Matrix-binding proteins of Staphylococcus aureus: functional analysis of mutant and hybrid molecules.
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.
Hartford
Orla
O
Department of Microbiology, Moyne Institute of Preventive Medicine, Trinity College, Dublin 2, Ireland1.
McDevitt
Damien
D
Albert B. Alkek Institute of Biosciences and Technology, Texas A&M University, Houston, TX 77030, USA2.
Foster
Timothy J
TJ
Department of Microbiology, Moyne Institute of Preventive Medicine, Trinity College, Dublin 2, Ireland1.
D018829:Adhesins, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001422:Bacterial Adhesion; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism; D003094:Collagen / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005340:Fibrinogen / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid; D013211:Staphylococcus aureus / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
D018829:Adhesins, Bacterial; D001426:Bacterial Proteins; D002352:Carrier Proteins; D011993:Recombinant Fusion Proteins; C048165:adhesin, Staphylococcus aureus; D005340:Fibrinogen; D003094:Collagen