Biomedical subjects

Jan-Ingmar Flock

Publications and source records attributed to Jan-Ingmar Flock.

17 recordsLinked to original sources

PubMed2005

Dual effects of extracellular adherence protein from Staphylococcus aureus on peripheral blood mononuclear cells.

Extracellular adherence protein (Eap) has been suggested as an important virulence factor of Staphylococcus aureus because it enhances bacterial adherence and internalization into eukaryotic cells, interference with T cells, and neutrophil adherence to endothelial cells. We demonstrate that Eap has dual effects on peripheral blood mononuclear cells, depending on its concentration. At low concentrations (up to 9 microg/mL), Eap induces a proliferative response; at higher concentrations, it causes a significant inhibition of T cell proliferation induced by S. aureus supernatants toxic shock syndrome toxin-1 or phytohemagglutinin. A marked increase in apoptotic (i.e., Annexin V and propidium iodide positive) T and B cells could be demonstrated after exposure to the inhibitory concentration of Eap. Human anti-Eap antibodies prepared from polyspecific immunoglobulin G (IgG) blocked the immunomodulatory effects of Eap. Our results demonstrate novel immunomodulatory activities of Eap and identify potential mechanisms of action of intravenous IgG therapy in the treatment of S. aureus infections.

Bacterial Proteins↗

PubMed2005

Studies of fibronectin-binding proteins of Streptococcus equi.

Streptococcus equi subsp. equi is the causative agent of strangles, a disease of the upper respiratory tract in horses. The initiation of S. equi subsp. equi infection is likely to involve cell surface-anchored molecules mediating bacterial adhesion to the epithelium of the host. The present study describes the cloning and characterization of FNEB, a fibronectin-binding protein with cell wall-anchoring motifs. FNEB can thus be predicted as cell surface located, contrary to the two previously characterized fibronectin-binding proteins in S. equi subsp. equi, FNE and SFS. Assays of antibody titers in horses and in experimentally infected mice indicate that the protein is immunogenic and expressed in vivo during S. equi subsp. equi infection. Using Western ligand blotting, it was shown that FNEB binds to the N-terminal 29-kDa fragment of fibronectin, while SFS and FNE both bind to the adjacent 40-kDa fragment. S. equi subsp. equi is known to bind fibronectin to a much lower degree than the closely related S. equi subsp. zooepidemicus, but the binding is primarily directed to the 29-kDa fragment. Inhibition studies using S. equi subsp. equi cells indicate that FNEB mediates cellular binding to fibronectin in this species.

Adhesins, Bacterial↗

PubMed2005

Extracellular fibrinogen binding protein, Efb, from Staphylococcus aureus as an antiplatelet agent in vivo.

Staphylococcus aureus produces and secretes a protein, extracellular fibrinogen binding protein (Efb), which contributes to virulence in wound infection. We have previously shown that Efb is a potent inhibitor of platelet function in vitro. We confirm here that this is also the case in vivo. Pre-treatment with Efb resulted in a significant prolongation of bleeding time in a mouse model. Furthermore, Efb was capable of rescuing animals from death caused by the administration of potent platelet agonists. This antiplatelet effect may explain the retardation of wound healing associated with Efb in S. aureus wound infections. These results are important not only in terms of understanding S. aureus pathogenesis, and consequently identifying new treatment strategies, but also with regard to the development of potential, novel antiplatelet agents for the prevention of thrombosis.

Animals↗

PubMed2004

Identification of a novel collagen-like protein, SclC, in Streptococcus equi using signal sequence phage display.

Strangles is a serious disease in horses caused by Streptococcus equi subspecies equi. In this study, genes encoding putative extracellular proteins in this subspecies have been identified using signal sequence phage display. Among these, one showed similarities to the SclB protein, a member of the collagen-like proteins of Streptococcus pyogenes. The novel gene denoted sclC encodes a protein, SclC, of 302 amino acids, containing typical features found in cell wall-anchored proteins in Gram-positive bacteria. Based on similarities to the S. pyogenes collagen-like proteins the mature SclC protein can be divided into various domains: an N-terminal non-repetitive region (A), a highly repetitive collagen-like region (CL), and a C-terminal proline-rich wall-associated region (W). Using PCR, the sclC gene was detected in all studied strains of S. equi subsp. equi and S. equi subsp. zooepidemicus. Further, antibodies against recombinant SclC were detected in a collection of sera from horses with no history of strangles as well as horses previously infected with S. equi subsp. equi. Interestingly, the sera from convalescence horses were found to have significantly increased antibody titers against the SclC protein indicating that this protein is expressed during infection of S. equi subsp. equi.

Amino Acid Sequence↗

PubMed2004

The extracellular adherence protein from Staphylococcus aureus inhibits neutrophil binding to endothelial cells.

Extracellular adherence protein (Eap) from Staphylococcus aureus inhibits the adherence of neutrophils to nonstimulated and tumor necrosis factor alpha-stimulated endothelial cells in both static adhesion assays and flow adhesion assays. Consequently, Eap also impaired their transendothelial migration. During an S. aureus infection, Eap may thus serve to reduce inflammation by inhibiting neutrophil adhesion and extravasation.

Bacterial Proteins↗

PubMed2004

The fibrinogen binding protein of Staphylococcus epidermidis is a target for opsonic antibodies.

Antibodies against the fibrinogen binding protein (Fbe) of Staphylococcus epidermidis significantly increased macrophage phagocytosis. Antibodies against autolysin E were opsonic but to a lesser extent. Antibodies against a novel, putatively surface-located antigen were unable to enhance phagocytosis. The severity of systemic infection of mice with S. epidermidis was reduced if the bacteria were preopsonized with anti-Fbe prior to administration. Fbe is thus a strong candidate for protein vaccination against S. epidermidis infection, and antibodies against Fbe can be used to prevent or treat infections caused by S. epidermidis.

Animals↗

PubMed2004

Recombinant Streptococcus equi proteins protect mice in challenge experiments and induce immune response in horses.

Horses that have undergone infection caused by Streptococcus equi subspecies equi (strangles) were found to have significantly increased serum antibody titers against three previously characterized proteins, FNZ (cell surface-bound fibronectin binding protein), SFS (secreted fibronectin binding protein), and EAG (alpha2-macroglobulin, albumin, and immunoglobulin G [IgG] binding protein) from S. equi. To assess the protective efficacy of vaccination with these three proteins, a mouse model of equine strangles was utilized. Parts of the three recombinant proteins were used to immunize mice, either subcutaneously or intranasally, prior to nasal challenge with S. equi subsp. equi. The adjuvant used was EtxB, a recombinant form of the B subunit of Escherichia coli heat-labile enterotoxin. It was shown that nasal colonization of S. equi subsp. equi and weight loss due to infection were significantly reduced after vaccination compared with a mock-vaccinated control group. This effect was more pronounced after intranasal vaccination than after subcutaneous vaccination; nearly complete eradication of nasal colonization was obtained after intranasal vaccination (P < 0.001). When the same antigens were administered both intranasally and subcutaneously to healthy horses, significant mucosal IgA and serum IgG antibody responses against FNZ and EAG were obtained. The antibody response was enhanced when EtxB was used as an adjuvant. No adverse effects of the antigens or EtxB were observed. Thus, FNZ and EAG in conjunction with EtxB are promising candidates for an efficacious and safe vaccine against strangles.

Adhesins, Bacterial↗

PubMed2004

Extracellular fibrinogen binding protein, Efb, from Staphylococcus aureus binds to platelets and inhibits platelet aggregation.

S. aureus produces and secretes a protein, extracellular fibrinogen binding protein (Efb), which contributes to virulence in wound infection. We have shown here that Efb is a potent inhibitor of platelet aggregation. Efb can bind specifically to platelets by two mechanisms; 1) to fibrinogen naturally bound to the surface of activated platelets and 2) also directly to a surface localized component on the platelets. This latter binding of Efb is independent of fibrinogen. The specific binding of Efb to the putative receptor on the platelet surface results in a stimulated, non-functional binding of fibrinogen in a dose dependent manner, distinct from natural binding of fibrinogen to platelets. The natural binding of fibrinogen to GPIIb/IIIa on activated platelets could be blocked by a monoclonal antibody against this integrin, whereas the Efb-mediated fibrinogen binding could not be blocked. The enhanced Efb-dependent fibrinogen binding to platelets is of a nature that does not promote aggregation of the platelets; instead it inhibits aggregation. The anti-thrombotic action of Efb may explain the effect of Efb on wound healing, which is delayed in the presence of Efb.

Bacterial Proteins↗

PubMed2003

GST-Fbe can recognize beta-chains of fibrin(ogen) on explanted materials.

Staphylococcus epidermidis, a coagulase-negative staphylococcus (CoNS), is one of the leading pathogens of nosocomial infections, particularly associated with foreign body infections. Adherence of S. epidermidis to fibrinogen deposited on the surfaces of implants is important for the development of foreign body infections. A gene (fbe) encoding a fibrinogen-binding protein from S. epidermidis (Fbe) was identified by shotgun phage display. A portion of fbe was cloned into a GST-fusion vector. Affinity to glutathione-Sepharose by the GST-tag and affinity to fibrinogen-Sepharose by the Fbe part were applied to purify the recombinant Fbe. The purity and efficacy of the methods used in protein purification was compared. Furthermore, the potential physiological role of Fbe was studied by the interaction between GST-Fbe and components extracted from explanted materials in vitro.

Animals↗

PubMed2003

The adhesive and immunomodulating properties of the multifunctional Staphylococcus aureus protein Eap.

Adherence of Staphylococcus aureus to the host tissue is an important step in the initiation of pathogenesis. At least 10 adhesins produced by S. aureus have been described and it is becoming clear that the expression of these adhesins and their interactions with eukaryotic cells involve complex processes. Some of these, such as the fibronectin-binding proteins (FnBPs) and Clumping Factor A, are well characterized. However, in the last 10 years a number of novel S. aureus adhesins have been described. Functional analyses of these proteins, one of which is Eap (extracellular adherence protein, also known as Map and p70), are revealing important information on the pathogenesis of staphylococcal disease. More than 10 years after the first report of Eap, we are beginning to understand that this protein, which has a broad spectrum of functions, may be a critical factor in the pathogenesis of S. aureus. This review will focus on the interactions of Eap with eukaryotic cells, plasma proteins and the extracellular matrix as well as on the recently recognized role of Eap as an important mediator in the immune response to staphylococcal infection.

Adhesins, Bacterial↗

PubMed2003

Extracellular adherence protein from Staphylococcus aureus enhances internalization into eukaryotic cells.

In this study we have shown that Eap (extracellular adherence protein) plays a role in the internalization process of Staphylococcus aureus into eukaryotic cells. Eap is a protein that is mostly extracellularly and to a lesser extent is bound to the bacterial surface as a result of rebinding. Eap is able to bind to several plasma proteins, such as fibronectin, fibrinogen, and prothrombin. It has the capacity to form oligomers and is able to agglutinate S. aureus. A mutant strain, Newman mAH12 (eap:: Ery(r)), with a deficient eap gene was used in the present study. We have demonstrated that (i) strain Newman mAH12 could adhere to and become internalized to a higher extent by eukaryotic cells than the isogenic mutant, (ii) strain Newman mAH12 complemented with the eap gene displayed restoration of the internalization level, (iii) externally added Eap enhanced the internalization of laboratory and clinical S. aureus strains as well as of S. carnosus (a coagulase-negative species devoid of proteins important for internalization), and (iv) antibodies against Eap were able to block the internalization process in strain Newman mAH12 and clinical isolates. Eap, with its broad binding capacity and its surface localization, thus seems to contribute to the internalization of S. aureus into eukaryotic cells. We therefore propose a novel internalization pathway for S. aureus in which Eap plays an enhancing role.

Adhesins, Bacterial↗

PubMed2002

Staphylococcus aureus extracellular adherence protein serves as anti-inflammatory factor by inhibiting the recruitment of host leukocytes.

Staphylococcus aureus is a human pathogen that secretes proteins that contribute to bacterial colonization. Here we describe the extracellular adherence protein (Eap) as a novel anti-inflammatory factor that inhibits host leukocyte recruitment. Due to its direct interactions with the host adhesive proteins intercellular adhesion molecule 1 (ICAM-1), fibrinogen or vitronectin, Eap disrupted beta(2)-integrin and urokinase receptor mediated leukocyte adhesion in vitro. Whereas Eap-expressing S. aureus induced a 2 3-fold lower neutrophil recruitment in bacterial peritonitis in mice as compared with an Eap-negative strain, isolated Eap prevented beta(2)-integrin-dependent neutrophil recruitment in a mouse model of acute thioglycollate-induced peritonitis. Thus, the specific interactions with ICAM-1 and extracellular matrix proteins render Eap a potent anti-inflammatory factor, which may serve as a new therapeutic substance to block leukocyte extravasation in patients with hyperinflammatory pathologies.

Animals↗

PubMed2002

A novel von Willebrand factor binding protein expressed by Staphylococcus aureus.

When a shotgun phage-display library of Staphylococcus aureus Newman was affinity selected (panned) against recombinant von Willebrand factor (vWf), a novel von Willebrand factor binding protein (vWbp) was found. Experimental data indicate that the interaction between vWbp and vWf is very specific and mediated by a region of 26 aa residues in the C-terminal part of vWbp. vWbp has an N-terminal secretory signal sequence but no cell wall anchoring motif, suggesting a soluble extracellular location. Mature vWbp could be purified from the culture supernatant and the identity of the protein was confirmed by N-terminal sequencing. vWbp migrates with an apparent molecular mass of 66 kDa and the deduced protein consists of 482 aa. The gene encoding vWbp, named vwb, was present in all S. aureus strains investigated.

Amino Acid Sequence↗

PubMed2002

Insertional inactivation of Eap in Staphylococcus aureus strain Newman confers reduced staphylococcal binding to fibroblasts.

To initiate invasive infection, Staphylococcus aureus must adhere to host substrates, such as the extracellular matrix or eukaryotic cells, by virtue of different surface proteins (adhesins). Recently, we identified a 60-kDa cell-secreted extracellular adherence protein (Eap) of S. aureus strain Newman with broad-spectrum binding characteristics (M. Palma, A. Haggar, and J. I. Flock, J. Bacteriol. 181:2840-2845, 1999), and we have molecularly confirmed Eap to be an analogue of the previously identified major histocompatibility complex class II analog protein (Map) (M. Hussain, K. Becker, C. von Eiff, G. Peter, and M. Herrmann, Clin. Diagn. Lab. Immunol. 8:1281-1286, 2001). Previous analyses of the Eap/Map function performed with purified protein did not allow dissection of its precise role in the complex situation of the staphylococcal whole cell presenting several secreted and wall-bound adhesins. Therefore, the role of Eap was investigated by constructing a stable eap::ermB deletion in strain Newman and by complementation of the mutant. Patterns of extracted cell surface proteins analyzed both by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by Western ligand assays with various adhesive matrix molecules clearly confirmed the absence of Eap in the mutant. However, binding and adhesion tests using whole staphylococcal cells demonstrated that both the parent and mutant strains bound equally well to fibronectin- and fibrinogen-coated surfaces, possibly due to their recognition by other staphylococcal adhesins. Furthermore, Eap mediated staphylococcal agglutination of both wild-type and mutant cells. In contrast, the mutant adhered to a significantly lesser extent to cultured fibroblasts (P < 0.001) than did the wild type, while adherence was restorable upon complementation. Furthermore, adherence to both epithelial cells (P < 0.05) and fibroblasts (not significant) could be blocked with antibodies against Eap, whereas preimmune serum was not active. In conclusion, Eap may contribute to pathogenicity by promoting adhesion of whole staphylococcal cells to complex eukaryotic substrates.

Adhesins, Bacterial↗

PubMed2002

Antibiotic properties of novel synthetic temporin A analogs and a cecropin A-temporin A hybrid peptide.

Temporin A, 18 analogs, and a cecropin A-temporin A hybrid peptide were tested with antibiotic sensitive and resistant bacteria, fungi, human erythrocytes, and in clotting assays. Several peptides were active in these assays, and some analogs (D-TA, W1-TA, and Con-L4,G10) may be useful lead compounds for further antibiotics development. The activity of temporin A was found to be dependent upon several of its structural features, including amino acid composition and sequence, chirality, helicity, and positive charge.

Anti-Bacterial Agents↗

PubMed1999

Experimental endocarditis in the rat secondary to septic arthritis induced by Staphylococcus aureus.

OBJECTIVE: To develop a modified model for experimental infective endocarditis (IE) in the rat. The goal was to induce a primary infectious focus in the temporomandibular joint (TMJ) of a rat. Hematogenous translocation of the bacteria to the traumatized aortic valve was desired. METHODS: Catheterization of the right carotid artery through the aortic valve was performed 7 days after induction of arthritis, which was done by intra-articular injection of glucocorticosteroid (triamcinolone acetonide, 1 mg) and intravenous challenge with 107 CFU Staphylococcus aureus. RESULTS: TMJ arthritis could be induced by intra-articular triamcinolone acetonide followed by intravenous bacterial challenge. Joints not given glucocorticosteroid were not affected. Only rats with arthritis developed IE subsequent to catheterization as a result of bacteremia generated from the arthritis. CONCLUSIONS: The present model may serve as a complement to the conventional method for induction of IE, in which a high intravenous challenge has to be given. In the present model, IE was instead the result of a continuous low level of bacteremia from an infectious focus in the TMJ. This model mimics the natural development of IE in patients, and may assist as a setting for prophylactic and therapeutic trials.

Journal Article↗

PubMed1998

Antianaerobic activity of a cecropin---melittin peptide.

OBJECTIVE: Several small, 15-residue peptides that contain portions of the amino acid sequences of both cecropin A and melittin have previously been shown to have broad-spectrum antibacterial activities against aerobic microorganisms, with no undesirable hemolytic properties. It would also be useful to know what effect these hybrid peptides have on anaerobic bacteria. METHODS: The minimum inhibitory concentrations of one hybrid, CA(1--7)M(2--9)NH2, were compared with those of seven other antimicrobial agents against 111 clinical anaerobic strains; Bacteroides fragilis, 24 strains; other Bacteroides fragilis group, 14 strains; other Bacteroides species, 13 strains; Fusobacterium nucleatum, six strains; Clostridium difficile, 22 strains; Clostridium perfringens, 10 strains, Propionibacterium spp., nine strains; and anaerobic cocci, 13 strains. RESULTS: Ninety per cent of strains belonging to the B. fragilis group, fusobacteria, propionibacteria and peptostreptococci were inhibited by 4 mg/L CA(1--7)M(2--9)NH2, and the antimicrobial activity was approximately in the same range as that of chloramphenicol. CONCLUSION: This investigation showed that the antimicrobial spectrum of this cecropin---melittin hybrid also includes anaerobic organisms.

Journal Article↗