| Using a DNA probe to the surface-located fibrinogen-binding protein (clumping factor; ClfA) of S. aureus southern blotting experiments revealed several other loci in the S. aureus Newman genome. One of these loci is analyzed here. It also encodes a fibrinogen-binding protein, which we have called ClfB. Recombinant biotinylated ClfB protein bound to fibrinogen in Western ligand blots. This protein was only detectable on cells that were grown to the early exponential phase. Using a clfB mutant isolated by allelic replacement alone and in combination with a clfA mutation, the ClfB protein was shown to mediate (i) clumping of exponential phase cells in a solution of fibrinogen, (ii) adherence of exponential-phase bacteria to immobilized fibrinogen in vitro, and (iii) bacterial adherence to ex vivo human haemodialysis tubing, suggesting that it could contribute to the pathogenicity of biomaterial-related infections. We hypothesize that the ClfB A-region is comprised of three subdomains, which we have named N1, N2, and N3, respectively. Far UV Circular Dichroism spectra showed that each subdomain is composed mainly of β-sheets with little or no discernable α-helices. Heat induced unfolding/refolding and gel permeation chromatography indicated that the subdomains were globular and folded as discreet units. N123, N12 and N23 all bound to fibrinogen, but N23 had a higher affinity for fibrinogen than that observed for the full-length A-region; N123 or for N12. However, an extended N-terminus of N23 was required for ligand binding. Addition of recombinant N23 effectively inhibited C1fB-mediated bacterial adherence to fibrinogen, N123 caused some reduction in bacterial attachment, whereas N12 was essentially inactive. Antibodies raised against the central N2 domain of the A-region were the most effective at inhibiting bacterial adhesion to immobilized fibrinogen, although anti-N3 or anti-N1 antibodies also caused some reduction in ClfB-mediated adherence to fibrinogen. Crystallographic studies showed the fibrinogen-binding region, N23, consists of two separate domains, termed N2 and N3, which have an immunoglobin-like fold. This confirms the previous hypothesis that these domains are independently folded units, consisting of majority β-sheet structure. Additionally, the extreme N- and C-termini were shown to be critical to the binding of N23 to immobilized fibrinogen. |
