Structure of the Streptococcus pneumoniae Surface Protein and Adhesin PfbA

PfbA (plasmin- and fibronectin-binding protein A) is an extracellular Streptococcus pneumoniae cell-wall attached surface protein that binds to fibronectin, plasmin, and plasminogen. Here we present a structural analysis of the surface exposed domains of PfbA using a combined approach of X-ray crystallography and small-angle X-ray scattering (SAXS). The crystal structure of the PfbA core domain, here called PfbAβ, determined to 2.28 Å resolution revealed an elongated 12-stranded parallel β-helix fold, which structure-based comparisons reveal is most similar to proteins with carbohydrate modifying activity. A notable feature of the PfbAβ is an extensive cleft on one face of the protein with electrochemical and spatial features that are analogous to structurally similar carbohydrate-active enzymes utilizing this feature for substrate accommodation. Though this cleft displays a combination of basic amino acid residues and solvent exposed aromatic amino acids that are distinct features for recognition of carbohydrates, no obvious arrangement of amino acid side chains that would constitute catalytic machinery is evident. The pseudo-atomic SAXS model of a larger fragment of PfbA suggests that it has a relatively well-ordered structure with the N-terminal and core domains of PfbA adopting an extend organization and reveals a novel structural class of surface exposed pneumococcal matrix molecule adhesins.     

Sequence analysis and characterization of a novel fibronectin-binding repeat domain from the surface of Streptococcus pneumoniae

Streptococcus pneumoniae open reading frame SP0082 encodes a surface protein that contains four copies of a novel conserved repeat domain that bears no significant sequence similarity to proteins of known function. Homologous sequences from other streptococci contain two to six of these repeats, designated the SSURE (streptococcal surface repeat) domain. To investigate the functional role(s) of this domain, the third SSURE repeat of SP0082 sequence has been expressed in Escherichia coli, purified to homogeneity and characterized by biochemical and immunological methods. The expressed protein fragment was found to bind to fibronectin, but not to collagen or submaxillary mucin. Anti-SSURE antibodies recognized the corresponding protein on the surface of pneumococcal cells. These data identify S. pneumoniae SP0082 protein and its homologs in other streptococci as fibronectin-binding surface adhesins. The SSURE domain is likely to contain a novel protein fold, which was tentatively modeled using ab initio modeling methods.     

The pavA gene of Streptococcus pneumoniae encodes a fibronectin-binding protein that is essential for virulence

Streptococcus pneumoniae colonizes the nasopharynx in up to 40% of healthy subjects, and is a leading cause of middle ear infections (otitis media), meningitis and pneumonia. Pneumococci adhere to glycosidic receptors on epithelial cells and to immobilized fibronectin, but the bacterial adhesins mediating these reactions are largely uncharacterized. In this report we describe a novel pneumococcal protein PavA, which binds fibronectin and is associated with pneumococcal adhesion and virulence. The pavA gene, present in 64 independent isolates of S. pneumoniae tested, encodes a 551 amino acid residue polypeptide with 67% identical amino acid sequence to Fbp54 protein in Streptococcus pyogenes. PavA localized to the pneumococcal cell outer surface, as demonstrated by immunoelectron microscopy, despite lack of conventional secretory or cell-surface anchorage signals within the primary sequence. Full-length recombinant PavA polypeptide bound to immobilized human fibronectin in preference to fluid-phase fibronectin, in a heparin-sensitive interaction, and blocked binding of wild-type pneumococcal cells to fibronectin. However, a C-terminally truncated PavA' polypeptide (362 aa residues) failed to bind fibronectin or block pneumococcal cell adhesion. Expression of pavA in Enterococcus faecalis JH2-2 conferred > sixfold increased cell adhesion levels to fibronectin over control JH2-2 cells. Isogenic mutants of S. pneumoniae, either abrogated in PavA expression or producing a 42 kDa C-terminally truncated protein, showed up to 50% reduced binding to immobilized fibronectin. Inactivation of pavA had no effects on growth rate, cell morphology, cell-surface physico-chemical properties, production of pneumolysin, autolysin, or surface proteins PspA and PsaA. Isogenic pavA mutants of encapsulated S. pneumoniae D39 were approximately 104-fold attenuated in virulence in the mouse sepsis model. These results provide evidence that PavA fibronectin-binding protein plays a direct role in the pathogenesis of pneumococcal infections.     

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.     

Streptococcus mutans autolysin AtlA is a fibronectin-binding protein and contributes to bacterial survival in the bloodstream and virulence for infective endocarditis

Streptococcus mutans , a commensal of the human oral cavity, can survive in the bloodstream and cause infective endocarditis (IE). However, the virulence factors associated with this manifestation of disease are not known. Here, we demonstrate that AtlA, an autolysin of S. mutans is a newly identified fibronectin (Fn) binding protein and contributes to bacterial resistance to phagocytosis and survival in the bloodstream. Interestingly, prior exposure to plasma at low concentrations was sufficient to enhance bacterial survival in the circulation. Calcium ions at physiological plasma concentrations induced maturation of AtlA from the 104–90 kDa isoform resulting in increased Fn binding and resistance to phagocytosis. An isogenic mutant strain defective in AtlA expression exhibited reduced survival and virulence when tested in a rat model of IE compared with the wild-type and complemented strains. The data presented suggest that plasma components utilized by S. mutans enhanced survival in the circulation and AtlA is a virulence factor associated with infective endocarditis.     

Identification and characterization of a novel fibronectin-binding protein gene from Streptococcus equi subspecies zooepidemicus strain VTU211

This work describes the cloning and sequencing of genes encoding fibronectin-binding proteins from Streptococcus equi subspecies zooepidemicus strain VTU211. A gene encoding a cell-wall protein FNZ was amplified and sequenced. In the same bacterial strain, a second gene termed fnz2 was now discovered, encoding another fibronectin-binding protein (FNZ2). The complete amino acid sequence encoded by fnz2 was deduced and compared to that deduced from fnz. The sequence comparison of the fnz and fnz2 predicted that fibronectin-binding activity is localizing a domain in the C terminal part of FNZ2, since this domain is composed of three repeats, which contain a motif similar to what has earlier been found in other fibronectin-binding proteins in streptococci. Three parts of fnz2 [fnz2(1-8), fnz2(2-4), and fnz2(4-3)] were amplified using polymerase chain reaction and ligated into an expression vector, and recombinant FNZ2 proteins were produced in Escherichia coli. Fibronectin bound to the FNZ2(1-8) [amino acids 212-396] and FNZ2(2-4) (amino acids 36-448) but not to the FNZ2(4-3) (amino acids 36-191) in a Western ligand blot, showing that repeat domain of FNZ2 protein was sufficient for binding of fibronectin. Purified FNZ2(2-4) protein was also shown to display collagen-binding activity to collagen-coated microtiter wells. These results show that recombinant FNZ2 has fibronectin- and collagen-binding activities.     

Hic, a novel surface protein of Streptococcus pneumoniae that interferes with complement function

The important human pathogen Streptococcus pneumoniae was found to absorb factor H, an inhibitor of complement, from human plasma. We identified the gene encoding a novel surface protein, factor H-binding inhibitor of complement (Hic), in the pspC locus of type 3 pneumococci. Unlike PspC proteins in other serotypes, Hic is anchored to the cell wall by means of an LPXTG motif, and the overall sequence homology to various PspC proteins is low. However, the NH(2)-terminal region showed significant homology to the NH(2)-terminal region of several PspC proteins. A fragment of Hic, covering this homologous region, was expressed as a glutathione S-transferase (GST) fusion protein. GST:Hic(39-261) bound radiolabeled factor H and inhibited binding of factor H to pneumococci of different serotypes. Interaction kinetics between GST:Hic(39-261) and factor H were studied with surface plasmon resonance and showed a high affinity binding (K(A) = 5 x 10(7), K(D) = 2.3 x 10(-)(8)). Mutant pneumococci lacking Hic showed no absorption of factor H in human plasma and no binding of radiolabeled factor H, suggesting that Hic is responsible for factor H-binding in type 3 pneumococci. Factor H-dependent inhibition of the alternative pathway was not diminished by the presence of GST:Hic(39-261). In addition, an intrinsic inhibitory effect of Hic is suggested.     

Sucrose metabolism contributes to in vivo fitness of Streptococcus pneumoniae

We characterized two sucrose-metabolizing systems -sus and scr- and describe their roles in the physiology and virulence of Streptococcus pneumoniae in murine models of carriage and pneumonia. The sus and scr systems are regulated by LacI family repressors SusR and ScrR respectively. SusR regulates an adjacent ABC transporter (susT1/susT2/susX) and sucrose-6-phosphate (S-6-P) hydrolase (susH). ScrR controls an adjacent PTS transporter (scrT), fructokinase (scrK) and second S-6-P hydrolase (scrH). sus and scr play niche-specific roles in virulence. The susH and sus locus mutants are attenuated in the lung, but dispensable in nasopharyngeal carriage. Conversely, the scrH and scr locus mutants, while dispensable in the lung, are attenuated for nasopharyngeal colonization. The scrH/susH double mutant is more attenuated than scrH in the nasopharynx, indicating SusH can substitute in this niche. Both systems are sucrose-inducible, with ScrH being the major in vitro hydrolase. The scrH/susH mutant does not grow on sucrose indicating that sus and scr are the only sucrose-metabolizing systems in S. pneumoniae. We propose a model describing hierarchical regulation of the scr and sus systems by the putative inducer, S-6-P. The transport and metabolism of sucrose or a related disaccharide thus contributes to S. pneumoniae colonization and disease.     

Molecular characterization of a novel fibronectin-binding protein of Streptococcus pyogenes strains isolated from toxic shock-like syndrome patients

Group A Streptococcus pyogenes has surface-located fibronectin (Fn)-binding proteins known to be a major virulence factor, which adheres to and invades host cells. We present a novel Fn-binding protein of group A streptococcus serotype M3 and M18 strains isolated from patients with toxic shock-like syndrome (TSLS). By searching the whole genome sequence of an M3 strain from a TSLS patient, an open reading frame was found among the putative surface proteins. It possessed an LPXTG motif and Fn-binding repeat domains in the C-terminal region and was designated as FbaB (Fn-binding protein of group A streptococci type B). The fbaB gene was found in all M3 and M18 strains examined, although not in other M serotypes. Furthermore, FbaB protein was expressed on the cell surface of TSLS strains but not on non-TSLS ones. Enzyme-linked immunosorbent assay and ligand blotting revealed that recombinant FbaB exhibits a strong Fn-binding ability. An FbaB-deficient mutant strain showed 6-fold lower adhesion and invasion efficiencies to HEp-2 cells than the wild type. Moreover, mortality was decreased in mice infected with the mutant strain in comparison to the wild type. These data suggest that FbaB is etiologically involved in the development of invasive streptococcal diseases.     

Domain structure and conserved epitopes of Sfb protein, the fibronectin-binding adhesin of Streptococcus pyogenes

Streptococcus pyogenes expresses a fibronectin-binding surface protein (Sfb protein) which mediates adherence to human epithelial cells. The nucleotide sequence of the sfb gene was determined and the primary sequence of the Sfb protein was analysed. The protein consists of 638 amino acids and comprises five structurally distinct domains. The protein starts with an N-terminal signal peptide followed by an aromatic domain. The central part of the protein is formed by four proline-rich repeats which are flanked by non-repetitive spacer sequences. A second repeat region, consisting of four repeats that are distinct from the proline repeats and have been shown to form the fibronectin-binding domain, is located in the Cterminal part of the protein. The protein ends with a typical cell wall and membrane anchor region. Comparative sequence analysis of the N-terminal aromatic domain revealed similarities with carbohydrate-binding sites of other proteins. The proline repeat region of the Sfb protein shares characteristic features with proline-rich repeats of functionally distinct surface proteins from pathogenic Gram-positive cocci. Immunoelectron microscopy revealed an even distribution of the fibronectin-binding domain of Sfb protein on the surface of streptococcal cells. Analyses of 38 sfb genes originating from different S. pyogenes isolates revealed primary sequence variability in regions coding for the N-termini of mature Sfb proteins, whereas sequences coding for the central and C-terminal repeats were highly conserved. The repeat sequences are postulated to act as target sites for intragenic recombination events that result in variable numbers of repeats within the different sfb genes. A model of the Sfb protein is presented.     

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 SfbII, which is distinct from the previously described Sfbl proteins. The sfbII gene originated from a λ EMBL3 library of chromosomal DNA from group A streptococcal strain A75 and coded for a 113kDa 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.     

The Streptococcus pneumoniae beta-galactosidase is a surface protein

The beta-galactosidase gene of Streptococcus pneumoniae, bgaA, encodes a putative 2,235-amino-acid protein with the two amino acid motifs characteristic of the glycosyl hydrolase family of proteins. In addition, an N-terminal signal sequence and a C-terminal LPXTG motif typical of surface-associated proteins of gram-positive bacteria are present. Trypsin treatment of cells resulted in solubilization of the enzyme, documenting that it is associated with the cell envelope. In order to obtain defined mutants suitable for lacZ reporter experiments, the bgaA gene was disrupted, resulting in a complete absence of endogenous beta-galactosidase activity. The results are consistent with beta-galactosidase being a surface protein that seems not to be involved in lactose metabolism but that may play a role during pathogenesis.     

Contribution of novel choline-binding proteins to adherence, colonization and immunogenicity of Streptococcus pneumoniae

The surface of Streptococcus pneumoniae is decorated with a family of choline-binding proteins (CBPs) that are non-covalently bound to the phosphorylcholine of the teichoic acid. Two examples (PspA, a protective antigen, and LytA, the major autolysin) have been well characterized. We identified additional CPBs and characterized a new CBP, CbpA, as an adhesin and a determinant of virulence. Using choline immobilized on a solid matrix, a mixture of proteins from a pspA -deficient strain of pneumococcus was eluted in a choline-dependent fashion. Antisera to these proteins passively protected mice challenged in the peritoneum with a lethal dose of pneumococci. The predominant component of this mixture, CbpA, is a 75-kDa surface-exposed protein that reacts with human convalescent antisera. The deduced sequence from the corresponding gene showed a chimeric architecture with a unique N-terminal region and a C-terminal domain consisting of 10 repeated choline-binding domains nearly identical to PspA. A cbpA -deficient mutant showed a >50% reduction in adherence to cytokine-activated human cells and failed to bind to immobilized sialic acid or lacto-N-neotetraose, known pneumococcal ligands on eukaryotic cells. Carriage of this mutant in an animal model of nasopharyngeal colonization was reduced 100-fold. There was no difference between the parent strain and this mutant in an intraperitoneal model of sepsis. These data for CbpA extend the important functions of the CBP family to bacterial adherence and identify a pneumococcal vaccine candidate.     

Interaction between M-like protein and macrophage thioredoxin facilitates antiphagocytosis for Streptococcus equi ssp. zooepidemicus

Streptococcus equi ssp. zooepidemicus (S. zooepidemicus, S.z) is one of the common pathogens that can cause septicemia, meningitis, and mammitis in domesticated species. M-like protein (SzP) is an important virulence factor of S. zooepidemicus and contributes to bacterial infection and antiphagocytosis. The interaction between SzP of S. zooepidemicus and porcine thioredoxin (TRX) was identified by the yeast two-hybrid and further confirmed by co-immunoprecipitation. SzP interacted with both reduced and the oxidized forms of TRX without inhibiting TRX activity. Membrane anchored SzP was able to recruit TRX to the surface, which would facilitate the antiphagocytosis of the bacteria. Further experiments revealed that TRX regulated the alternative complement pathway by inhibiting C3 convertase activity and associating with factor H (FH). TRX alone inhibited C3 cleavage and C3a production, and the inhibitory effect was additive when FH was also present. TRX inhibited C3 deposition on the bacterial surface when it was recruited by SzP. These new findings indicated that S. zooepidemicus used SzP to recruit TRX and regulated the alternative complement pathways to evade the host immune phagocytosis.     

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.     

Inhibition of sortase-mediated Staphylococcus aureus adhesion to fibronectin via fibronectin-binding protein by sortase inhibitors

The sortase enzymes are a family of Gram-positive transpeptidases responsible for anchoring surface protein virulence factors to the peptidoglycan cell wall layer. In Staphylococcus aureus, deletion of the sortase isoforms results in marked reduction in virulence and infection potential, making it an important antivirulence target. Recombinant sortase A (SrtA) and sortase B (SrtB) were incubated with peptide substrate containing either the LPETG or NPQTN motifs. (Z)-3-(2,5-dimethoxyphenyl)-2-(4-methoxyphenyl) acrylonitrile, β-sitosterol-3-O-glucopyranoside, berberine chloride, and psammaplin A1 showed potent inhibitory activity against SrtA and SrtB. These compounds also exhibited potent inhibitory activity against S. aureus cell adhesion to fibronectin. The fibronectin-binding activity data highlight the potential of these compounds for the treatment of S. aureus infections via inhibition of sortase activity.     

Identification of a novel protein, PriB, in Klebsiella pneumoniae

PriB is a primosomal protein required for the reinitiation of replication in bacteria. Here, we report the identification and characterization of a novel PriB protein in Klebsiella pneumoniae (KPN_04595; KpPriB). Unlike the well-studied Escherichia coli PriB protein (EcPriB), which exists as a homodimer comprising 104-aa polypeptides, KpPriB forms a monomer of only 55 aa, due to the absence of the 49 aa N-terminus in KpPriB. Although this N-terminal region (1–49 aa) in EcPriB contains several important residues, such as K18, R34, and W47, which are crucial for ssDNA binding, we found that KpPriB binds ssDNA, but not ssRNA, with comparable affinity as that for EcPriB. Results from filter-binding assays demonstrate that the KpPriB–ssDNA interaction is cooperative and salt-sensitive. Substituting the residue K33 in KpPriB with alanine, the position corresponding to the classic ssDNA-binding residue K82 of EcPriB located in loop L₄₅, significantly reduced ssDNA-binding activity and cooperativity. These results reveal that the 1–49 aa region of the classical PriB protein is unnecessary for ssDNA binding. On the basis of these findings, the structure–function relationships of KpPriB are discussed.