Identification of the ligand-binding domain of the surface-located fibrinogen receptor (clumping factor) of Staphylococcus aureus.
The ability of Staphylococcus aureus to bind to fibrinogen and fibrin is believed to be an important factor in the initiation of foreign-body and wound infections. Recently, we reported the cloning and sequencing of the gene clfA encoding the fibrinogen receptor (clumping factor, ClfA) of S. aureus strain Newman and showed that the gene product was responsible for the clumping of bacteria in soluble fibrinogen and for the adherence of bacteria to solid-phase fibrinogen. This was confirmed here by showing that antibodies raised against purified Region A inhibited both of these properties. Also, immunofluorescent microscopic analysis of wild-type Newman and a clfA::Tn917 mutant of Newman with anti-ClfA Region A sera confirmed that Region A is exposed on the bacterial cell surface. Furthermore, polystyrene beads coated with the Region A protein formed clumps in soluble fibrinogen showing that the ClfA protein alone is sufficient for the clumping phenotype. Western immunoblotting with anti-ClfA Region A antibodies identified the native ClfA receptor as a 185 kDa protein that was released from the cell wall of S. aureus by lysostaphin treatment. A single extensive ligand-binding site was located within Region A of the ClfA protein. Truncated ClfA proteins were expressed in Escherichia coli. Lysates of E. coli and proteins that had been purified by affinity chromatography were tested for (i) their ability to bind fibrinogen in Western ligand blotting experiments, (ii) for their ability to inhibit clumping of bacteria in fibrinogen solution and adherence of bacteria to solid-phase fibrinogen, and (iii) for their ability to neutralize the blocking activity of anti-ClfA Region A antibody. These tests allowed the ligand-binding domain to be localized to a 218-residue segment (residues 332-550) within Region A.
D001422:Bacterial Adhesion; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D003030:Coagulase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D004926:Escherichia coli; D005340:Fibrinogen / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D005798:Genes, Bacterial; D008024:Ligands; D008969:Molecular Sequence Data; D010957:Plasmids; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000737:chemistry* / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013211:Staphylococcus aureus / Q000235:genetics / Q000502:physiology*
D003030:Coagulase; D017931:DNA Primers; D008024:Ligands; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D011993:Recombinant Fusion Proteins; D005340:Fibrinogen