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 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.

A two-domain mechanism for group A streptococcal adherence through protein F to the extracellular matrix.

“…Our analysis revealed that a single RD2 repeat unit of 37 amino acids was incapable of binding Fn, even when expressed within the surface-exposed region of an unrelated streptococcal protein. This is in contrast to another report that a single RD2 repeat could block the binding of Fn to GAS and adherence of the bacteria to cultured epithelial cells (Talay et al, 1992). The reason for this discrepancy is not clear, but it may result from the use of a different technique for the construction of the fusion protein with protein F. Our results demonstrate instead that an active unit of the RD2 repeat consists of 44 amino acids located on a fragment composed of two adjacent repeats.…”

“…Perhaps the best evidence for the role of a specific surface protein in promoting the ability of GAS to bind Fn has come from studies of a family of highly homologous proteins which include sfb (Talay et al, 1991) and protein F (Hanski and Caparon, 1992). These proteins bind Fn when expressed in Escherichia coli (Hanski and Caparon, 1992;Talay et al, 1992), can inhibit the binding of Fn to intact streptococcal cells (Hanski and Caparon, 1992;Talay et al, 1992) and can inhibit the adherence of GAS to several epithelial cells (Talay et al, 1992). Insertional inactivation of the gene encoding protein F (prtF), generated a mutant which lost Fn binding activity and the ability to adhere to respiratory epithelial cells (Hanski and Caparon, 1992).…”

Multiple binding of type 3 streptococcal M protein to human fibrinogen, albumin and fibronectin

“…In contrast, Caperon et al [5] showed that M ÷ and M-mutants of type 6 streptococci did not differ in their adherence to epithelial cells. Furthermore, fibronectin binding proteins from streptococci have been cloned [11,33] and a C-terminal binding peptide with a molecular mass of 40 kDa was found to be different from M protein [34]. Another protein, group A streptococcal glyceraldehyde-3-phosphate-dehydrogenase was shown to be a multifunctional protein also with affinity to fibronectin [24].…”

“…Fibronectin (Fn) is one of the cellular matrix components which is a target molecule for bacterial adhesion [2]. Fibronectin binding proteins from group A streptococci other than M protein have been cloned [11,33] and sequence comparison of a group A streptococcal fibronectin binding peptide [34] with FnBP from Staphylococcus aureus [30] revealed similarities in the four repeat binding regions.…”

SdrI ofStaphylococcus saprophyticus is a multifunctional protein: localization of the fibronectin-binding site

“…Fibronectin has a modular structure and is composed of type 1, type 2 and type 3 (F1, F2 and F3) modules. FnBPs from S. aureus, Streptococcus pyogenes and Borrelia burgdorferi (Signas et al, 1989;Talay et al, 1992;Probert & Johnson, 1998) all bind the N-terminal domain, i.e. the small 30-kDa fragment of fibronectin, through a tandem b-zipper (n), SA5 (), SA6 () and PM81 (') were incubated in the wells for 1 h at room temperature.…”