title: Fibronectin-binding protein of Streptococcus pyogenes: sequence of the binding domain involved in adherence of streptococci to epithelial cells.
abstract: The sequence of the fibronectin-binding domain of the fibronectin-binding protein of Streptococcus pyogenes (Sfb protein) was determined, and its role in streptococcal adherence was investigated by use of an Sfb fusion protein in adherence studies. A 1-kb DNA fragment coding for the binding domain of Sfb protein was cloned into the expression vector pEX31 to produce an Sfb fusion protein consisting of the N-terminal part of MS2 polymerase and a C-terminal fragment of the streptococcal protein. Induction of the vector promoter resulted in hyperexpression of fibronectin-binding fusion protein in the cytoplasm of the recombinant Escherichia coli cells. Sequence determination of the cloned 1-kb fragment revealed an in-frame reading frame for a 268-amino-acid peptide composed of a 37-amino-acid sequence which is completely repeated three times and incompletely repeated a fourth time. Cloning of one repeat into pEX31 resulted in expression of small fusion peptides that show fibronectin-binding activity, indicating that one repeat contains at least one binding domain. Each repeat exhibits two charged domains and shows high homology with the 38-amino-acid D3 repeat of the fibronectin-binding protein of Staphylococcus aureus. Sequence comparison with other streptococcal ligand-binding surface proteins, including M protein, failed to reveal significant homology, which suggests that Sfb protein represents a novel type of functional protein in S. pyogenes. The Sfb fusion protein isolated from the cytoplasm of recombinant cells was purified by fast protein liquid chromatography. It showed a strong competitive inhibition of fibronectin binding to S. pyogenes and of the adherence of bacteria to cultured epithelial cells. In contrast, purified streptococcal lipoteichoic acid showed only a weak inhibition of fibronectin binding and streptococcal adherence. These results demonstrate that Sfb protein is directly involved in the fibronectin-mediated adherence of S. pyogenes to epithelial cells.
authors.affiliation: Department of Microbiology, Technical University/GBF-National Research Centre for Biotechnology, Braunschweig, Germany.
authors.forename: S R
authors.initials: SR
authors.lastname: Talay
authors.forename: P
authors.initials: P
authors.lastname: Valentin-Weigand
authors.forename: P G
authors.initials: PG
authors.lastname: Jerlström
authors.forename: K N
authors.initials: KN
authors.lastname: Timmis
authors.forename: G S
authors.initials: GS
authors.lastname: Chhatwal
mesh_terms: D000595:Amino Acid Sequence; D000942:Antigens, Bacterial; D001422:Bacterial Adhesion; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins / Q000737:chemistry; D001483:Base Sequence; D002352:Carrier Proteins; D003001:Cloning, Molecular; D004848:Epithelium / Q000382:microbiology; D005353:Fibronectins / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D017469:Receptors, Fibronectin; D011971:Receptors, Immunologic / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D012091:Repetitive Sequences, Nucleic Acid; D013297:Streptococcus pyogenes / Q000737:chemistry*
chemical_list: D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002352:Carrier Proteins; D005353:Fibronectins; D017469:Receptors, Fibronectin; D011971:Receptors, Immunologic; D011993:Recombinant Fusion Proteins; C014689:streptococcal M protein