Extended binding site on fibronectin for the functional upstream domain of protein F1 of Streptococcus pyogenes

The 49-residue functional upstream domain (FUD) of Streptococcus pyogenes F1 adhesin interacts with fibronectin (FN) in a heretofore unknown manner that prevents assembly of a FN matrix. Biotinylated FUD (b-FUD) bound to adsorbed FN or its recombinant N-terminal 70-kDa fibrin- and gelatin-binding fragment (70K). Binding was blocked by FN or 70K, but not by fibrin- or gelatin-binding subfragments of 70K. Isothermal titration calorimetry showed that FUD binds with K(d) values of 5.2 and 59 nM to soluble 70K and FN, respectively. We tested sets of FUD mutants and epitope-mapped monoclonal antibodies (mAbs) for ability to compete with b-FUD for binding to FN or to block FN assembly by cultured fibroblasts. Deletions or alanine substitutions throughout FUD caused loss of both activities. mAb 4D1 to the (2)FNI module had little effect, whereas mAb 7D5 to the (4)FNI module in the fibrin-binding region, 5C3 to the (9)FNI module in the gelatin-binding region, or L8 to the G-strand of (1)FNIII module adjacent to (9)FNI caused loss of binding of b-FUD to FN and decreased FN assembly. Conversely, FUD blocked binding of 7D5, 5C3, or L8, but not of 4D1, to FN. Circular dichroism indicated that FUD binds to 70K by β-strand addition, a possibility supported by modeling based on crystal structures of peptides bound to (2)FNI-(5)FNI of the fibrin-binding domain and (8)FNI-(9)FNI of the gelatin-binding domain. Thus, the interaction likely involves an extensive anti-parallel β-zipper in which FUD interacts with the E-strands of (2)FNI-(5)FNI and (8)FNI-(9)FNI.     

Roles of integrins and fibronectin in the entry of Streptococcus pyogenes into cells via protein F1

Entry of group A streptococcus (GAS) into cells has been suggested as an important trait in GAS pathogenicity. Protein F1, a fibronectin (Fn) binding protein, mediates GAS adherence to cells and the extracellular matrix, and efficient cell internalization. We demonstrate that the cellular receptors responsible for protein F1-mediated internalization of GAS are integrins capable of Fn binding. In HeLa cells, bacterial entry is blocked by anti-β1 integrin monoclonal antibody. In the mouse cell line GD25, a β1 null mutant, the αvβ3 integrin promotes GAS entry. Internalization of these cells by GAS is blocked by a peptide that specifically binds to αvβ3 integrin. In both cell lines, entry of GAS requires the occupancy of protein F1 by Fn. Neither the 29 kDa nor the 70 kDa N-terminal fragments or the 120 kDa cell-binding fragment of Fn promote bacterial entry. Fn-coated beads are taken up efficiently by HeLa cells. Both the entry of GAS via protein F1 and the uptake of Fn-coated beads are blocked by anti-β1 antibody but are unaffected by a large excess of soluble Fn. Internalization of HeLa cells by bacteria bearing increasing amounts of prebound Fn to protein F1 reveals a sigmoidal ultrasensitive curve. These suggest that the ability of particles to interact via Fn with multiple integrin sites plays a central role in their ability to enter cells.     

Specific interactions between F1 adhesin of Streptococcus pyogenes and N-terminal modules of fibronectin

Protein F1 is a surface protein of Streptococcus pyogenes that mediates high affinity binding to fibronectin (Fn) and facilitates S. pyogenes adherence and penetration into cells. The smallest portion of F1 known to retain the full binding potential of the intact protein is a stretch of 49 amino acids known as the functional upstream domain (FUD). Synthetic and recombinant versions of FUD were labeled with fluorescein isothiocyanate and used in fluorescence anisotropy experiments. These probes bound to Fn or the 70-kDa fragment of Fn with dissociation constants of 8-30 nm. Removal of the N-terminal seven residues of FUD did not cause a change in binding affinity. Further N- or C-terminal truncations resulted in complete loss of binding activity. Analysis of recombinant versions of the 70-kDa fragment that lacked one or several type I modules indicates that residues 1-7 of the 49-mer bind to type I modules I1 and I2 of the 27-kDa subfragment and the C-terminal residues bind to modules I4 and I5. Fluorescein isothiocyanate-labeled 49-mer also bound with lower affinity to large Fn fragments that lack the five type I modules of the 27-kDa fragment but contain the other seven type 1 modules of Fn. These results indicate that, although FUD has a general affinity for type I modules, high affinity binding of FUD to Fn is mediated by specific interactions with N-terminal type I modules.     

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.     

Interactions with fibronectin attenuate the virulence of Streptococcus pyogenes

Fibronectin-binding surface proteins are found in many bacterial species. Most strains of Streptococcus pyogenes, a major human pathogen, express the fibronectin-binding protein F1, which promotes bacterial adherence to and entry into human cells. In this study, the role of fibronectin in S. pyogenes virulence was investigated by introducing the protein F1 gene in an S. pyogenes strain lacking this gene. Furthermore, transgenic mice lacking plasma fibronectin were used to examine the relative contribution of plasma and cellular fibronectin to S. pyogenes virulence. Unexpectedly, protein F1-expressing bacteria were less virulent to normal mice, and virulence was partly restored when these bacteria were used to infect mice lacking plasma fibronectin. Dissemination to the spleen of infected mice was less efficient for fibronectin-binding bacteria. These bacteria also disseminated more efficiently in mice lacking plasma fibronectin, demonstrating that plasma fibronectin bound to the bacterial surface downregulates S. pyogenes virulence by limiting bacterial spread. From an evolutionary point of view, these results suggest that reducing virulence by binding fibronectin adds selective advantages to the bacterium.     

Protein F: an adhesin of Streptococcus pyogenes binds fibronectin via two distinct domains

The binding of Streptococcus pyogenes to fibronectin (FN) enables the adherence of this pathogen to target epithelial cells, which is the first necessary step for initiation of infection. Binding is mediated by a bacterial surface protein termed protein F. Here we provide the complete structure of protein F and identify two domains responsible for binding to fibronectin. The first domain is located towards the C-terminal end of the molecule and is composed of five repeats of 37 amino acids that are completely repeated four times and a fifth time partially. The second domain is adjacent to the first domain and is located on the /V-terminal side of it. It is composed of a single stretch of 43 amino acids. Protein F expressed in Escherichia coli completely blocked the binding of fibronectin to S. pyogenes. However, mutant proteins that contained only one or the other of the two domains were only capable of partial blockage of binding. Complete blockage of binding of fibronectin could be achieved when a protein extract containing the N-terminal domain was mixed in a binding reaction with a protein extract containing the C-terminal domain. Similarly, a purified recombinant protein containing the two domains only, blocked the binding completely. In contrast, a purified recombinant protein containing just the C-terminal domain, blocked the binding partially. A clone exclusively expressing the C-terminal domain, completely blocked the binding of the 30 kDa N-terminal fragment of fibronectin to S. pyogenes, whereas a clone expressing the N-terminal domain failed to block the binding of this FN fragment. Thus, the two FN-binding domains of protein F are necessary for maximal bacterial binding and act in concert to enhance the binding to fibronectin. The possibility that the two domains bind to two different regions on the fibronectin molecule is discussed.     

Synthesis of a Streptococcus pyogenes vaccine candidate based on the M protein PL1 epitope

Group A streptococcus is a Gram-positive bacteria that causes a range of infectious diseases. Targeting the bacteria, a new self-adjuvanting vaccine candidate, incorporating a carbohydrate carrier and an amino acid-based adjuvant, was synthesised utilising carbohydrate chemistry and solid-phase peptide synthesis procedures. Characterisation of the candidate was achieved using reverse-phase HPLC and electrospray ionisation mass spectrometry. The successful synthesis and characterisation of the vaccine candidate may contribute to the discovery of a therapeutically and clinically viable vaccine against group A streptococcus.     

Soluble M1 protein of Streptococcus pyogenes triggers potent T cell activation

Streptococcus pyogenes of the M1 serotype is commonly associated with large outbreaks of invasive streptococcal infections and development of streptococcal toxic shock syndrome (STSS). The pathogenesis behind these infections is believed to involve bacterial superantigens that induce potent inflammatory responses, but the reason why strains of the M1 serotype are over-represented in STSS is still not understood. In the present investigation, we show that a highly purified soluble form of the M1 protein from S. pyogenes , which lacks the membrane-spanning region, is a potent inducer of T cell proliferation and release of Th1 type cytokines. M1 protein-evoked T cell proliferation was HLA class II-dependent but not MHC-restricted, did not require intracellular processing and was Vβ-restricted. Extensive mass spectrometry studies indicated that there were no other detectable proteins in the preparation. Taken together, our data demonstrate that soluble M1 protein is a novel streptococcal superantigen, which likely contributes to the excessive T cell activation and hyperinflammatory response seen in severe invasive streptococcal infections.     

Binding of the low-density lipoprotein by streptococcal collagen-like protein Scl1 of Streptococcus pyogenes

Several bacterial genera express proteins that contain collagen-like regions, which are associated with variable (V) non-collagenous regions. The streptococcal collagen-like proteins, Scl1 and Scl2, of group A Streptococcus (GAS) are members of this 'prokaryotic collagen' family, and they too contain an amino-terminal non-collagenous V region of unknown function. Here, we use recombinant rScl constructs, derived from several Scl1 and Scl2 variants, and affinity chromatography to identify Scl ligands present in human plasma. First, we show that Scl1, but not Scl2, proteins from different GAS serotypes bind the same ligand identified as apolipoprotein B (ApoB100), which is a major component of the low-density lipoprotein (LDL). Scl1 binding to purified ApoB100 and LDL is specific and concentration-dependent. Furthermore, the non-collagenous V region of the Scl1 protein is responsible for LDL/ApoB100 binding because only those rScls, constructed by domain swapping, which contain the V region from Scl1 proteins, were able to bind to ApoB100 and LDL ligands, and this binding was inhibited by antibodies directed against the Scl1-V region. Electron microscopy images of Scl1-LDL complexes showed that the globular V domain of Scl1 interacted with spherical particles of LDL. Importantly, live M28-type GAS cells absorbed plasma LDL on the cell surface and this binding depended on the surface expression of the Scl1.28, but not Scl2.28, protein. Phylogenetic analysis showed that the non-collagenous globular domains of Scl1 and Scl2 evolved independently to form separate lineages, which differ in amino acid sequence, and these differences may account for the variations in binding patterns of Scl1 and Scl2 proteins. Present studies provide insight into the structure-function relationship of the Scl proteins and also underline the importance of lipoprotein binding by GAS.     

Type 1 M protein of Streptococcus pyogenes. N-terminal sequence and peptic fragments

Limited proteolysis of the surface of type 1 Streptococcus pyogenes by pepsin gives rise to fragment Pep M1 of Mr 20270 as the main product which covers the N-terminal part of the M protein. The amino acid sequence was determined of the N-terminal region of the M protein representing the most exposed part of the molecule on the surface fibrils of streptococcal cells, which seems to be very important for the differentiation of the individual serological types. The sequence differs from the homologous N-terminal sequences of types 5, 6 and 24, and shows a homology with sequences repeating in the chain of type 24. Fragment Pep M1 binds to fibrinogen; the absence of its 30 N-terminal amino acid residues, however, abolishes this interaction which is believed to play a role in the virulence of S. pyogenes.     

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.     

Kinetic and functional analysis of the heparin-binding domain of fibronectin

We have previously shown that the heparin-binding domain of fibronectin (FN-HBD) enhances cell adhesion and proliferation of osteoblasts. Here we demonstrated that FN-HBD binds to heparin with a K_D of 5 μM. Although, FN-HBD itself produces a modest effect on cell adhesion in the absence of central cell-binding domain (CCBD), FN-HBD significantly enhances cell adhesion and spreading activities by a cooperative mechanism of CCBD in MG63 cells (P < 0.05).     

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.     

Intrinsic tryptophan fluorescence measurements suggest that polylactosaminyl glycosylation affects the protein conformation of the gelatin-binding domain from human placental fibronectin

Glycosylation can affect the physical and biochemical properties of the polypeptide chain in glycoproteins. Asparagine-N-linked polylactosaminyl glycosylation of the chymotryptic 44-kDa gelatin-binding domain from human placental fibronectin confers protease resistance [Zhu, B. C. R., Fisher, S. F., Panda, H., Calaycay, J., Shively, J. E. & Laine, R. A. (1984) J. Biol. Chem. 259, 3962-3970] and weaken the binding to gelatin [Zhu, B. C. R. & Laine, R. A. (1985) J. Biol. Chem. 260, 4041-4045]. Intrinsic tryptophan fluorescence of the gelatin-binding domain was used to probe glycosylation-dependent protein conformation changes. In gelatin-binding fragments containing incrementally smaller polylactosamine oligosaccharides, the fluorescence intensity progressively decreased and the emission spectrum shifted about 7 nm to the blue. Removal of the polylactosamine chains from a highly glycosylated fragment with endo-beta-galactosidase from Escherichia freundii also quenched the protein fluorescence. The fluorescence lifetimes did not appear to be affected by the extent of glycosylation, suggesting static quenching of the tryptophan emission in the low glycosylated fragments. Acrylamide quenching studies showed that the accessibility of the tryptophans to small solutes was not altered by glycosylation. The steady-state emission anisotropy increased with decreasing polylactosamine chain length. The results indicate that the polylactosamine chains alter the tryptophan environments in the gelatin-binding domain, probably by changing the polypeptide conformation. These putative protein conformation changes may be partially responsible for the altered gelatin binding, protease resistance, and cell adhesion functions of fetal tissue fibronectin.