The dipeptide repeat region of the fibrinogen‐binding protein (clumping factor) is required for functional expression of the fibrinogen‐binding domain on the Staphylococcus aureus cell surface

Abstract

SummaryClumping factor of Staphylococcus aureus is a fibrinogen-binding protein that is located on the bacterial cell surface. The protein has an unusual repeat domain (region R) comprising mainly the dipeptide aspartate and serine. To determine if region R has a role in the surface display of the fibrinogen-binding region A domain, deletions lacking the region R encoding region of the clfA gene were generated. To determine the minimum length of region R required for wild-type levels of ClfA expression, variants with truncated region R domains were constructed. S. aureus cells expressing mutated clfA genes were tested for (i) proteins released by lysostaphin treatment that reacted with antisera specific for region A, (ii) clumping in soluble fibrinogen, (iii) adherence to immobilized fibrinogen and (iv) expression of the ClfA antigen on the cell surface by fluorescent activated cell sorting analysis. Each construct expressed three major immunoreactive proteins, two of which were putative Nterminal degradation products. Region R residues greater than 40 were required between region A and W (72 residues between region A and the LPDTG sorting signal) for wild-type levels of clumping in fibrinogen. A stepwise decrease in clumping titre was observed as the distance between region A and LPDTG was decreased from 72 to 4 residues. Similarly, a decrease in binding of anti-ClfA serum and in binding to fibrinogen-coated plastic surfaces was observed with cells expressing ClfA with 40 region R residues or less. Nevertheless, low levels of adherence to fibrinogen and binding to anti-ClfA serum occurred with ClfA derivatives that lacked region R altogether. This indicates that a small proportion of the ClfA molecules are linked to peptidoglycan very close to the cell surface but that residues greater than 72 are needed to allow sufficient ClfA molecules to span the entire cell wall and to display the biologically active A domain in a form that can participate fully in fibrinogen binding.

Bacterial fibronectin-binding proteins and endothelial cell surface fibronectin mediate adherence of Staphylococcus aureus to resting human endothelial cells

“…Transductants resistant to 2 µg tetracycline ml − " and 10 µg erythromycin ml − " were selected. The lack of adherence to purified human fibronectin at 10 µg ml − " (Sigma) was confirmed by microtitre adherence assay using an established method (data not shown) (Hartford et al, 1997).…”

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

“…The SX dipeptide repeats of SasA resemble the SD dipeptide repeats of the Clf-Sdr family. The length of the SX repeats varies considerably from strain to strain, as do the SD repeats , and it seems reasonable to think that they have the same function, namely to project the ligand-binding region from the cell surface (Hartford et al, 1997). Despite the strain-to-strain length variation of the SD and SX repeats, the underlying frequency of recombination between the DNA repeats is actually quite low.…”

“…The ClfA and ClfB A regions are composed of independently folded subdomains consisting primarily of b-sheets and coils with a small amount of a-helix (Perkins et al, 2001;Deivanayagam et al, 2002). Repeat regions occur as dipeptides in SD repeats of Sdr proteins (Josefsson et al, 1998a) that act as a stalk to project the N-terminal A region away from the bacterial cell surface (Hartford et al, 1997) or larger tandem repeated domains which vary in size and identity between proteins. The B repeats of Sdr proteins bind Ca 2+ and form rigid rod-like structures (Josefsson et al, 1998b) while the D repeats of the FnbpA and FnbpB proteins are flexible and bind fibronectin (McGavin et al, 1993;House-Pompeo et al, 1996).…”

The predicted secretome of Lactobacillus plantarum WCFS1 sheds light on interactions with its environment

“…In the ClfB protein of S. aureus, a long Ser-Asp repeat has been shown to play a role in the adherence of this bacterium to fibrinogen (Hartford et al, 1997). The Ser-Asp region in ClfB is over 300 residues in size and separates the fibronectin-binding domain from its Cterminal peptidoglycan anchor; it probably functions as a spacer (Hartford et al, 1997). In lp_1303a the repeat separates a C-terminal transmembrane anchor from a bacterial Ig-like domain, which is found in a variety of bacterial adhesion proteins (Pfam: PF02368).…”