Binding of Streptococcus pneumoniae Endopeptidase O (PepO) to Complement Component C1q Modulates the Complement Attack and Promotes Host Cell Adherence

The Gram-positive species Streptococcus pneumoniae is a human pathogen causing severe local and life-threatening invasive diseases associated with high mortality rates and death. We demonstrated recently that pneumococcal endopeptidase O (PepO) is a ubiquitously expressed, multifunctional plasminogen and fibronectin-binding protein facilitating host cell invasion and evasion of innate immunity. In this study, we found that PepO interacts directly with the complement C1q protein, thereby attenuating the classical complement pathway and facilitating pneumococcal complement escape. PepO binds both free C1q and C1 complex in a dose-dependent manner based on ionic interactions. Our results indicate that recombinant PepO specifically inhibits the classical pathway of complement activation in both hemolytic and complement deposition assays. This inhibition is due to direct interaction of PepO with C1q, leading to a strong activation of the classical complement pathway, and results in consumption of complement components. In addition, PepO binds the classical complement pathway inhibitor C4BP, thereby regulating downstream complement activation. Importantly, pneumococcal surface-exposed PepO-C1q interaction mediates bacterial adherence to host epithelial cells. Taken together, PepO facilitates C1q-mediated bacterial adherence, whereas its localized release consumes complement as a result of its activation following binding of C1q, thus representing an additional mechanism of human complement escape by this versatile pathogen.     

Complement Regulator Factor H Mediates a Two-step Uptake of Streptococcus pneumoniae by Human Cells

Streptococcus pneumoniae, a human pathogen, recruits complement regulator factor H to its bacterial cell surface. The bacterial PspC protein binds Factor H via short consensus repeats (SCR) 8–11 and SCR19–20. In this study, we define how bacterially bound Factor H promotes pneumococcal adherence to and uptake by epithelial cells or human polymorphonuclear leukocytes (PMNs) via a two-step process. First, pneumococcal adherence to epithelial cells was significantly reduced by heparin and dermatan sulfate. However, none of the glycosaminoglycans affected binding of Factor H to pneumococci. Adherence of pneumococci to human epithelial cells was inhibited by monoclonal antibodies recognizing SCR19–20 of Factor H suggesting that the C-terminal glycosaminoglycan-binding region of Factor H mediates the contact between pneumococci and human cells. Blocking of the integrin CR3 receptor, i.e. CD11b and CD18, of PMNs or CR3-expressing epithelial cells reduced significantly the interaction of pneumococci with both cell types. Similarly, an additional CR3 ligand, Pra1, derived from Candida albicans, blocked the interaction of pneumococci with PMNs. Strikingly, Pra1 inhibited also pneumococcal uptake by lung epithelial cells but not adherence. In addition, invasion of Factor H-coated pneumococci required the dynamics of host-cell actin microfilaments and was affected by inhibitors of protein-tyrosine kinases and phosphatidylinositol 3-kinase. In conclusion, pneumococcal entry into host cells via Factor H is based on a two-step mechanism. The first and initial contact of Factor H-coated pneumococci is mediated by glycosaminoglycans expressed on the surface of human cells, and the second step, pneumococcal uptake, is integrin-mediated and depends on host signaling molecules such as phosphatidylinositol 3-kinase.     

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.     

Identification and characterization of a cell surface protein of Prevotella intermedia 17 with broad-spectrum binding activity for extracellular matrix proteins

Prevotella intermedia binds and invades a variety of host cells. This binding is most probably mediated through cell surface proteins termed adhesins. To identify proteins binding to the host extracellular matrix (ECM) component, fibronectin, and study the molecular mechanism underlying bacterial colonization, we applied proteomic approaches to perform a global investigation of P. intermedia strain 17 outer membrane proteins. 2-DE followed by Far Western Blot analysis using fibronectin as a probe revealed a 29-kDa fibronectin-binding protein, designated here AdpB. The molecular identity of the protein was determined using PMF followed by a search of the P. intermedia 17 protein database. Database searches revealed the similarity of AdpB to multiple bacterial outer membrane proteins including the fibronectin-binding protein from Campylobacter jejuni. A recombinant AdpB protein bound fibronectin as well as other host ECM components, including fibrinogen and laminin, in a saturable, dose-dependent manner. Binding of AdpB to immobilized fibronectin was also inhibited by soluble fibronectin, laminin, and fibrinogen, indicating the binding was specific. Finally, immunoelectron microscopy with anti-AdpB demonstrated the cell surface location of the protein. This is the first cell surface protein with a broad-spectrum ECM-binding abilities identified and characterized in P. intermedia 17.     

Streptococcus pneumoniae Phosphoglycerate Kinase Is a Novel Complement Inhibitor Affecting the Membrane Attack Complex Formation

The Gram-positive bacterium Streptococcus pneumoniae is a major human pathogen that causes infections ranging from acute otitis media to life-threatening invasive disease. Pneumococci have evolved several strategies to circumvent the host immune response, in particular the complement attack. The pneumococcal glycolytic enzyme phosphoglycerate kinase (PGK) is both secreted and bound to the bacterial surface and simultaneously binds plasminogen and its tissue plasminogen activator tPA. In the present study we demonstrate that PGK has an additional role in modulating the complement attack. PGK interacted with the membrane attack complex (MAC) components C5, C7, and C9, thereby blocking the assembly and membrane insertion of MAC resulting in significant inhibition of the hemolytic activity of human serum. Recombinant PGK interacted in a dose-dependent manner with these terminal pathway proteins, and the interactions were ionic in nature. In addition, PGK inhibited C9 polymerization both in the fluid phase and on the surface of sheep erythrocytes. Interestingly, PGK bound several MAC proteins simultaneously. Although C5 and C7 had partially overlapping binding sites on PGK, C9 did not compete with either one for PGK binding. Moreover, PGK significantly inhibited MAC deposition via both the classical and alternative pathway at the pneumococcal surface. Additionally, upon activation plasmin(ogen) bound to PGK cleaved the central complement protein C3b thereby further modifying the complement attack. In conclusion, our data demonstrate for the first time to our knowledge a novel pneumococcal inhibitor of the terminal complement cascade aiding complement evasion by this important pathogen.     

Polymeric Immunoglobulin Receptor-mediated Invasion of Streptococcus pneumoniae into Host Cells Requires a Coordinate Signaling of SRC Family of Protein-tyrosine Kinases,ERK, and c-Jun N-terminal Kinase

Streptococcus pneumoniae are commensals of the human nasopharynx with the capacity to invade mucosal respiratory cells. PspC, a pneumococcal surface protein, interacts with the human polymeric immunoglobulin receptor (pIgR) to promote bacterial adherence to and invasion into epithelial cells. Internalization of pneumococci requires the coordinated action of actin cytoskeleton rearrangements and the retrograde machinery of pIgR. Here, we demonstrate the involvement of Src protein-tyrosine kinases (PTKs), focal adhesion kinase (FAK), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) but not p38 mitogen-activated protein kinases (MAPK) in pneumococcal invasion via pIgR. Pharmacological inhibitors of PTKs and MAPKs and genetic interference with Src PTK and FAK functions caused a significant reduction of pIgR-mediated pneumococcal invasion but did not influence bacterial adhesion to host cells. Furthermore, pneumococcal ingestion by host cells induces activation of ERK1/2 and JNK. In agreement with activated JNK, its target molecule and DNA-binding protein c-Jun was phosphorylated. We also show that functionally active Src PTK is essential for activation of ERK1/2 upon pneumococcal infections. In conclusion, these data illustrate the importance of a coordinated signaling between Src PTKs, ERK1/2, and JNK during PspC-pIgR-mediated uptake of pneumococci by host epithelial cells.     

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.     

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.     

Use of green fluorescent protein in visualisation of pneumococcal invasion of broncho-epithelial cells in vivo

The pneumococcus is the principle cause of bacterial pneumonia and also a major cause of bacterial meningitis. The mechanisms and sites of pneumococcal adherence and invasion of the respiratory tract in vivo are not clear however. We have made pneumococci expressing green fluorescent protein (GFP) and used it to trace pneumococcal adherence and invasion in vivo. By using GFP pneumococci we have shown bacterial adherence and invasion of broncho-epithelial cells in vivo by 4 h post-infection, with increases in pneumococcal invasiveness by 24 h. Using confocal image analysis we have shown varying levels of pneumococcal penetration and internalisation into host cells, as well as translocation through epithelial layers. To our knowledge this is the first report of pneumococcal invasion and cellular translocation in vivo.     

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.     

Two distinct pathways for the invasion of Streptococcus pyogenes in non-phagocytic cells

Adherence to and invasion of epithelial cells represent important pathogenic mechanisms of Streptococcus pyogenes . A fibronectin-binding surface protein of S. pyogenes , SfbI protein, has been implicated in both adherence and invasion processes. Invasion of SfbI-containing strains has been suspected to be responsible for the failure of antibiotics treatment to eradicate S. pyogenes . In this study, we tested the adherence and invasion properties of two well-characterized clinical isolates: A40, which expresses SfbI; and A8, which is SfbI negative and is unable to bind fibronectin. In strain A40, SfbI was the main factor required for attachment and invasion by using fibronectin as a bridging molecule and the α₅β₁ integrin as cellular receptor. The uptake process was characterized by the generation of large membrane invaginations at the bacteria–cell interface without evidence of actin recruitment or cellular injury. A40 cells were located in phagosomes and, only 24 h after infection, a consistent part of the bacterial population reached the cytoplasm. In contrast, uptake of strain A8 required major rearrangements of cytoskeletal proteins underneath attached bacteria. In A8, a proteinaceous moiety was involved, which does not interact with α₅β₁ or need any known bridging molecule. Bacterial attachment stimulated elongation and massive recruitment of neighbouring microvilli, which fused to surround streptococcal chains. They led to the generation of large pseudopod-like structures, which engulfed bacteria that were rapidly released and replicated in the cytoplasm. The identification of two completely different uptake pathways reported here provided further evidence regarding the diversity of S. pyogenes isolates and might contribute towards understanding the pathogenesis and persistence of S. pyogenes .     

Identification of a 47 kDa fibronectin-binding protein expressed by Borrelia burgdorferi isolate B31

The attachment of pathogenic microorganisms to host cells and tissues is often mediated through the expression of surface receptors recognizing components of the extracellular matrix (ECM). Here, we investigate the ability of Borrelia spirochaetes to bind the ECM constituent, fibronectin. Borrelia lysates were separated by SDS–PAGE, transferred to nitrocellulose and probed with alkaline phosphatase-labelled fibronectin (fibronectin-AP). Five of six Borrelia species and four of eight B. burgdorferi sensu lato isolates expressed one or more fibronectin-binding proteins. Borrelia burgdorferi isolate B31 expressed a 47 kDa (P47) fibronectin-binding protein that was localized to the outer envelope based on susceptibility to proteinase K. The interaction of P47 with fibronectin was specific, and the region of fibronectin bound by P47 mapped to the gelatin/collagen binding domain. P47 was purified by affinity chromatography, digested with endoproteinase Lys-C, and the peptide fragments analysed by liquid chromatography/tandem mass spectroscopy. A search of protein databases disclosed that the P47 peptide mass profile matched that predicted for the bbk32 gene product of B. burgdorferi isolate B31. The bbk32 gene was cloned into Escherichia coli , and the ability of recombinant BBK32 to bind fibronectin and inhibit the attachment of B. burgdorferi was demonstrated. The identification of BBK32 as a receptor for fibronectin binding may enhance our understanding of the pathogenesis and chronic nature of Lyme disease.     

The role played by the group A streptococcal negative regulator Nra on bacterial interactions with epithelial cells

Group A streptococci (GAS) specifically attach to and internalize into human epithelial host cells. In some GAS isolates, fibronectin-binding proteins were identified as being responsible for these virulence traits. In the present study, the previously identified global negative regulator Nra was shown to control the binding of soluble fibronectin probably via regulation of protein F2 and/or SfbII expression in the serotype M49 strain 591. According to results from a conventional invasion assay based on the recovery of viable intracellular bacteria, the increased fibronectin binding did not affect bacterial adherence to HEp-2 epithelial cells, but was associated with a reduction in the internalization rates. However, when examined by confocal and electron microscopy techniques, the nra-mutant bacteria were shown to exhibit higher adherence and internalization rates than the corresponding wild type. The mutant bacteria escaped from the phagocytic vacuoles much faster, promoting consistent morphological changes which resulted in severe host cell damage. The apoptotic and lytic processes observed in nra-mutant infected host cells were correlated with an increased expression of the genes encoding superantigen SpeA, the cysteine protease SpeB, and streptolysin S in the nra-mutant bacteria. Adherence and internalization rates of a nra/speB-double mutant at wild-type levels indicated that the altered speB expression in the nra mutant contributed to the observed changes in both processes. The Nra-dependent effects on bacterial virulence were confined to infections carried out with stationary growth phase bacteria. In conclusion, the obtained results demonstrated that the global GAS regulator Nra modulates virulence genes, which are involved in host cell damage. Thus, by helping to achieve a critical balance of virulence factor expression that avoids the injury of target cells, Nra may facilitate GAS persistence in a safe intracellular niche.     

A Processed Multidomain Mycoplasma hyopneumoniae Adhesin Binds Fibronectin,Plasminogen, and Swine Respiratory Cilia

Porcine enzootic pneumonia is a chronic respiratory disease that affects swine. The etiological agent of the disease, Mycoplasma hyopneumoniae, is a bacterium that adheres to cilia of the swine respiratory tract, resulting in loss of cilia and epithelial cell damage. A M. hyopneumoniae protein P116, encoded by mhp108, was investigated as a potential adhesin. Examination of P116 expression using proteomic analyses observed P116 as a full-length protein and also as fragments, ranging from 17 to 70 kDa in size. A variety of pathogenic bacterial species have been shown to bind the extracellular matrix component fibronectin as an adherence mechanism. M. hyopneumoniae cells were found to bind fibronectin in a dose-dependent and saturable manner. Surface plasmon resonance was used to show that a recombinant C-terminal domain of P116 bound fibronectin at physiologically relevant concentrations (K_D 24 ± 6 nm). Plasmin(ogen)-binding proteins are also expressed by many bacterial pathogens, facilitating extracellular matrix degradation. M. hyopneumoniae cells were found to also bind plasminogen in a dose-dependent and saturable manner; the C-terminal domain of P116 binds to plasminogen (K_D 44 ± 5 nm). Plasminogen binding was abolished when the C-terminal lysine of P116 was deleted, implicating this residue as part of the plasminogen binding site. P116 fragments adhere to the PK15 porcine kidney epithelial-like cell line and swine respiratory cilia. Collectively these data suggest that P116 is an important adhesin and virulence factor of M. hyopneumoniae.     

PavB is a surface‐exposed adhesin of Streptococcus pneumoniae contributing to nasopharyngeal colonization and airways infections

The genomic analysis of Streptococcus pneumoniae strains identified the Pneumococcal adherence and virulence factor B (PavB), whose repetitive sequences, designated Streptococcal Surface REpeats (SSURE), interact with human fibronectin. Here, we showed the gene in all tested pneumococci and identified that the observed differences in the molecular mass of PavB rely on the number of repeats, ranging from five to nine SSURE. PavB interacted with fibronectin and plasminogen in a dose‐dependent manner as shown by using various SSURE peptides. In addition, we identified PavB as colonization factor. Mice infected intranasally with ΔpavB pneumococci showed significantly increased survival times compared with wild‐type bacteria. Importantly, the pavB‐mutant showed a delay in transmigration to the lungs as observed in real‐time using bioluminescent pneumococci and decreased colonization rates in a nasopharyngeal carriage model. In co‐infection experiments the wild‐type out‐competed the pavB‐mutant and infections of epithelial cells demonstrated that PavB contributes to adherence to host cell. Blocking experiments suggested a function of PavB as adhesin, which was confirmed by direct binding of SSURE peptides to host cells. Finally, PavB may represent a new vaccine candidate as SSURE peptides reacted with human sera. Taken together, PavB is a surface‐exposed adhesin, which contributes to pneumococcal colonization and infections of the respiratory airways.     

The A domain of fibronectin-binding protein B of Staphylococcus aureus contains a novel fibronectin binding site

The fibronectin-binding proteins FnBPA and FnBPB are multifunctional adhesins than can also bind to fibrinogen and elastin. In this study, the N2N3 subdomains of region A of FnBPB were shown to bind fibrinogen with a similar affinity to those of FnBPA (2 μM). The binding site for FnBPB in fibrinogen was localized to the C-terminus of the γ-chain. Like clumping factor A, region A of FnBPB bound to the γ-chain of fibrinogen in a Ca(2+)-inhibitable manner. The deletion of 17 residues from the C-terminus of domain N3 and the substitution of two residues in equivalent positions for crucial residues for fibrinogen binding in clumping factor A and FnBPA eliminated fibrinogen binding by FnBPB. This indicates that FnBPB binds fibrinogen by the dock-lock-latch mechanism. In contrast, the A domain of FnBPB bound fibronectin with K(D) = 2.5 μM despite lacking any of the known fibronectin-binding tandem repeats. A truncate lacking the C-terminal 17 residues (latching peptide) bound fibronectin with the same affinity, suggesting that the FnBPB A domain binds fibronectin by a novel mechanism. The substitution of the two residues required for fibrinogen binding also resulted in a loss of fibronectin binding. This, combined with the observation that purified subdomain N3 bound fibronectin with a measurable, but reduced, K(D) of 20 μM, indicates that the type I modules of fibronectin bind to both the N2 and N3 subdomains. The fibronectin-binding ability of the FnBPB A domain was also functional when the protein was expressed on and anchored to the surface of staphylococcal cells, showing that it is not an artifact of recombinant protein expression.     

Fibronectin-binding 36 kDa protein in human fibroblasts

A 36 kDa fibronectin-binding protein was identified from electrophoretically separated proteins of the deoxycholate-soluble fraction of cultured fibroblasts by blotting with fibronectin and using poly- or monoclonal antibodies and immunoperoxidase staining to detect the bound fibronectin. The 36 kDa protein was purified by preparative electrophoresis and used to raise specific antibodies. Solid-phase 36 kDa protein bound plasma and fibroblast fibronectins equally well. The 36 kDa protein is an amphipathic protein with pI 5.9. It is monomeric with a tendency to dimerize and appears to be distinct from the cell surface fibronectin receptors which interact with the Arg-Gly-Asp recognition site in the fibronectin molecule.     

Mhp107 is a member of the multifunctional adhesin family of Mycoplasma hyopneumoniae

Mycoplasma hyopneumoniae is the causative pathogen of porcine enzootic pneumonia, an economically significant disease that disrupts the mucociliary escalator in the swine respiratory tract. Expression of Mhp107, a P97 paralog encoded by the gene mhp107, was confirmed using ESI-MS/MS. To investigate the function of Mhp107, three recombinant proteins, F1(Mhp107), F2(Mhp107), and F3(Mhp107), spanning the N-terminal, central, and C-terminal regions of Mhp107 were constructed. Colonization of swine by M. hyopneumoniae requires adherence of the bacterium to ciliated cells of the respiratory tract. Recent studies have identified a number of M. hyopneumoniae adhesins that bind heparin, fibronectin, and plasminogen. F1(Mhp107) was found to bind porcine heparin (K(D) ～90 nM) in a dose-dependent and saturable manner, whereas F3(Mhp107) bound fibronectin (K(D) ～180 nM) at physiologically relevant concentrations. F1(Mhp107) also bound porcine plasminogen (K(D) = 24 nM) in a dose-dependent and physiologically relevant manner. Microspheres coated with F3(Mhp107) mediate adherence to porcine kidney epithelial-like (PK15) cells, and all three recombinant proteins (F1(Mhp107)-F3(Mhp107)) bound swine respiratory cilia. Together, these findings indicate that Mhp107 is a member of the multifunctional M. hyopneumoniae adhesin family of surface proteins and contributes to both adherence to the host and pathogenesis.     

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.