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

Department of Microbiology, Moyne Institute of Preventive Medicine, Trinity College, Dublin 2, Ireland¹
Albert B. Alkek Institute of Biosciences and Technology, Texas A&M University, Houston, TX 77030, USA²

Author for correspondence: Timothy J. Foster. Tel: +353 1 6082014. Fax: +353 1 6799294. e-mail: tfoster{at}tcd.ie

The fibrinogen-binding protein ClfA and the collagen-binding protein Cna are surface-associated adhesins of Staphylococcus aureus. ClfA has a dipeptide repeat region R composed mainly of serine and aspartate residues, more than 40 of which are required along with the 28-residue region W, the LPXTG motif and region M to display the ligand-binding region A on the cell surface in a functional form. Cna has a 61-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 necessary 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 expressed 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 failed to support fibrinogen binding by S. aureus cells, despite the fact that ClfA region A was detected on the bacterial surface by immunoblotting. In contrast, the ClfA region A–Cna region B hybrid expressed as a recombinant protein in Escherichia coli did bind fibrinogen in Western ligand blots and in an ELISA-type assay. It is concluded that Cna region B cannot support functional display of ClfA region A on the bacterial cell surface. However, the ClfA dipeptide repeat region R and region WM did promote functional surface expression of the Cna collagen-binding domain in a hybrid Cna–ClfA protein.

Keywords: Staphylococcus aureus, clumping factor, collagen-binding protein, dipeptide repeat, fibrinogen-binding protein

^a Present address: SmithKline Beecham, 21250 South Collegeville Road, PO Box 5089, Collegeville, PA 19426-0989, USA.

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