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

Microbiology. 1999 Sep;145 ( Pt 9):2497-2505. doi: 10.1099/00221287-145-9-2497.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adhesins, Bacterial / chemistry
  • Adhesins, Bacterial / genetics
  • Adhesins, Bacterial / metabolism*
  • Bacterial Adhesion
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Blotting, Western
  • Carrier Proteins / metabolism
  • Collagen / metabolism
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme-Linked Immunosorbent Assay / methods
  • Fibrinogen / metabolism*
  • Polymerase Chain Reaction / methods
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / metabolism
  • Repetitive Sequences, Amino Acid
  • Staphylococcus aureus / genetics
  • Staphylococcus aureus / metabolism
  • Staphylococcus aureus / physiology*

Substances

  • Adhesins, Bacterial
  • Bacterial Proteins
  • Carrier Proteins
  • Recombinant Fusion Proteins
  • adhesin, Staphylococcus aureus
  • Fibrinogen
  • Collagen