Biomedical subjects

Simone Bergmann

Publications and source records attributed to Simone Bergmann.

11 recordsLinked to original sources

PubMed2006

Versatility of pneumococcal surface proteins.

Surface-exposed proteins are key players during the infectious process of pathogenic bacteria. The cell surface of the Gram-positive human pathogen Streptococcus pneumoniae is decorated not only by typical Gram-positive surface proteins, but also by a family of proteins that recognizes the phosphorylcholine of the lipoteichoic and teichoic acids, namely the choline-binding proteins, and by non-classical surface proteins that lack a leader peptide and membrane-anchor motif. A comprehensive understanding of how microbial proteins subvert host immunity or host protein functions is a prerequisite for the development of novel therapeutic strategies to combat pneumococcal infections. This article reviews recent progress in the investigation of the versatility and sophistication of the virulence functions of surface-exposed pneumococcal proteins.

Bacterial Outer Membrane Proteins↗

PubMed2006

Streptococcus pneumoniae enolase is important for plasminogen binding despite low abundance of enolase protein on the bacterial cell surface.

Enolase represents one of the anchorless surface proteins of Streptococcus pneumoniae and has previously been identified as a plasminogen-binding protein, endowing this pathogen with host proteolytic activity. In this study the mAb 245,C-6 (IgG1) was produced in a BALB/c mouse after immunizing with a protein fraction from S. pneumoniae. The mAb reacted with recombinant pneumococcal enolase both under non-denaturing and denaturing conditions. The epitope for the mAb was mapped to residues (55)DKSRYGGLG(63) of pneumococcal enolase using a peptide array. By applying the previously reported structure of enolase, this epitope was localized in a surface-exposed loop in each of the monomers of the octameric enolase. Previous immunoelectron microscopic studies, using polyclonal rabbit antibodies against enolase, depicted enolase on the cell surface but did not quantify the amount of surface-exposed enolase on viable pneumococci. Here, flow cytometry revealed no binding of mAb 245,C-6 to viable pneumococci, including TIGR4 and its non-encapsulated isogenic mutant, and only a minor increase of fluorescence intensity was measured when the polyclonal anti-enolase antibodies were used. In contrast, control antibodies recognizing the choline-binding proteins (CBPs) PspA and PspC showed high reactivities. The non-encapsulated TIGR4 did not show increased levels of antibody binding for mAb 245,C-6 or polyclonal anti-enolase antibodies, but revealed increased binding of polyclonal antibodies reacting with PspA or PspC. These results suggest that, compared to other surface-exposed proteins such as CBPs, the amount of enolase under the selected conditions is low. Flow cytometry, however, with FITC-labelled plasminogen demonstrated that the amount of surface-exposed enolase is important for plasminogen binding and, therefore, is also important for pneumococcal pathogenesis.

Antibodies, Bacterial↗

PubMed2005

PavA of Streptococcus pneumoniae modulates adherence, invasion, and meningeal inflammation.

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.

Adhesins, Bacterial↗

PubMed2005

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.

Amino Acid Motifs↗

PubMed2004

Plasmin(ogen)-binding alpha-enolase from Streptococcus pneumoniae: crystal structure and evaluation of plasmin(ogen)-binding sites.

Alpha-enolases are ubiquitous cytoplasmic, glycolytic enzymes. In pathogenic bacteria, alpha-enolase doubles as a surface-displayed plasmin(ogen)-binder supporting virulence. The plasmin(ogen)-binding site was initially traced to the two C-terminal lysine residues. More recently, an internal nine-amino acid motif comprising residues 248 to 256 was identified with this function. We report the crystal structure of alpha-enolase from Streptococcus pneumoniae at 2.0A resolution, the first structure both of a plasminogen-binding and of an octameric alpha-enolase. While the dimer is structurally similar to other alpha-enolases, the octamer places the C-terminal lysine residues in an inaccessible, inter-dimer groove restricting the C-terminal lysine residues to a role in folding and oligomerization. The nine residue plasminogen-binding motif, by contrast, is exposed on the octamer surface revealing this as the primary site of interaction between alpha-enolase and plasminogen.

Aspartic Acid↗

PubMed2004

The cell wall subproteome of Listeria monocytogenes.

The surface subproteome of Listeria monocytogenes that includes many proteins already known to be involved in virulence and interaction with host cells has been characterized. A new method for the isolation of a defined surface proteome of low complexity has been established based on serial extraction of proteins by different salts at high concentration, and in all 55 proteins were identified by N-terminal sequencing and mass spectrometry. About 16% of these proteins are of unknown function and three proteins have no orthologue in the nonpathogenic L. innocua and might be involved in virulence mechanisms. Remarkably, a relatively high number of proteins with a function in the cytoplasmic compartment was identified in this surface proteome. These proteins had neither predicted or detectable signal peptides nor could any modification be observed except removal of the N-terminal methionine. Enolase (Lmo2455) is one of these proteins. It was shown to be present in the cell wall of the pathogen by immunoelectron microscopy and, along with heat shock factor DnaK (Lmo1473), elongation factor TU (Lmo2653), and glyceraldehyde-3-phosphate dehydrogenase (Lmo2459), it was found to be able to bind human plasminogen in overlay blots and surface plasmon resonance (SPR) experiments. The KD values of these interactions were determined by SPR measurements. The data indicate a possible role of these proteins as receptors for human plasminogen on the bacterial cell surface. The potential role of this recruitment of a host protease for extracellular invasion mechanisms is discussed.

Aminopeptidases↗

PubMed2004

Glyceraldehyde-3-phosphate dehydrogenase of Streptococcus pneumoniae is a surface-displayed plasminogen-binding protein.

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.

Base Sequence↗

PubMed2004

Characterization of plasmin(ogen) binding to Streptococcus pneumoniae.

BACKGROUND & OBJECTIVES: The proteolytic activity of plasmin promotes migration of pathogenic bacteria through the human extracellular matrix. The human pathogen Streptococcus pneumoniae binds both human plasminogen and plasmin via the surface displayed alpha-enolase designated Eno. Electron microscopic studies verified the surface exposition of the glycolytic enzyme alpha-enolase and moreover, its ability to reassociate to the cell surface. Carboxyterminal lysine residues of recently described eukaryotic and prokaryotic plasminogen-binding proteins such as SEN of S. pyogenes are involved in interaction with lysine binding sites of kringle domains of plasminogen. In this study, the role of carboxy terminal lysyl residue of eno in plasminogen binding is further analysed. METHODS: Site-directed mutagenesis of eno gene was done using DNA primers with Hind III-restriction enzyme sites for cloning. Purified Eno fusion proteins were separated by SDS-PAGE and human plasminogen binding assay was performed. Radioiodinated ligand binding was done by competitive inhibition assay. RESULTS: Binding assays performed under reduced conditions indicated also a role of the C-terminal lysyl residues of Eno for plasmin(ogen) binding. Binding of pneumococci to radioiodinated plasminogen was competitively inhibited in the presence of plasminogen, kringle 1-3 (LBS 1) and the lysineanalogon epsilon-amino caproic acid indicating the crucial role of lysine-binding sites of plasminogen. However, binding analysis of plasminogen and LBS 1 to wild type Eno and carboxy terminal modified Eno proteins did not reveal any difference in plasminogen-binding activity under native conditions. INTERPRETATION & CONCLUSION: The present results suggested the presence of a further plasminogenbinding motif in Eno. This hypothesis was confirmed by plasminogen-binding activity of reassociated C-terminal modified enolase to the pneumococcal surface and indicated, therefore, the presence of a further binding motif in Eno for plasminogen binding.

Bacterial Adhesion↗

PubMed2003

Ectodomains 3 and 4 of human polymeric Immunoglobulin receptor (hpIgR) mediate invasion of Streptococcus pneumoniae into the epithelium.

Streptococcus pneumoniae binds to the ectodomain of the human polymeric Ig receptor (pIgR), also known as secretory component (SC), via a hexapeptide motif in the choline-binding protein SpsA. The SpsA-pIgR interaction mediates adherence and internalization of the human pathogen into epithelial cells. In this study the results of SpsA binding to human, mouse, and chimeric SC strongly supported the human specificity of this unique interaction and suggested that binding sites in the third and fourth Ig-like domain of human SC (D3 and D4, respectively) are involved in SpsA-pIgR complex formation. Binding of SpsA to SC-derived synthetic peptides indicated surface-located potential binding motifs in D3 and D4. Adherence and uptake of pneumococci or SpsA-coated latex beads depended on the SpsA hexapeptide motif as well as SpsA-binding sites in D3 and D4 of human pIgR. The involvement of D3 and D4 in adherence and invasion was demonstrated by the lack of binding of SpsA-coated latex beads to transfected epithelial cells expressing mutated pIgR. Finally, blocking experiments with chimeric human-mouse SC as well as synthetic peptides indicated the participation of D3 and a key role of D4 in pneumococcal invasion.

Amino Acid Motifs↗

PubMed2003

Molecular cloning of an alpha-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 host's 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.

Amino Acid Sequence↗

PubMed2003

Identification of a novel plasmin(ogen)-binding motif in surface displayed alpha-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 alpha-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 alpha-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.

Amino Acid Motifs↗