Matrix-binding proteins of Staphylococcus aureus: functional analysis of mutant and hybrid molecules

The fibrinogen-binding protein ClfA and the collagen-binding protein Cna ar e surface-associated adhesins of Staphylococcus aureus, ClfA has a dipeptid e repeat region R composed mainly of serine and aspartate residues, more th an 40 of which are required along with the 28-residue region W, the LPXTG m otif and region M to display the ligand-binding region A on the cell surfac e in a functional form. Cna has a Ol-residue region W and at least one 187- residue region B linking the collagen-binding region A to peptidoglycan. A cna mutant of S, aureus lacking region B was shown to bind collagen at the same level as wild-type Cna(+) cells, indicating that region B is not neces sary for ligand binding. Furthermore, altering the number of B repeats did not influence the level of collagen binding. In order to study the ability of C-terminal domains of Cna and ClfA to support functional ligand-binding activity of different adhesins, chimeric proteins were constructed and expr essed in S, aureus, Surprisingly, the presence of a single Cna B domain and a nonapeptide linker located between ClfA region A and Cna region WM faile d to support fibrinogen binding by S. aureus cells, despite the fact that C lfA region A was detected on the bacterial surface by immunoblotting. In co ntrast, the ClfA region A-Cna region B hybrid expressed as a recombinant pr otein in Escherichia coli did bind fibrinogen in Western ligand blots and i n an ELISA-type assay. It is concluded that Cna region B cannot support fun ctional display of ClfA region A on the bacterial cell surface. However, th e ClfA dipeptide repeat region R and region WM did promote functional surfa ce expression of the Cna collagen-binding domain in a hybrid Cna-ClfA prote in.