating that the binding site for this molecule is not located between amino acids 88 and 406 of the B␤-chain.…”

Bacterial c-di-GMP Is an Immunostimulatory Molecule

“…To evaluate the possibility that c-di-GMP could also act as an adjuvant, the cyclic dinucleotide was coinjected into mice with the recombinant ClfA Ag, a surface adhesion protein of S. aureus (33). Following vaccination with two i.m.…”

The N-terminal A Domain of Fibronectin-binding Proteins A and B Promotes Adhesion of Staphylococcus aureus to Elastin

“…They also share 25% identity to the A domains of ClfA and ClfB. Indeed, the FnBPA and FnBPB A domains bind to the C terminus of the ␥-chain of fibrinogen at the same site as ClfA and the platelet integrin (20,27). 10 fibronectin binding domains extend throughout domains B, C, and D of FnBPA (28,29), although most binding studies have been performed with the D repeats, such as D1D2D3 (30), which share 95% identity between both FnBPA and FnBPB.…”