Streptococcal Fibronectin-binding Proteins Research Articles

Group B Streptococcal Colonization, Molecular Characteristics, and Epidemiology.

Streptococcus agalactiae or group B streptococcus (GBS) is a leading cause of serious neonatal infections. GBS is an opportunistic commensal constituting a part of the intestinal and vaginal physiologic flora and maternal colonization is the principal route of GBS transmission. GBS is a pathobiont that converts from the asymptomatic mucosal carriage state to a major bacterial pathogen causing severe invasive infections. At present, as many as 10 serotypes (Ia, Ib, and II–IX) are recognized. The aim of the current review is to shed new light on the latest epidemiological data and clonal distribution of GBS in addition to discussing the most important colonization determinants at a molecular level. The distribution and predominance of certain serotypes is susceptible to variations and can change over time. With the availability of multilocus sequence typing scheme (MLST) data, it became clear that GBS strains of certain clonal complexes possess a higher potential to cause invasive disease, while other harbor mainly colonizing strains. Colonization and persistence in different host niches is dependent on the adherence capacity of GBS to host cells and tissues. Bacterial biofilms represent well-known virulence factors with a vital role in persistence and chronic infections. In addition, GBS colonization, persistence, translocation, and invasion of host barriers are largely dependent on their adherence abilities to host cells and extracellular matrix proteins (ECM). Major adhesins mediating GBS interaction with host cells include the fibrinogen-binding proteins (Fbs), the laminin-binding protein (Lmb), the group B streptococcal C5a peptidase (ScpB), the streptococcal fibronectin binding protein A (SfbA), the GBS immunogenic bacterial adhesin (BibA), and the hypervirulent adhesin (HvgA). These adhesins facilitate persistent and intimate contacts between the bacterial cell and the host, while global virulence regulators play a major role in the transition to invasive infections. This review combines for first time epidemiological data with data on adherence and colonization for GBS. Investigating the epidemiology along with understanding the determinants of mucosal colonization and the development of invasive disease at a molecular level is therefore important for the development of strategies to prevent invasive GBS disease worldwide.

Immunization with a streptococcal multiple-epitope recombinant protein protects mice against invasive group A streptococcal infection.

Streptococcus pyogenes (group A Streptococcus; GAS) causes clinical diseases, including pharyngitis, scarlet fever, impetigo, necrotizing fasciitis and streptococcal toxic shock syndrome. A number of group A streptococcus vaccine candidates have been developed, but only one 26-valent recombinant M protein vaccine has entered clinical trials. Differing from the design of a 26-valent recombinant M protein vaccine, we provide here a vaccination using the polyvalence epitope recombinant FSBM protein (rFSBM), which contains four different epitopes, including the fibronectin-binding repeats domain of streptococcal fibronectin binding protein Sfb1, the C-terminal immunogenic segment of streptolysin S, the C3-binding motif of streptococcal pyrogenic exotoxin B, and the C-terminal conserved segment of M protein. Vaccination with the rFSBM protein successfully prevented mortality and skin lesions caused by several emm strains of GAS infection. Anti-FSBM antibodies collected from the rFSBM-immunized mice were able to opsonize at least six emm strains and can neutralize the hemolytic activity of streptolysin S. Furthermore, the internalization of GAS into nonphagocytic cells is also reduced by anti-FSBM serum. These findings suggest that rFSBM can be applied as a vaccine candidate to prevent different emm strains of GAS infection.

Accelerating the development of a group A Streptococcus vaccine: an urgent public health need.

Group A Streptococcus (GAS) infections cause substantial worldwide morbidity and mortality, mostly associated with suppurative complications such as pharyngitis, impetigo, and non-suppurative immune syndromes such as acute rheumatic fever, rheumatic heart disease, and acute post-streptococcal glomerulonephritis. Deaths occur mostly in children, adolescents, and young adults in particular pregnant women in low- and middle-income countries. GAS strains are highly variable, and a GAS vaccine would need to overcome the issue of multiple strains. Several approaches have been used multivalent vaccines using N-terminal polypeptides of different M protein; conserved M protein vaccines with antigens from the conserved C-repeat portion of the M protein; incorporation selected T- and B-cell epitopes from the C-repeat region in a synthetic polypeptide or shorter single minimal B-cell epitopes from this same region; and non-M protein approaches utilizing highly conserved motives of streptococcal C5a peptidase, GAS carbohydrate and streptococcal fibronectin-binding proteins. A GAS vaccine represents urgent need for this neglected disease and should therefore deserve the greatest attention of international organizations, donors, and vaccine manufacturers.

Identification of a Group B Streptococcal Fibronectin Binding Protein, SfbA, That Contributes to Invasion of Brain Endothelium and Development of Meningitis

Group B Streptococcus (GBS) is currently the leading cause of neonatal meningitis. This is due to its ability to survive and multiply in the bloodstream and interact with specialized human brain microvascular endothelial cells (hBMEC), which constitute the blood-brain barrier (BBB). The exact mechanism(s) of GBS-BBB penetration is still largely unknown. We and others have shown that GBS interacts with components of the extracellular matrix. In this study, we demonstrate that GBS of representative serotypes binds immobilized and cell surface fibronectin and identify a putative fibronectin binding protein, streptococcal fibronectin binding protein A (SfbA). Allelic replacement of sfbA in the GBS chromosome resulted in a significant decrease in ability to bind fibronection and invade hBMEC compared with the wild-type (WT) parental strain. Expression of SfbA in the noninvasive strain Lactococcus lactis was sufficient to promote fibronectin binding and hBMEC invasion. Furthermore, the addition of an antifibronectin antibody or an RGD peptide that blocks fibronectin binding to integrins significantly reduced invasion of the WT but not the sfbA-deficient mutant strain, demonstrating the importance of an SfbA-fibronectin-integrin interaction for GBS cellular invasion. Using a murine model of GBS meningitis, we also observed that WT GBS penetrated the brain and established meningitis more frequently than did the ΔsfbA mutant strain. Our data suggest that GBS SfbA plays an important role in bacterial interaction with BBB endothelium and the pathogenesis of streptococcal meningitis.

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

In the article by Rohde et al. (2011), Figure 6B contained within Figure 6 is incorrect. The correct version of the figure is given below. The authors apologise for this error.

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.

Inhibition of Streptococcus pyogenes adherence to HaCaT cells by a peptide corresponding to the streptococcal fibronectin-binding protein, SfbI, is strain dependent

Streptococcus pyogenes (group A streptococcus, GAS) is a human-specific pathogen, which employs a large number of adhesins for colonization. Fibronectin-binding proteins (FBPs) play a major role in GAS adhesion to host cells. SfbI, a major streptococcal FBP, has been well studied. A peptide (peptide-MSG) based on this adhesin inhibits fibronectin (Fn)-binding by the pathogen. To test whether this peptide also inhibits adherence of GAS to host cells, adhesion assays were performed with strains possessing different combinations of genes for three distinct FBPs. Peptide-MSG inhibited GAS adherence to human keratinocytes (HaCaT) in a strain dependent manner. There is no consistent pattern between the effect and the ability to express one or more of the FBPs. A single peptide may be insufficient to prevent GAS adherence to host cells.

Molecular genetic analysis of a group A Streptococcus operon encoding serum opacity factor and a novel fibronectin-binding protein, SfbX.

The group A Streptococcus (GAS) sof gene encodes the serum opacity factor protein, which is capable of opacifying mammalian sera and binding at least two host proteins, fibronectin and fibrinogen. The sof gene exists in approximately 50% of clinical isolates, and there is a classical association of so-called nephritogenic strains with the opacity factor-positive phenotype. In both a type emm49 strain and a type emm12 strain, the sequences upstream of the 5' end of sof and downstream of the putative terminator were determined to be nearly identical to a region in the M type 1 genome approximately 10 kb upstream of the emm1 gene. This close genetic linkage is likely reflected in the strict correlation of opacity factor phenotype with specific emm genotypes. A new fibronectin-binding protein gene, sfbX, was discovered immediately downstream of sof in emm12 and emm49 strains and in several other sof-positive strains. The sof and sfbX genes were found to be expressed on the same transcription unit, which was correlated with the putative promoter and rho-independant terminator sequences that flank these two genes. The sfbX genes from different emm types are predicted to encode approximately 650-residue surface-bound proteins sharing 89 to 92% sequence identity. SfbX residues approximately 1 to 480 are not highly similar to those of other known proteins, with the closest match being the Staphylococcus aureus coagulase protein. The remaining portions of these proteins (residues 481 to 650) contain four putative fibronectin-binding repeats highly similar to those of other streptococcal fibronectin-binding proteins and a potential LP(X)SG cell wall anchor motif. Targeted in-frame allelic-exchange mutagenesis, complementation, and heterologous-expression studies found that serum opacification is encoded by sof alone and that sfbX encodes a fibronectin-binding function. A recombinant SfbX protein was found to bind immobilized fibronectin and to partially inhibit GAS adherence to fibronectin. The sfbX gene was found to be present only in sof-positive strains, and together these genes could influence the spectrum of tissues colonized by sof-positive GAS.

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.

Streptococcal erythrogenic toxin B abrogates fibronectin-dependent internalization of Streptococcus pyogenes by cultured mammalian cells.

Streptococcus pyogenes secretes several proteins that influence host-pathogen interactions. A tissue-culture model was used to study the influence of the secreted cysteine protease streptococcal erythrogenic toxin B (SPE B) on the interaction between S. pyogenes strain NZ131 (serotype M49) and mammalian cells. Inactivation of the speB gene enhanced fibronectin-dependent uptake of the pathogen by Chinese hamster ovary (CHO-K1) cells compared to that in the isogenic wild-type strain. Preincubation of the NZ131 speB mutant with purified SPE B protease significantly inhibited fibronectin-dependent uptake by both CHO-K1 and CHO-pgs745 cells. The effect was attributed to an abrogation of fibronectin binding to the surface of the bacteria that did not involve either the M49 protein or the streptococcal fibronectin-binding protein SfbI. In contrast, pretreatment of the NZ131 speB mutant with SPE B did not influence sulfated polysaccharide-mediated uptake by CHO-pgs745 cells. The results indicate that the SPE B protease specifically alters bacterial cell surface proteins and thereby influences pathogen uptake.

Detection of bacterial virulence genes associated with infective endocarditis in infected root canals.

The aim of this study was to examine whether bacteria associated with root canals possess genes that might predispose to bacterial colonization of the endocardium. Oligonucleotides were designed from DNA sequences encoding the functional binding regions of streptococcal fibronectin-binding protein (FnBP) and staphylococcal fibrinogen-binding protein (FgBP). The specificity and cross-reactivity of the oligonucleotide primers were investigated; streptococcal primers were tested for recognition of FnBP genes in other strains of streptococci, and the staphylococcal primers for detection of FgBP from other staphylococci. Interspecies specificity of these primers was also investigated. In a pilot clinical study, the pulp chambers of 16 nonvital teeth without sinus tracts, were opened aseptically. Root canal samples were collected, along with samples from the gingival sulcus and anterior nares. From these samples DNA was extracted, subjected to polymerase chain reaction (PCR) and analysed by agarose gel electrophoresis. Using the streptococcal FnBP primers, PCR bands were amplified from eight root canal samples, eight gingival samples and three nasal samples. With the staphylococcal primers, PCR bands were amplified from seven root canals, 11 gingival and nine nasal samples. This study showed that PCR could be used to detect bacteria in root canals that possess genes with homology to functional regions of those encoding FnBP or FgBP. If bacteria in root canals possess FnBP or FgBP, they may have the potential to cause infective endocarditis.

Distribution and Antigenicity of Fibronectin Binding Proteins (SfbI and SfbII) of Streptococcus pyogenes Clinical Isolates from the Northern Territory, Australia

Fibronectin binding proteins play an important role in the adherence and invasion of group A streptococci (GAS). Genotypically distinct GAS isolates were screened for the presence and expression of two streptococcal fibronectin binding protein genes, sfbI and sfbII. Of the tested strains, 64 and 36% were shown to harbor and express the sfbI and sfbII genes, respectively. All sfbII-positive strains tested were also positive for sfbI, but only 28% of the sfbII-negative strains were positive for sfbI. High levels of immunoglobulin G antibodies to both SfbI and SfbII were found in sera from 80 subjects with defined streptococcal infections.

Differential effects of the streptococcal fibronectin-binding protein, FBP54, on adhesion of group A streptococci to human buccal cells and HEp-2 tissue culture cells.

We have previously demonstrated that fibronectin mediates streptococcal adhesion to host cells and that streptococci interact primarily with the N-terminal domain of fibronectin. FBP54 is a 54-kDa protein from group A streptococci that binds fibronectin. In this report, we show that the N-terminal domain of fibronectin reacts with FBP54 and preferentially blocks streptococcal adhesion to buccal epithelial cells. FBP54 blocked adhesion to human buccal epithelial cells by 80% in a dose-related fashion. In contrast, FBP54 had little effect on adhesion of group A streptococci to HEp-2 tissue culture cells. The fibronectin-binding domain of FBP54 has been localized to the first 89 N-terminal residues of the protein. Experiments using affinity-purified antibodies to this region indicated that the N terminus of FBP54 is exposed on the surface of streptococci in a manner that can interact with immobilized receptors. Analysis of sera from patients with post-streptococcal glomerulonephritis and acute rheumatic fever indicated that FBP54 is expressed in vivo and is immunogenic in the human host. These data indicate that FBP54 is a streptococcal adhesin that is expressed in the human host and that preferentially mediates adhesion to certain types of human cells.

Identification of a fibronectin-binding protein (GfbA) in pathogenic group G streptococci

Attachment to eukaryotic cell surfaces is an essential step in the establishment of colonization and infection by bacterial pathogens. This report examines the adherence capabilities of pathogenic group G streptococci and demonstrates that certain group G streptococcal clinical isolates express a fibronectin-binding protein. This protein, termed GfbA for group G streptococcal fibronectin-binding protein, mediates adherence to human skin fibroblasts (HSF). The gene encoding this protein, gfbA, was isolated, and the complete DNA sequence of gfbA was determined. From this sequence GfbA was predicted to be a 580-amino-acid protein (molecular weight = 64,979) with significant amino acid identity to the group A streptococcal fibronectin-binding proteins SfbI and protein F (PrtF) (76 and 78% identity, respectively). GfbA contains regions with notable identity to the fibronectin-binding repeat domains of PrtF. gfbA(+) strains were able to bind to HSF, and preincubation of the gfbA(+) strains with fibronectin blocked this adherence. In addition, gfbA(+) strains were able to bind radiolabeled fibronectin, and this binding was inhibited with addition of excess unlabeled fibronectin. gfbA-negative strains were not able to bind either the HSF or radiolabeled fibronectin. DNA homologous to gfbA was found in 36% of the group G streptococcal isolates examined. Since not all group G streptococcal strains examined contained gfbA, this suggests there might be other tissue-specific adherence molecules expressed by these pathogenic strains.

Characterization of a novel fibronectin-binding surface protein in group A streptococci.

Streptococcus pyogenes interacts with host fibronectin via distinct surface components. One of these components is the Sfbl protein (streptococcal fibronectin-binding protein, now specified as class I), an adhesin that represents a protein family with characteristic features. Here we present the complete structure of a novel fibronectin-binding protein of S. pyogenes, designated Sfbll, which is distinct from the previously described Sfbl proteins. The sfbll gene originated from a lambda EMBL3 library of chromosomal DNA from group A streptococcal strain A75 and coded for a 113 kDa protein exhibiting features of membrane-anchored surface proteins of Gram-positive cocci. The expression of biologically active fusion proteins allowed the determination of the location of the fibronectin-binding domain within the C-terminal part of the protein. It consisted of two and a half repeats which share common motifs with fibronectin-binding repeats of other streptococcal and staphylococcal proteins. Purified recombinant fusion protein containing this domain competitively inhibited the binding of fibronectin to the parental S. pyogenes strain. Furthermore, polyclonal antibodies against the binding domain specifically blocked the Sfbll receptor site on the streptococcal surface. No cross-reactivity could be detected between anti-Sfbll antibodies and the sfbl gene product, and vice versa, indicating that the two proteins do not share common immunogenic epitopes. Southern hybridization experiments performed with specific sfbll gene probes revealed the presence of the sfbll gene in more than 55% of 93 streptococcal isolates tested. The majority of the strains also harboured the sfbl gene, and 86% carried at least one of the two sfb genes.

Identification of a Fibronectin-Binding Protein as Adhesin of Streptococcus pyogenes

The role of streptococcal fibronectin-binding protein (Sfb protein) in the adherence of Streptococcus pyogenes to epithelial cells was analyzed by using a recombinant Sfb fusion protein which was constructed by fusion of the binding domain of Sfb protein to MS2 polymerase. Sfb fusion protein was overexpressed in Escherichia coli, purified from E. coli lysates by FPLC and analysed in binding experiments with 125I-labelled fibronectin and adherence studies with HEp2 epithelial cells. Lysates from E. coli expressing the fusion protein as well as purified Sfb protein competitively inhibited fibronectin binding and epithelial cell adherence of S. pyogenes, whereas streptococcal lipoteichoic acid (LTA), previously suggested to be the adhesin of S. pyogenes, had no effects. Southern blot analysis of chromosomal DNA from various streptococci revealed that the gene coding for the binding domain of Sfb protein was also present in other S. pyogenes strains. These results indicate that fibronectin-binding protein is the major adhesin of S. pyogenes.