Identification of residues in the Staphylococcus aureus fibrinogen-bindingMSCRAMM clumping factor A (ClfA) that are important for ligand binding

Clumping factor A (ClfA) is a cell surface-associated protein of Staphyloco ccus aureus that promotes binding of this pathogen to both soluble and immo bilized fibrinogen (Fg), Previous studies have localized the Fg-binding act ivity of ClfA to residues 221-559 within the A region of this protein. In a ddition, the C-terminal part of the A region (residues 484-550) has been im plicated as being important for Fg binding, In this study, we further inves tigate the involvement of this part of ClfA in the interaction of this prot ein with Fg, Polyclonal antibodies generated against a recombinant protein encompassing residues 500-559 of the A region inhibited the interaction of both S, aureus and recombinant ClfA with immobilized Fg in a dose-dependent manner. Using site-directed mutagenesis, two adjacent residues, Glu(526) a nd Val(527), were identified as being important for the activity of ClfA S. aureus expressing ClfA containing either the E526A or V5275 substitution e xhibited a reduced ability to bind to soluble Fg and to adhere to immobiliz ed Fg, Furthermore, bacteria expressing ClfA containing both substitutions were almost completely defective in Fg binding. The E526A and V527S substit utions were also introduced into recombinant ClfA (rClfA-(221-559)) express ed in Escherichia coli, The single mutant rClfA-(221-559) proteins showed a significant reduction in affinity for both immobilized Fg and a synthetic fluorescein-labeled C-terminal gamma -chain peptide compared with the wild- type protein, whereas the double mutant rClfA-(221-559) protein was almost completely defective in binding to either species. Substitution of Glu(526) and/or Val(527) did not appear to alter the secondary structure of rClfA-( 221-559) as determined by far-UV circular dichroism spectroscopy. These dat a suggest that the C terminus of the A region may contain at least part of the Fg-binding site of ClfA and that Glu(526) and Val(527) may be involved in ligand recognition.