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 ligandbinding 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.

The beta antigen of the lbc protein complex of Group B streptococci is a cell-surface receptor which binds the Fc region of human immunoglobulin A (IgA). Determination of the nucleotide sequence of the beta antigen gene shows that it encodes a preprotein having a molecular weight of 130,963 daltons and a polypeptide of 1164 amino acid residues that is typical of other Gram-positive cell-wall proteins. There is a long signal sequence of 37 amino acids at the N-terminus. Four of the five C-terminal amino acid residues are basic and are preceded by a hydrophobic stretch that appears to anchor the C-terminus in the cell membrane. To the N-terminal side of this hydrophobic stretch is a putative cell-wall-spanning region containing proline-rich repeated sequences. An unusual feature of these repeated sequences is a three-residue periodicity, whereby every first residue is a proline, the second residue is alternating positively or negatively charged, and the third residue is uncharged. The IgA-binding activity was approximately localized by expressing subfragments of the beta antigen as fusion proteins. Two distinct but adjacent DNA segments specified peptides that bound IgA, which indicates that the IgA-binding activity is located in two distinct regions of the protein.

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The ␤-antigen of the c protein complex of group B streptococci contains two immunoglobulin A (IgA)binding domains called A and B. A 73-amino-acid segment in domain A is responsible for most of the IgA-binding activity. To identify the IgA binding motif, the 73-amino-acid domain was divided into 60 14amino-acid overlapping peptides spot synthesized onto a cellulose membrane. A 20-residue putative antigenic epitope was identified and expressed as a fusion protein. The fusion protein was purified by fast protein liquid chromatography and used to raise rabbit antiserum. By use of a membrane with spot-synthesized peptide amino acids of decreasing length (from 14 to 6 amino acids), the major antigenic epitope recognized by the anti-fusion protein antibodies was mapped to motif MLKKIE. Anti-fusion protein antibodies inhibited the binding of IgA to group B streptococci. This inhibition could be blocked by the peptide containing the motif MLKKIE. These results indicate that the motif MLKKIE is located in the IgA-binding site. The IgA-binding domain of ␤-antigen from three group B streptococcal strains reacted with the anti-fusion protein antibodies, and their coding sequences gave positive signals in Southern hybridization. The sequences of ␤-antigen from these strains were amplified by PCR, and sequence analysis showed them to be identical. The results indicate that the motif MLKKIE is required for IgA binding and is present in different group B streptococcal strains.