Sven Hammerschmidt

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.

SPSA, A NOVEL PNEUMOCOCCAL SURFACE PROTEIN WITH SPECIFIC BINDING TO SECRETORY IMMUNOGLOBULIN A AND SECRETORY COMPONENT

The interaction of pathogenic bacteria with host serum and matrix proteins is a common strategy to enhance their virulence. Streptococcus pneumoniae colonizes the human upper respiratory tract in healthy individuals and is also able to cause invasive diseases. Here, we describe a novel pneumococcal surface protein, SpsA, capable of binding specifically to human secretory immunoglobulin A (SIgA). The dissociation constant of SIgA binding to SpsA was 9.3 × 10−9 M. Free secretory component (SC) also binds to S. pneumoniae, whereas serum IgA does not, suggesting that pneumococcal binding to SIgA is mediated by the SC. To our knowledge, this is the first defined interaction of SC with a prokaryotic protein. The spsA gene encodes a polypeptide of 523 amino acids with a predicted molecular mass of 59 151 Da. The SIgA‐ or SC‐binding domain is located in the N‐terminal part of SpsA and exhibits no significant homology to any other proteins. The purified SIgA‐binding domain of SpsA could completely inhibit the binding of SIgA to pneumococci. SpsA was expressed by 73% of the tested S. pneumoniae isolates and was substantially conserved between different serotypes. The interaction between S. pneumoniae and SC via SpsA represents a novel biological interaction that might increase virulence by the impairment of bacterial clearance.

Illustration of Pneumococcal Polysaccharide Capsule during Adherence and Invasion of Epithelial Cells

ABSTRACT The capsular polysaccharide of Streptococcus pneumoniae represents an important virulence factor and protects against phagocytosis. In this study the amount of capsular polysaccharide present on the bacterial surface during the infection process was illustrated by electron microscopic studies. After infection of A549 cells (type II pneumocytes) and HEp-2 epithelial cells a modified fixation method was used that allowed visualization of the state of capsule expression. This modified fixation procedure did not require the use of capsule-specific antibodies. Visualization of pneumococci in intimate contact and invading cells demonstrated that pneumococci were devoid of capsular polysaccharide. Pneumococci not in contact with the cells did not show alterations in capsular polysaccharide. After infection of the cells, invasive pneumococci of different strains and serotypes were recovered. Single colonies of these recovered pneumococci exhibited an up-to-105-fold-enhanced capacity to adhere and an up-to-104-fold-enhanced capacity to invade epithelial cells. Electron microscopic studies using a lysine-ruthenium red (LRR) fixation procedure or cryo-field emission scanning electron microscopy revealed a reduction in capsular material, as determined in detail for a serotype 3 pneumococcal strain. The amount of polysaccharide in the serotype 3 capsule was also determined after intranasal infection of mice. This study illustrates for the first time the phenotypic variation of the polysaccharide capsule in the initial phase of pneumococcal infections. The modified LRR fixation allowed monitoring of the state of capsule expression of pathogens during the infectious process.

Capsule phase variation in Neisseria meningitidis serogroup B by slipped‐strand mispairing in the polysialyltransferase gene (siaD): correlation with bacterial invasion and the outbreak of meningococcal disease

A mechanism of capsular polysaccharide phase variation in Neisseria meningitidis is described. Meningococcal cells of an encapsulated serogroup B strain were used in invasion assays. Only unencapsulated variants were found to enter epithelial cells. Analysis of one group of capsule‐deficient variants indicated that the capsular polysaccharide was re‐expressed at a frequency of 10−3. Measurement of enzymatic activities involved in the biosynthesis of the α‐2,8 polysialic acid capsule showed that polysialyltransferase (PST) activity was absent in these capsule‐negative variants. Nucleotide sequence analysis of siaD revealed an insertion or a deletion of one cytidine residue within a run of (dC)7 residues at position 89, resulting in a frameshift and premature termination of translation. We analysed unencapsulated isolates from carriers and encapsulated case isolates collected during an outbreak of meningococcal disease. Further paired blood‐culture isolates and unencapsulated nasopharyngeal isolates from patients with meningococcal meningitis were examined. In all unencapsulated strains analysed we found an insertion or deletion within the oligo‐(dC) stretch within siaD, resulting in a frameshift and loss of capsule formation. All encapsulated isolates, however, had seven dC residues at this position, indicating a correlation between capsule phase variation and bacterial invasion and the out‐break of meningococcal disease.

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.

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.

Modulation of cell surface sialic acid expression in Neisseria meningitidis via a transposable genetic element.

Cell surface‐located sialic acids of the capsule and the lipooligosaccharide (LOS) are both pivotal virulence factors in Neisseria meningitidis, promoting survival and dissemination of this pathogen which can cause both sepsis and meningitis. With the aid of a unique set of isogenic meningococcal mutants defective in the expression of cell surface‐located sialic acids, we have demonstrated that encapsulation hinders the primary event in the development of the disease, but the spontaneous switching of encapsulated wild‐type bacteria to a capsule‐negative phenotype promotes meningococcal adherence and invasion into mucosal epithelial cells. Genetic analysis of the capsule‐negative, invasive bacteria revealed a unique mechanism for modulation of capsule expression based on the reversible inactivation of an essential sialic acid biosynthesis gene, siaA, by insertion/excision of a naturally occurring insertion sequence element, IS1301. Inactivation of siaA regulates both capsule expression and endogenous LOS sialylation. This is the first example of an insertion sequence element‐based genetic switch mechanism in the pathogenic bacterium and is an important step in the understanding of bacterial virulence.

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.