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.

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-10 5 -fold-enhanced capacity to adhere and an up-to-10 4 -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.

SummaryThe 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.

No abstract

SummaryThe interaction of Streptococcus pneumoniae with human plasmin(ogen) represents a mechanism to enhance bacterial virulence by capturing surfaceassociated proteolytic activity in the infected host. Plasminogen binds to surface displayed pneumococcal a a a a -enolase (Eno) and is subsequently activated to the serine protease plasmin by host-derived tissue plasminogen activator (tPA) or urokinase (uPA).

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.

A clinically important adverse drug reaction, heparin-induced thrombocytopenia (HIT), is induced by antibodies specific for complexes of the chemokine platelet factor 4 (PF4) and the polyanion heparin. Even heparin-naive patients can generate anti-PF4/heparin IgG as early as day 4 of heparin treatment, suggesting preimmunization by antigens mimicking PF4/heparin complexes. These antibodies probably result from bacterial infections, as (1) PF4 bound charge-dependently to various bacteria, (2) human heparin-induced anti-PF4/heparin antibodies cross-reacted with PF4-coated Staphylococcus aureus and Escherichia coli, and (3) mice developed anti-PF4/heparin antibodies during polymicrobial sepsis without heparin application. Thus, after binding to bacteria, the endogenous protein PF4 induces antibodies with specificity for PF4/polyanion complexes. These can target a large variety of PF4-coated bacteria and enhance bacterial phagocytosis in vitro. The same antigenic epitopes are expressed when pharmacologic heparin binds to platelets augmenting formation of PF4 complexes. Boosting of preformed B cells by PF4/ heparin complexes could explain the early occurrence of IgG antibodies in HIT. We also found a continuous, rather than dichotomous, distribution of anti-PF4/heparin IgM and IgG serum concentrations in a cross-sectional population study (n ‫؍‬ 4029), indicating frequent preimmunization to modified PF4. PF4 may have a role in bacterial defense, and HIT is probably a misdirected antibacterial host defense mechanism. (Blood. 2011;117(4): 1370-1378) IntroductionThe chemokine platelet factor 4 (PF4, CXCL4) is stored within platelet ␣-granules 1 and released during platelet activation. Although its biologic role is poorly understood, PF4 commands attention in clinical medicine because it binds charge-dependently to the anticoagulant heparin, one of the most frequently used anticoagulants in clinical medicine, thereby neutralizing heparin's anticoagulant effect, 2,3 but also forming highly antigenic multimolecular complexes. [4][5][6] The resulting antibody response 7 induces the most frequent immune-mediated adverse drug reaction involving human blood cells, heparin-induced thrombocytopenia (HIT). 8 The pathogenic antibodies bind to PF4/heparin complexes at the platelet surface, and the resulting immune complexes induce Fcreceptor-mediated platelet activation and enhanced thrombin generation. 8 In a subset of patients, this causes thrombocytopenia and triggers paradoxical thrombosis, which is aggravated by continuation of heparin treatment.The immune response of HIT has several atypical features. Even in patients who receive heparin for the first time, there is rapid induction (as early as 4 days) of anti-PF4/heparin antibodies of IgG isotype. Moreover, IgG antibodies are the predominant class of immunoglobulins formed. 9,10 We and others discovered that up to 50% of patients after cardiopulmonary bypass surgery and 20% to 30% of orthopedic surgery patients (many of whom have not been previously exposed to heparin) devel...

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 alpha-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 outbreak of meningococcal disease.