The Fibrinogen-binding MSCRAMM (Clumping Factor) of Staphylococcus aureus Has a Ca2+-dependent Inhibitory Site

Abstract

The clumping factor (ClfA) is a cell surface-associated protein of Staphylococcus aureus that promotes binding of fibrinogen or fibrin to the bacterial cell. Previous studies have shown that ClfA and the platelet integrin ␣ IIb ␤ 3 recognize the same domain at the extreme C terminus of the fibrinogen ␥-chain. ␣ IIb ␤ 3 interaction with this domain is known to occur in close proximity to a Ca 2؉ -binding EF-hand structure in the ␣-subunit. Analysis of the primary structure of ClfA indicated the presence of a potential Ca Staphylococcus aureus causes a wide range of opportunistic infections that range from superficial skin infections to lifethreatening diseases including endocarditis, pneumonia, and septicemia. Adherence of bacteria to host matrix components that is mediated by bacterial surface adhesins is the initial critical event in the pathogenesis of most infections. The extracellular matrix (ECM) 1 contains numerous glycoproteins and proteoglycans assembled into insoluble matrices that serve as substrata for the adhesion and migration of tissue cells. These processes involve integrins, a family of heterodimeric (␣␤) cellsurface receptors that recognize specific ECM proteins. It has become increasingly evident that bacteria, including S. aureus, also utilize the ECM as substrata for their adhesion by way of a family of adhesins called MSCRAMM (microbial surface components recognizing adhesive matrix molecules) (1) that specifically recognize host matrix components. One important component of the ECM, also occurring in soluble form in blood plasma, is fibrinogen, a 340-kDa hexamer composed of 2␣-, 2␤-, and 2␥-chains linked by disulfide bonds. This protein is recognized by several integrins including the platelet integrin ␣ IIb ␤ 3 . Activation of platelets and integrin ␣ IIb ␤ 3 results in fibrinogen-dependent aggregation in vitro and the formation of platelet-fibrin thrombi in vivo.

Characterization of novel LPXTG-containing proteins of Staphylococcus aureus identified from genome sequences

“…The PCR fragments were cloned into pQE30 at the BamHI and HindIII sites and the resulting plasmids transformed into Escherichia coli TOPP-3 (Stratagene) for protein expression. The recombinant proteins were purified using Ni 2+ -chelate chromatography (O'Connell et al, 1998). The SasF40-211 construct was used to immunize a rabbit to generate anti-SasF antibodies.…”

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

“…The PCR products were cloned into the Nterminal six-histidine tag expression vector pQE-30 (Qiagen). Recombinant proteins were expressed and purified by Ni 2ϩ chelate chromatography followed by Q-Sepharose chromatography as described previously (34). SDS-PAGE analysis showed a single major band of Ͼ95% purity Antibodies-Specific pathogen-free rabbits were immunized with rFnBPA and rFnBPB .…”

The Elastin-binding Protein of Staphylococcus aureus(EbpS) Is Expressed at the Cell Surface as an Integral Membrane Protein and Not as a Cell Wall-associated Protein

“…rEbpS1-267 was insoluble and formed inclusion bodies, which were dissolved in 8 M urea, 100 mM NaH 2 PO 4 , 10 mM Tris-HCl, pH 8.0, buffer and purified as described previously (18). rEbpS343-486 was soluble and was purified as described previously (28).…”