Simone Bergmann

alpha-Enolase of Streptococcus pneumoniae is a plasmin(ogen)-binding protein displayed on the bacterial cell surface.

Binding of human plasminogen to Streptococcus pneumoniae and its subsequent activation promotes penetration of bacteria through reconstituted basement membranes. In this study, we have characterized a novel pneumococcal surface protein with a molecular mass of 47 kDa, designated Eno, which specifically binds human plasmin(ogen), exhibits α‐enolase activity and is necessary for viability. Using enzyme assays, we have confirmed the α‐enolase activity of both pneumococcal surface‐displayed Eno and purified recombinant Eno protein. Immunoelectron microscopy indicated the presence of Eno in the cytoplasm as well as on the surface of encapsulated and unencapsulated pneumococci. Plasminogen‐binding activity was demonstrated with whole pneumococcal cells and purified Eno protein. Binding of activated plasminogen was also shown for Eno; however, the affinity for plasmin is significantly reduced compared with plasminogen. Results from competitive inhibition assays indicate that binding is mediated through the lysine binding sites in plasmin(ogen). Carboxypeptidase B treatment and amino acid substitutions of the C‐terminal lysyl residues of Eno indicated that the C‐terminal lysine is pivotal for plasmin(ogen)‐binding activity. Eno is ubiquitously distributed among pneumococcal serotypes, and binding experiments suggested the reassociation of secreted Eno to the bacterial cell surface. The reassociation was also confirmed by immunoelectron microscopy. The results suggest a mechanism of plasminogen activation for human pathogens that might contribute to their virulence potential in invasive infectious processes.

Glyceraldehyde-3-Phosphate Dehydrogenase of Streptococcus pneumoniae Is a Surface-Displayed Plasminogen-Binding Protein

ABSTRACT The recruitment of plasminogen endows the bacterial cell surface of Streptococcus pneumoniae with proteolytic activity. In this study we demonstrate specific plasmin- and plasminogen-binding activity for the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is located in the cytoplasm as well as on the surface of pneumococci. GAPDH exhibits a high affinity for plasmin and a significantly lower affinity for plasminogen.

Identification of a novel plasmin(ogen)‐binding motif in surface displayed α‐enolase of Streptococcus pneumoniae

The interaction of Streptococcus pneumoniae with human plasmin(ogen) represents a mechanism to enhance bacterial virulence by capturing surface‐associated proteolytic activity in the infected host. Plasminogen binds to surface displayed pneumococcal α‐enolase (Eno) and is subsequently activated to the serine protease plasmin by host‐derived tissue plasminogen activator (tPA) or urokinase (uPA). The C‐terminal lysyl residues of Eno at position 433 and 434 were identified as a binding site for the kringle motifs of plasmin(ogen) which contain lysine binding sites. In this report we have identified a novel internal plamin(ogen)‐binding site of Eno by investigating the protein–protein interaction. Plasmin(ogen)‐binding activity of C‐terminal mutated Eno proteins used in binding assays as well as surface plasmon resonance studies suggested that an additional binding motif of Eno is involved in the Eno‐plasmin(ogen) complex formation. The analysis of spot synthesized synthetic peptides representing Eno sequences identified a peptide of nine amino acids located between amino acids 248–256 as the minimal second binding epitope mediating binding of plasminogen to Eno. Binding of radiolabelled plasminogen to viable pneumococci was competitively inhibited by a synthetic peptide FYDKERKVYD representing the novel internal plasmin(ogen)‐binding motif of Eno. In contrast, a synthetic peptide with amino acid substitutions at critical positions in the internal binding motif identified by systematic mutational analysis did not inhibit binding of plasminogen to pneumococci. Pneumococcal mutants expressing α‐enolase with amino acid substitutions in the internal binding motif showed a substantially reduced plasminogen‐binding activity. The virulence of these mutants was also attenuated in a mouse model of intranasal infection indicating the significance of the novel plasminogen‐binding motif in the pathogenesis of pneumococcal diseases.

Fibrinolysis and host response in bacterial infections

The plasminogen activation system is part of the fibrinolysis which is tightly regulated and protected against dysfunction by various activators and inhibitors. However, microorganisms including bacteria, fungi and also parasites have been proven to interact in a specific manner with components of the fibrinolytic pathways. Pathogenic bacteria are capable to subvert the function of proteases, activators or inhibitors for their own benefits including dissemination within the host and evasion of host inflammatory immune response. Here, we provide a state of the art overview of the divers strategies employed by bacteria to interact with components of the fibrinolytic system and to exploit the system for invasion. Moreover, the role of factors of the fibrinolytic cascade in inflammatory host response due to different bacterial infections will be presented.

PavA of Streptococcus pneumoniae Modulates Adherence, Invasion, and Meningeal Inflammation

ABSTRACT Pneumococcal adherence and virulence factor A (PavA) is displayed to the cell outer surface of Streptococcus pneumoniae and mediates pneumococcal binding to immobilized fibronectin. PavA, which lacks a typical gram-positive signal sequence and cell surface anchorage motif, is essential for pneumococcal virulence in a mouse infection model of septicemia. In this report the impact of PavA on pneumococcal adhesion to and invasion of eukaryotic cells and on experimental pneumococcal meningitis was investigated. In the experimental mouse meningitis model, the virulence of the pavA knockout mutant of S. pneumoniae D39, which did not show alterations of subcellular structures as indicated by electron microscopic studies, was strongly decreased. Pneumococcal strains deficient in PavA showed substantially reduced adherence to and internalization of epithelial cell lines A549 and HEp-2. Similar results were obtained with human brain-derived microvascular endothelial cells and human umbilical vein-derived endothelial cells. Attachment and internalization of pneumococci were not significantly affected by preincubation or cocultivations of pneumococci with anti-PavA antisera. Pneumococcal adherence was also not significantly affected by the addition of PavA protein. Complementation of the pavA knockout strain with exogenously added PavA polypeptide did not restore adherence of the mutant. These data suggest that PavA affects pneumococcal colonization by modulating expression or function of important virulence determinants of S. pneumoniae.

The nine residue plasminogen-binding motif of the pneumococcal enolase is the major cofactor of plasmin-mediated degradation of extracellular matrix, dissolution of fibrin and transmigration

The glycolytic enzyme alpha-enolase represents one of the nonclassical cell surface plasminogen-binding proteins of Streptococcus pneumoniae. In this study we investigated the impact of an internal plasminogen-binding motif of enolase on degradation of extracellular matrix and pneumococcal transmigration. In the presence of host-derived plasminogen activators (PA) tissue-type PA or urokinase PA and plasminogen S. pneumoniae expressing wild-type enolase efficiently degraded Matrigel or extracellular matrix (ECM). In contrast, amino acid substitutions in the nine residue plasminogen-binding motif of enolase significantly reduced degradation of ECM or Matrigel by mutated pneumococci. Similarly, recombinant wild-type enolase but not a mutated enolase derivative that lacks plasminogen-binding activity efficiently degraded ECM and Matrigel, respectively. In particular, bacterial cell enolase-bound plasmin potentiated dissolution of fibrin or laminin and transmigration of pneumococci through a fibrin matrix. In conclusion, these results provide evidence that the enolase is the major plasminogen-binding protein of pneumococci and that the nine residue plasminogen-binding motif of enolase is the key cofactor for plasmin-mediated pneumococcal degradation and transmigration through host ECM.

Integrin-linked kinase is required for vitronectin-mediated internalization of Streptococcus pneumoniae by host cells

By interacting with components of the human host, including extracellular matrix (ECM) proteins, Streptococcus pneumoniae has evolved various strategies for colonization. Here, we characterized the interaction of pneumococci with the adhesive glycoprotein vitronectin and the contribution of this protein to pneumococcal uptake by host cells in an integrin-dependent manner. Specific interaction of S. pneumoniae with the heparin-binding sites of purified multimeric vitronectin was demonstrated by flow cytometry analysis. Host-cell-bound vitronectin promoted pneumococcal adherence to and invasion into human epithelial and endothelial cells. Pneumococci were trapped by microspike-like structures, which were induced upon contact of pneumococci with host-cell-bound vitronectin. αvβ3 integrin was identified as the major cellular receptor for vitronectin-mediated adherence and uptake of pneumococci. Ingestion of pneumococci by host cells via vitronectin required a dynamic actin cytoskeleton and was dependent on integrin-linked kinase (ILK), phosphatidylinositol 3-kinase (PI3K), and protein kinase B (Akt), as demonstrated by gene silencing or in inhibition experiments. In conclusion, pneumococci exploit the vitronectin–αvβ3-integrin complex as a cellular receptor for invasion and this integrin-mediated internalization requires the cooperation between the host signalling molecules ILK, PI3K and Akt.

Molecular cloning of an α-enolase from the human filarial parasite Onchocerca volvulus that binds human plasminogen

Enolase represents a multifunctional protein involved in basic energy metabolism and plasminogen binding and activation at the surface of prokaryotic pathogens. A complete cDNA of 1615 bp of an alpha-enolase from Onchocerca volvulus (Ov-ENO) was isolated using a PCR-based approach. The open reading frame encoded for 435 amino acids and the high degree of conservation included the crucial amino acid residues that participate in the formation of the catalytic site, Mg(2+) binding site, and a hydrophobic motif reported to relate to surface expression. A 1089-bp fragment was expressed in a N-terminal 6 x His-tag expression vector in Escherichia coli. By immunohistological analysis using anti-Ov-ENO rabbit antibodies, native enolase could be detected in most tissues of adult O. volvulus, microfilariae, and infective larvae. Intense staining was observed in the muscles, where the energy consumption is high. The purified recombinant protein fragment revealed plasminogen binding activity in a blot-overlay assay employing anti-plasminogen antibodies. In sera from individuals infected with O. volvulus, IgG antibodies reactive with recombinant Ov-ENO were demonstrated by immunoblot and enzyme-linked immunosorbent analyses. The plasminogen-binding property of O. volvulus alpha-enolase may support plasmin-mediated proteolysis including degradation of hosts extracellular matrix thereby promoting the migration of larval stages through tissues. The recognition by antibodies in sera of O. volvulus-infected persons indicate an involvement of the protein in the interaction between the parasite and the human host.

Cytosolic Proteins Contribute to Surface Plasminogen Recruitment of Neisseria meningitidis

Plasminogen recruitment is a common strategy of pathogenic bacteria and results in a broad-spectrum surface-associated protease activity. Neisseria meningitidis has previously been shown to bind plasminogen. In this study, we show by several complementary approaches that endolase, DnaK, and peroxiredoxin, which are usually intracellular proteins, can also be located in the outer membrane and act as plasminogen receptors. Internal binding motifs, rather than C-terminal lysine residues, are responsible for plasminogen binding of the N. meningitidis receptors. Recombinant receptor proteins inhibit plasminogen association with N. meningitidis in a concentration-dependent manner. Besides binding purified plasminogen, N. meningitidis can also acquire plasminogen from human serum. Activation of N. meningitidis-associated plasminogen by urokinase results in functional activity and allows the bacteria to degrade fibrinogen. Furthermore, plasmin bound to N. meningitidis is protected against inactivation by alpha(2)-antiplasmin.

Binding of Human Plasminogen to Bifidobacterium

Bifidobacteria constitute up to 3% of the total microbiota and represent one of the most important health-promoting bacterial groups of the human intestinal microflora. The presence of Bifidobacterium in the human gastrointestinal tract has been directly related to several health-promoting activities; however, to date, no information about the specific mechanisms of interaction with the host is available. In order to provide some insight into the molecular mechanisms involved in the interaction with the host, we investigated whether Bifidobacterium was able to capture human plasminogen on the cell surface. By using flow cytometry, we demonstrated a dose-dependent human plasminogen-binding activity for four strains belonging to three bifidobacterial species: Bifidobacterium lactis, B. bifidum, and B. longum. The binding of human plasminogen to Bifidobacterium was dependent on lysine residues of surface protein receptors. By using a proteomic approach, we identified five putative plasminogen-binding proteins in the cell wall fraction of the model strain B. lactis BI07. The data suggest that plasminogen binding to B. lactis is due to the concerted action of a number of proteins located on the bacterial cell surface, some of which are highly conserved cytoplasmic proteins which have other essential cellular functions. Our findings represent a step forward in understanding the mechanisms involved in the Bifidobacterium-host interaction.