Protein F2, a novel fibronectin-binding protein from Streptococcus pyogenes, possesses two binding domains

Binding of the group A streptococcus (GAS) to respiratory epithelium is mediated by the fibronectin (Fn)-binding adhesin, protein F1. Previous studies have suggested that certain GAS strains express Fn-binding proteins that are different from protein F1. In this study, we have cloned, sequenced, and characterized a gene ( prtF2 ) from GAS strain 100076 encoding a novel Fn-binding protein, termed protein F2. Insertional inactivation of prtF2 in strain 100076 abolishes its high-affinity Fn binding. prtF2 -related genes exist in most GAS strains that lack prtF1 (encoding protein F1) but bind Fn with high affinity. These observations suggest that protein F2 is a major Fn-binding protein in GAS. Protein F2 is highly homologous to Fn-binding proteins from Streptococcus dysgalactiae and Strep-tococcus equisimilis , particularly in its carboxy-terminal portion. Two domains are responsible for Fn binding by protein F2. One domain (FBRD) consists of three consecutive repeats, whereas the other domain (UFBD) resides on a non-repeated stretch of approximately 100 amino acids and is located 100 amino acids amino-terminal of FBRD. Each of these domains is capable of binding Fn when expressed as a separate protein. In strain 100076, protein F2 activity is regulated in response to alterations in the concentration of atmospheric oxygen.     

Serum opacity factor promotes group A streptococcal epithelial cell invasion and virulence

Serum opacity factor (SOF) is a bifunctional cell surface protein expressed by 40-50% of group A streptococcal (GAS) strains comprised of a C-terminal domain that binds fibronectin and an N-terminal domain that mediates opacification of mammalian sera. The sof gene was recently discovered to be cotranscribed in a two-gene operon with a gene encoding another fibronectin-binding protein, sfbX. We compared the ability of a SOF(+) wild-type serotype M49 GAS strain and isogenic mutants lacking SOF or SfbX to invade cultured HEp-2 human pharyngeal epithelial cells. Elimination of SOF led to a significant decrease in HEp-2 intracellular invasion while loss of SfbX had minimal effect. The hypoinvasive phenotype of the SOF(-) mutant could be restored upon complementation with the sof gene on a plasmid vector, and heterologous expression of sof49 in M1 GAS or Lactococcus lactis conferred marked increases in HEp-2 cell invasion. Studies using a mutant sof49 gene lacking the fibronectin-binding domain indicated that the N-terminal opacification domain of SOF contributes to HEp-2 invasion independent of the C-terminal fibronectin binding domain, findings corroborated by observations that a purified SOF N-terminal peptide could promote latex bead adherence to HEp-2 cells and inhibit GAS invasion of HEp-2 cells in a dose-dependent manner. Finally, the first in vivo studies to employ a single gene allelic replacement mutant of SOF demonstrate that this protein contributes to GAS virulence in a murine model of necrotizing skin infection.     

Borrelia burgdorferi binds fibronectin through a tandem beta-zipper, a common mechanism of fibronectin binding in staphylococci, streptococci, and spirochetes

BBK32 is a fibronectin-binding protein from the Lyme disease-causing spirochete Borrelia burgdorferi. In this study, we show that BBK32 shares sequence similarity with fibronectin module-binding motifs previously identified in proteins from Streptococcus pyogenes and Staphylococcus aureus. Nuclear magnetic resonance spectroscopy and isothermal titration calorimetry are used to confirm the binding sites of BBK32 peptides within the N-terminal domain of fibronectin and to measure the affinities of the interactions. Comparison of chemical shift perturbations in fibronectin F1 modules on binding of peptides from BBK32, FnBPA from S. aureus, and SfbI from S. pyogenes provides further evidence for a shared mechanism of binding. Despite the different locations of the bacterial attachment sites in BBK32 compared with SfbI from S. pyogenes and FnBPA from S. aureus, an antiparallel orientation is observed for binding of the N-terminal domain of fibronectin to each of the pathogens. Thus, these phylogenetically and morphologically distinct bacterial pathogens have similar mechanisms for binding to human fibronectin.     

Differences in the aromatic domain of homologous streptococcal fibronectin-binding proteins trigger different cell invasion mechanisms and survival rates

Group A streptococci (GAS, Streptococcus pyogenes) and Group G streptococci (GGS, Streptococcus dysgalactiae ssp. equisimilis) adhere to and invade host cells by binding to fibronectin. The fibronectin-binding protein SfbI from GAS acts as an invasin by using a caveolae-mediated mechanism. In the present study we have identified a fibronectin-binding protein, GfbA, from GGS, which functions as an adhesin and invasin. Although there is a high degree of similarity in the C-terminal sequence of SfbI and GfbA, the invasion mechanisms are different. Unlike caveolae-mediated invasion by SfbI-expressing GAS, the GfbA-expressing GGS isolate trigger cytoskeleton rearrangements. Heterologous expression of GfbA on the surface of a commensal Streptococcus gordonii and purified recombinant protein also triggered actin rearrangements. Expression of a truncated GfbA (lacking the aromatic domain) and chimeric GfbA/SfbI protein (replacing the aromatic domain of SfbI with the GfbA aromatic domain) on S. gordonii or recombinant proteins alone showed that the aromatic domain of GfbA is responsible for different invasion mechanisms. This is the first evidence for a biological function of the aromatic domain of fibronectin-binding proteins. Furthermore, we show that streptococci invading via cytoskeleton rearrangements and intracellular trafficking along the classical endocytic pathway are less persistence than streptococci entering via caveolae.     

Co-operative binding of human fibronectin to Sfbl protein triggers streptococcal invasion into respiratory epithelial cells

Streptococcal fibronectin binding protein I (SfbI) mediates adherence to and invasion of Streptococcus pyogenes into human epithelial cells. In this study, we analysed the binding activity of distinct domains of SfbI protein towards its ligand, the extracellular matrix component fibronectin, as well as the biological implication of the binding events during the infection process. By using purified recombinant SfbI derivatives as well as in vivo expressed SfbI domains on the surface of heterologous organism Streptococcus gordonii , we were able to dissociate the two major streptococcal target domains on the human fibronectin molecule. The SfbI repeat region exclusively bound to the 30 kDa N-terminal fragment of fibronectin, whereas the SfbI spacer region exclusively bound to the 45 kDa collagen-binding fragment of fibronectin. In the case of native surface-expressed SfbI protein, an induced fit mode of bacteria–fibronectin interaction was identified. We demonstrate that binding of the 30 kDa fibronectin fragment to the repeat region of SfbI protein co-operatively activates the adjacent SfbI spacer domain to bind the 45 kDa fibronectin fragment. The biological consequence arising from this novel mode of fibronectin targeting was analysed in eukaryotic cell invasion assays. The repeat region of SfbI protein is mediating adherence and constitutes a prerequisite for subsequent invasion, whereas the SfbI spacer domain efficiently triggers the invasion process of streptococci into the eukaryotic cell. Thus, we were able to dissect bacterial adhesion from invasion by manipulating one protein. SfbI protein therefore represents a highly evolved prokaryotic molecule that exploits the host factor fibronectin not only for extracellular targeting but also for its subsequent activation that leads to efficient cellular invasion.     

Interaction of Staphylococcus aureus fibronectin-binding protein with fibronectin: affinity, stoichiometry, and modular requirements

The repetitive D1, D2, and D3 elements of Staphylococcus aureus fibronectin-binding protein FnBPA each bind the N-terminal 29-kDa fragment (N29) of fibronectin with low micromolar dissociation constants (Kd), but in tandem they compose a high affinity domain, D1-3. An additional seven Fn-binding segments have been predicted in FnBPA in a region N-terminal of the D-repeats (Schwarz-Linek, U., Werner, J. M., Pickford, A. R., Gurusiddappa, S., Kim, J. H., Pilka, E. S., Briggs, J. A., Gough, T. S., Hook, M., Campbell, I. D., and Potts, J. R. (2003) Nature 423, 177-181). We have evaluated the requirements for high affinity binding of N29 to the D-repeat domain and determined the affinity and stoichiometry of N29 binding to segments that are N-terminal of the D-repeats in the related FnBPB adhesin. We confirmed that D1-3 has two equivalent high affinity sites (Kd, approximately 1 nm) and provided evidence for one or more lower affinity sites (Kd, approximately 0.5 microm). Bimodular D1-2 and D2-3 exhibit intermediate affinity sites with respective Kd values of 0.25 and 0.044 microm, as well as a low affinity site with a Kd value of 2.2-2.5 microm. We also identified two binding domains that are N-terminal of the D-repeats, designated DuB and DuA. Segments internal to these domains individually bound N29 with similar Kd values of approximately 2 microm, whereas the DuBA polypeptide possessing both segments and other intervening sites bound four molecules of N29 with much higher affinity (Kd, approximately 10 nm). DuBAD, a larger polypeptide harboring all of the known or predicted binding motifs in FnBPB, bound seven to eight molecules of N29, with a Kd of approximately 7 nm. Because most of the isolated binding segments display low affinity for N29 and lack motifs for binding of one or both of the 1F1 and 5F1 modules in the N-terminal domain of Fn, we propose that high affinity is achieved in part as a consequence of self-interaction between bound molecules of N29.     

Multiple domains are involved in the interaction of endothelial cell thrombospondin with fibronectin

Thrombospondin is a large multifunctional glycoprotein synthesized, secreted and incorporated into the extracellular matrix by several cell types in culture. It is also present in the blood platelet and is secreted following platelet activation. We have previously shown that thrombospondin co-distributes with fibronectin in the extracellular matrix and that it can bind directly to purified fibronectin. In order to elucidate the chemical aspects of thrombospondin incorporation into the extracellular matrix, we studied the interaction of endothelial cell thrombospondin and fibronectin. We find that endothelial cell thrombospondin has two distinct binding domains for fibronectin. One domain is on the 70-kDa core fragment, probably similar to that of platelet thrombospondin. The other domain is on the 27-kDa N-terminal fragment and is unique to endothelial cell thrombospondin. The dissociation constant of the intact endothelial-cell-derived molecule is 0.7 +/- 0.2 x 10(-7) M. Following fragmentation, the separate domains bind with somewhat lower affinity: the core domain binds with a Kd of 3.4 +/- 1.5 x 10(-7) M and the N-terminal domain binds with a Kd of 8.8 +/- 1.8 x 10(-7) M. Binding of the intact molecule is Ca2+-independent. By contrast, following tryptic fragmentation, binding of the 70-kDa fragment is practically lost. It can be restored, however, by removal of Ca2+, indicating that the binding site on this domain is either sequestered or becomes so following fragmentation. Heparin, which also binds to both fragments, competed with fibronectin binding to the 27-kDa fragment but not to the 70-kDa domain. The fact that heparin also competitively inhibits fibronectin binding of the intact molecule further supports sequestration of the fibronectin-binding domain on the 70-kDa core fragment. Our data suggest that endothelial-cell thrombospondin possesses two distinct binding sites for fibronectin, a low-affinity constitutively available one and a high-affinity one, possibly sequestered on the intact unbound molecule.     

Presence of fibronectin-binding protein gene prtF2 in invasive group A streptococci in tropical Australia is associated with increased internalisation efficiency

The fibronectin-binding proteins (FnBPs) PrtF1 and PrtF2 are considered to be major group A streptococcal virulence factors, mediating adherence to and internalisation of host cells. The present study investigated an association between the presence of prtF1 and prtF2 genes and internalisation efficiency in group A streptococci (GAS) isolated from patients with invasive disease. Of the 80 isolates tested, 58 (73%) had prtF1 and 71 (89%) possessed prtF2. Three isolates (4%) had neither gene, seven (9%) had prtF1 only, 19 (24%) had prtF2 only and 51 isolates (64%) had both prtF1 and prtF2. prtF2-positive isolates internalised up to three times more efficiently than isolates that had prtF1 alone (P<0.001), and 1.5-fold better than isolates that had neither gene. No significant association was found between internalisation efficiency and presence of the prtF1 gene. Analysis of the fibronectin-binding repeat domain (FBRD) of prtF2 revealed that this gene can contain 2, 3, 4 or 5 repeat regions and that five repeat regions conferred very high internalisation efficiency in invasive GAS isolates.     

The fibronectin-binding capacity and host cell adherence of Streptococcus pyogenes strains are discordant with each other

Surface exposed fibronectin-binding proteins (FBPs) play an important role in the adherence of Streptococcus pyogenes (group A streptococcus, GAS) to host cells. This pathogen expresses numerous FBPs, of which SfbI, SfbII and PrtF2 are major surface exposed FBPs. However, GAS strains differ in the genetic potential to express these proteins. To test whether this difference reflects in differences in fibronectin (Fn) binding, a set of circulating strains previously examined for adherence to host cells was used. The 68 distinct strains were isolated from throat, skin and blood. They were analyzed for (a) the presence of genes for SfbI, SfbII and PrtF2 and (b) the extent of Fn binding. The results suggest that strains possessing two or more of the genes for these FBPs bound Fn significantly more than strains possessing none or one of the genes. No correlation between the extent of Fn binding and the tissue site of isolation was found. Furthermore, together with our previous studies on adherence capacity of these GAS strains, we found no correlation between Fn binding ability and the avidity of the strains to adhere to epithelial cells. We suggest that while Fn binding is important for adhesion, for many GAS strains the extent of Fn binding is not the critical determinant of adherence.     

Fibronectin receptors of mononuclear phagocytes: Binding characteristics and biochemical isolation

Fibronectin receptors on mononuclear phagocytes are involved in the localization of monocytes at inflammatory sites and in the subsequent expression of macrophage-like phenotypes. In this study, we have investigated the hypothesis that proteolytically derived fragments of fibronectin may interfere with binding of fibronectin to monocytes in the extracellular matrix. We report on the reactivity of U937 cells with an 80-kDa tryptic fragment of fibronectin which contains the cell-binding domain but lacks the gelatin/collagen-binding domain. U937 cells attached to surfaces coated with the 80-kDa fragment as well as with intact fibronectin. Preincubation of the cells with the 80-kDa fragment inhibited attachment to both surfaces while intact fibronectin had little or no inhibitory effect. The Ki for inhibition of attachment (0.5 microM) was consistent with the Kd for binding of the 3H-labeled 80-kDa fragment (0.34 microM) to U937 cells in suspension. There were 4-5 x 10(5) 80-kDa binding sites per cell. The relatively high affinity of the 80-kDa fragment for the monocyte surface permitted the isolation and characterization of fibronectin-binding proteins from U937 cells and peripheral blood monocytes by affinity chromatography. When octylglucoside lysates of lactoperoxidase iodinated cells were applied to 80-kDa-Sepharose columns, a polypeptide complex of 152/125 kDa was eluted with the synthetic peptide GRGDSPC, but not with GRGESP. This complex resolved into a single diffuse band of 144 kDa upon reduction. Binding of the protein complex to the affinity column required divalent cations. The complex bound to wheat germ agglutinin and could be specifically eluted by N-acetylglucosamine. Similar cell-surface proteins were isolated from peripheral blood monocytes.     

Fc-mediated nonspecific binding between fibronectin-binding protein I of Streptococcus pyogenes and human immunoglobulins.

Fibronectin-binding protein I (SfbI) from Streptococcus pyogenes plays a key role in bacterial adhesion to, and invasion of, eukaryotic cells. In addition, SfbI exhibits a considerable potential as mucosal adjuvant and can trigger polyclonal activation of B cells. Here, we report that SfbI is also capable of binding human IgG in a nonimmune fashion. SfbI was reactive with IgG1, IgG2, IgG3, and IgG4 isotypes (type IIo IgG-binding profile). The affinity constant (Kd) of the SfbI-IgG interaction was in the range of 1-2 x 10(-5) M. Further studies demonstrated that the SfbI binding was mediated by the Fc component of the IgG molecule. Experiments performed using purified recombinant proteins spanning different domains of SfbI showed that the IgG-binding activity was restricted to the fibronectin-binding domains, and in particular to the fibronectin-binding repeats. Finally, the presence of recombinant SfbI resulted in an impairment of both phagocytosis of IgG-coated RBCs and Ab-dependent cell cytotoxicity by macrophages. These results demonstrated for the first time that, in addition to its major role during the colonization process, SfbI may also favor bacterial immune evasion after the onset of the infection by interfering with host clearance mechanisms.     

A 49-residue peptide from adhesin F1 of Streptococcus pyogenes inhibits fibronectin matrix assembly

F1 is an adhesin of Streptococcus pyogenes which binds the N-terminal 70-kDa region of fibronectin with high affinity. The fibronectin binding region of F1 is comprised of a 43-residue upstream domain and a repeat domain comprised of five tandem 37-residue sequences. We investigated the effects of these domains on the assembly of fibronectin matrix by human dermal fibroblasts, MG63 osteosarcoma cells, or fibroblasts derived from fibronectin-null stem cells. Subequimolar or equimolar concentrations of recombinant proteins containing both the upstream and repeat domains or just the repeat domain enhanced binding of fibronectin or its N-terminal 70-kDa fragment to cell layers; higher concentrations of these recombinant proteins inhibited binding. The enhanced binding did not result in greater matrix assembly and was caused by increased ligand binding to substratum. In contrast, recombinant or synthetic protein containing the 43 residues of the upstream domain and the first 6 residues from the repeat domain exhibited monophasic inhibition with an IC(50) of approximately 10 nm. Truncation of the 49-residue sequence at its N or C terminus caused loss of inhibitory activity. The 49-residue upstream sequence blocked incorporation of both endogenous cellular fibronectin and exogenous plasma fibronectin into extracellular matrix and inhibited binding of 70-kDa fragment to fibronectin-null cells in a fibronectin-free system. Inhibition of matrix assembly by the 49-mer had no effect on cell adhesion to substratum, cell growth, formation of focal contacts, or formation of stress fibers. These results indicate that the 49-residue upstream sequence of F1 binds in an inhibitory mode to N-terminal parts of exogenous and endogenous fibronectin which are critical for fibronectin fibrillogenesis.     

Characterization of fibronectin binding to Salmonella enteritidis strain 27655R

Abstract The optimal conditions for the binding of fibronectin to Salmonella enteritidis strain 27655R, and the cell-surface components involved in the binding, were identified. Cultivation on colonisation factor antigen (CFA) agar or in CFA broth at 33°C for 24 h were found to be optimal for the expression of fibronectin binding. Such cultures exhibited 88% and 70% binding of ¹²⁵I-labelled fibronectin and its 29-kDa N-terminal domain, respectively. The fibronectin binding was reversed by the addition of unlabelled fibronectin or its 29-kDa fragment. Scatchard plot analysis of the binding showed that the strain possessed one high-affinity ( K _d= 5.8 × 10⁻¹⁰ M) and one low-affinity ( K _d= 2 × 10⁻⁸ M) binding site. The fibronectin-binding could be inhibited by cell surface components of S. enteritidis 27655R released by 30 min treatment at 65°C or 95°C. Inhibition could also be achieved using purified fimbriae. A non-fimbriated mutant of strain 27655R showed a much reduced binding of fibronectin (15%). Electron microscopic analysis showed association of the gold-labelled 29-kDa N-terminal fragment with S. enteritidis 27655R fimbriae. In conclusion, the findings suggest that S. enteritidis (strain 27655R) possesses fibronectin-binding fimbriae.     

Identification of the domains of the fibronectin-binding protein I of Streptococcus pyogenes responsible for adjuvanticity

Intranasal administration of antigens coupled to full-length fibronectin-binding protein I (SfbI) of Streptococcus pyogenes results in the elicitation of improved humoral and cellular immune responses, at both systemic and mucosal levels. We want to evaluate if SfbI also exhibits adjuvant properties when co-administered with the antigen, as well as identify the minimal domain responsible for its adjuvanticity. To achieve this aim, mice were immunized by the intranasal route with the model antigen beta-galactosidase (beta-gal) co-administered with recombinant proteins spanning different portions of the SfbI protein. The obtained results demonstrated that the adjuvant properties of SfbI were maintained intact when admixed to the model antigen. Similar kinetics and absolute titers of beta-gal-specific IgG antibodies as well as a dominant IgG(1) isotype response pattern were observed using SfbI derivatives spanning either the aromatic and proline-rich (H10) or the fibronectin-binding (H12) domains, respectively. The use of all tested derivatives also stimulated the elicitation of efficient beta-gal-specific IgA responses in lung lavages (23-25% of the total IgA). The obtained results suggest that different sub-domains of the SfbI protein can be used as adjuvants for the development of mucosal vaccines.     

Paxillin phosphorylation: bifurcation point downstream of integrin-linked kinase (ILK) in streptococcal invasion

Efficient group A streptococcus (GAS) invasion of mammalian cells requires fibronectin (Fn) binding proteins, such as M1 and PrtF1/SfbI, that bridge bacteria to integrins and activate cellular signalling for ingestion. Previous studies of GAS invasion, mediated by both proteins, suggest a common signalling pathway. However, distinct cellular morphological changes at the port of bacterial entry suggest that different signals are also induced. Here we report that paxillin is phosphorylated in response to Fn-bound GAS that express either M1 or PrtF1/SfbI protein, but is not phosphorylated in response to a mutant deficient in both proteins. Inhibition of paxillin phosphorylation by a tyrosine kinase inhibitor, PP2, or by expression of a dominant negative form of paxillin significantly reduced invasion by M1+ but did not affect ingestion of PrtF1/SfbI+ strains. In contrast, another tyrosine inhibitor, genistein, did not significantly prevent paxillin phosphorylation and had no effect on ingestion of the M1+ strain, but reduced PrtF1/SfbI-mediated entry. This suggests that paxillin phosphorylation is induced by both proteins but only required for M1-mediated invasion. A bifurcation point, downstream of integrin-linked kinase (ILK) and phosphoinositide 3-kinase, likely accounts for the distinct morphological changes. Furthermore, ILK activity is indispensable for M1-induced paxillin recruitment and phosphorylation.     

Two distinct genotypes of prtF2, encoding a fibronectin binding protein, and evolution of the gene family in Streptococcus pyogenes

The group A Streptococcus (GAS) is an important pathogen that is responsible for a wide range of human diseases. Fibronectin binding proteins (FBPs) play an important role in promoting GAS adherence and invasion of host cells. The prtF2 gene encodes an FBP and is present in approximately 60% of GAS strains. In the present study we examined 51 prtF2-positive GAS strains isolated from the Northern Territory of Australia, and here we describe two genotypes of prtF2 which are mutually exclusive. The two genotypes have been identified previously as pfbp and fbaB. We show that these genotypes map to the same chromosomal location within the highly recombinatorial fibronectin-collagen-T antigen (FCT) locus, indicating that they arose from a common ancestor, and in this study these genotypes were designated the pfbp type and the fbaB type. Phylogenetic analysis of seven pfbp types, 14 fbaB types, and 11 prtF2-negative GAS strains by pulsed-field gel electrophoresis (PFGE) produced 32 distinct PFGE patterns. Interpretation of evolution based on the PFGE dendrogram by parsimony suggested that the pfbp type had a recent origin compared to the fbaB type. A comparison of multiple DNA sequences of the pfbp and fbaB types revealed a mosaic pattern for the amino-terminal region of the pfbp types. The fbaB type is generally conserved at the amino terminus but varies in the number of fibronectin binding repeats in the carboxy terminus. Our data also suggest that there is a possible association of the pfbp genotype with sof (84.2%), while the fbaB genotype was found in a majority of the GAS strains negative for sof (90.6%), indicating that these two prtF2 subtypes may be under different selective pressures.     

Comparison of fibronectin receptors from rat hepatocytes and fibroblasts

A cell-surface fibronectin receptor was isolated from primary rat hepatocytes by affinity chromatography on Sepharose conjugated with the cell-binding domain (105 kDa) of fibronectin. The receptor remained bound to the affinity column in the presence of 1 M NaCl but was eluted by 1.5 mM of glycl-arginyl-glycyl-aspartyl-seryl-cysteine peptide or by lowering the pH to 4. The eluted material migrated under nonreducing conditions in sodium dodecyl sulfate-polyacrylamide electrophoresis as two bands: the alpha- and beta-components had apparent Mrs of 155,000 and 115,000, respectively. After reduction the 155-kDa component gave rise to two peptides of Mrs 145,000 and 20,000, while the 115-kDa component shifted migration to an Mr of 130,000. Antibodies specifically recognizing the 155- and 115-kDa proteins from hepatocytes inhibited the attachment of these cells to fibronectin-coated dishes, whereas attachment to dishes coated with collagen or laminin was unaffected. A fibronectin receptor isolated from rat fibroblasts showed closely similar, but not identical, migration in sodium dodecyl sulfate-electrophoresis as the hepatocyte receptor. Furthermore, only the beta-subunit of the fibroblast receptor reacted with the antibodies. The results suggest that distinct alpha-subunits of the fibronectin receptors may be the basis for the different fibronectin-binding properties of these cells.