Characterization of the interaction between the Staphylococcus aureus clumping factor (ClfA) and fibrinogen.
The ability of Staphylococcus aureus to adhere to adsorbed fibrinogen and fibrin is believed to be an important step in the initiation of biomaterial 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 gamma 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 gamma chain were replaced by 20 unrelated residues. In addition, a synthetic peptide corresponding to the 17 C-terminal amino acids of the fibrinogen gamma 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 gamma chains, but failed to recognize recombinant fibrinogen gamma 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 gamma chains contains a binding site for the integrin alphaIIb beta3 (glycoprotein gpIIb/IIIa) receptor on platelets [Kloczewiak, M., Timmons, S., Bednarek, M. A., Sakon, M. & Hawiger, J. (1989) Biochemistry 28, 2915-1919; Farrell, D. H., Thiagarajan, P., Chung, D. W. & Davie, E. W. (1992) Proc. Natl. Acad. Sci. USA 89, 10729-10732; Hettasch, J. M., Bolyard, M. G. & Lord, S. T. (1992) Thromb. Haemostasis 68, 701-706]. We now show that Clf33 inhibits ADP-induced, 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 +/- 0.19 microM was experimentally determined using equilibrium binding data. It was also shown that the synthetic C-terminal gamma-chain peptide effectively inhibited this interaction.
McDevitt
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D
Department of Biochemistry and Biophysics, Institute of Biosciences and Technology, Texas A & M University, Houston 77030-3303, USA.
Nanavaty
T
T
House-Pompeo
K
K
Bell
E
E
Turner
N
N
McIntire
L
L
Foster
T
T
Höök
M
M
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D001422:Bacterial Adhesion; D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D005340:Fibrinogen / Q000378:metabolism*; D008969:Molecular Sequence Data; D010973:Platelet Adhesiveness / Q000187:drug effects; D010974:Platelet Aggregation / Q000187:drug effects; D013211:Staphylococcus aureus / Q000502:physiology*
D001426:Bacterial Proteins; D002352:Carrier Proteins; D000244:Adenosine Diphosphate; D005340:Fibrinogen