Clumping factor A

Abstract: The surface-located fibrinogen-binding protein (clumping factor; ClfA) of Staphylococcus aureus has an unusual dipeptide repeat linking the ligand binding domain to the wall-anchored region. Southern blotting experiments revealed several other loci in the S. aureus Newman genome that hybridized to a probe comprising DNA encoding the dipeptide repeat. One of these loci is analysed here. It also encodes a fibrinogen-binding protein, which we have called ClfB. The overall organization of ClfB is very similar to that of ClfA, and the proteins have considerable sequence identity in the signal sequence and wall attachment domains. However, the A regions are only 26% identical. Recombinant biotinylated ClfB protein bound to fibrinogen in Western ligand blots. ClfB reacted with the alpha- and beta-chains of fibrinogen in the ligand blots in contrast to ClfA, which binds exclusively to the gamma-chain. Analysis of proteins released from the cell wall of S. aureus Newman by Western immunoblotting using antibody raised against the recombinant A region of ClfB identified a 124 kDa protein as the clfB gene product. This protein was detectable only on cells that were grown to the early exponential phase. It was absent from cells from late exponential phase or stationary phase cultures. Using a clfB mutant isolated by allelic replacement alone and in combination with a clfA mutation, the ClfB protein was shown to promote (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. However, in wild-type exponential-phase S. aureus Newman cultures, ClfB activity was masked by the ClfA protein, and it did not contribute at all to interactions of cells from stationary-phase cultures with fibrinogen. ClfB-dependent bacterial adherence to immobilized fibrinogen was inhibited by millimolar concentrations of Ca2+ and Mn2+, which indicates that, like ClfA, ligand binding by ClfB is regulated by a low-affinity inhibitory cation binding site.

Abstract: The pathogenic role of staphylococcal coagulase and clumping factor was investigated in the rat model of endocarditis. The coagulase-producing and clumping factor-producing parent strain Staphylococcus aureus Newman and a series of mutants defective in either coagulase, clumping factor, or both were tested for their ability (i) to attach in vitro to either rat fibrinogen or platelet-fibrin clots and (ii) to produce endocarditis in rats with catheter-induced aortic vegetations. In vitro, the clumping factor-defective mutants were up to 100 times less able than the wild type strain to attach to fibrinogen and also significantly less adherent than the parents to platelet-fibrin clots. Coagulase-defective mutants, in contrast, were not altered in their in vitro adherence phenotype. The rate of in vivo infection was inoculum dependent. Clumping factor-defective mutants produced ca. 50% less endocarditis than the parent organisms when injected at inoculum sizes infecting, respectively, 40 and 80% (ID40 and ID80, respectively) of rats with the wild-type strain. This was a trend at the ID40 but was statistically significant at the ID80 (P < 0.05). Coagulase-defective bacteria were not affected in their infectivity. Complementation of a clumping factor-defective mutant with a copy of the wild-type clumping factor gene restored both its in vitro adherence and its in vivo infectivity. These results show that clumping factor plays a specific role in the pathogenesis of S. aureus endocarditis. Nevertheless, the rate of endocarditis with clumping factor-defective mutants increased with larger inocula, indicating the contribution of additional pathogenic determinants in the infective process.

Abstract: Staphylococcus aureus colonizes the moist squamous epithelium of the anterior nares. One of the adhesins likely to be responsible is the S. aureus surface protein G (SasG), which has sequence similarity with the proteins Pls (plasmin sensitive) of S. aureus and Aap (accumulation associated protein) of Staphylococcus epidermidis. Expression of SasG by a laboratory strain of S. aureus could not be detected by Western immunoblotting. To enable investigation of SasG, the gene was cloned into two expression vectors, the IPTG-inducible pMUTIN4 and the tetracycline-inducible pALC2073, and introduced into S. aureus. Expression of SasG masked the ability of exponentially grown S. aureus cells expressing protein A (Spa), clumping factor B (ClfB) and the fibronectin binding proteins A and B (FnBPA and FnBPB) to bind to IgG, cytokeratin 10 and fibronectin, respectively. SasG also masked binding to fibrinogen mediated by both ClfB and the FnBPs. Western immunoblotting showed no reduction in expression of the blocked adhesins following induction of SasG. SasG size variants with eight, six or five B repeats masked binding to the ligands, whereas variants with four, two or one repeats had no effect. SasG-expressing strains formed peritrichous fibrils (53.47±2.51 nm long) of varying density on the cell wall, which were labelled by immunogold negative staining with anti-SasG antibodies. SasG-expressing strains of S. aureus also formed biofilm independently of the polysaccharide intercellular adhesin (PIA). SasG variants with eight, six and five repeats formed biofilm, whereas variants with four, two or one repeats did not. It was concluded that the fibrillar nature of SasG explains its ability to mask binding of S. aureus microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) to their ligands and to promote formation of biofilm. In addition, the strong adhesion of SasG to desquamated nasal epithelial cells likely compensates for its blocking of the binding of S. aureus ClfB to cytokeratin 10, which is important in adhesion to squames by cells lacking SasG. Several clinical isolates expressed SasG at levels similar to those of SH1000 sasG : : pMUTIN4, indicating that the properties described in the laboratory strain SH1000 may be relevant in vivo.