Biomedical subjects

Jos A G van Strijp

Publications and source records attributed to Jos A G van Strijp.

15 recordsLinked to original sources

PubMed2007

Staphylococcal superantigen-like 5 binds PSGL-1 and inhibits P-selectin-mediated neutrophil rolling.

Staphylococcus aureus secretes several virulence factors interfering with host-cell functions. Staphylococcal superantigen-like (SSL) proteins are a family of 11 exotoxins with structural homology to superantigens but with generally unknown functions. Recently, we described that chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS(31-121)), a potent inhibitor of C5a-induced responses, is structurally homologous to the C-terminal domain of SSL5. Here, we identify P-selectin glycoprotein ligand-1 (PSGL-1), involved in the initial rolling of neutrophils along the endothelium, as a target for SSL5. SSL5 specifically bound to Chinese hamster ovary cells stably expressing PSGL-1 (CHO-PSGL-1), which was dependent of sulfation and sialylation. Furthermore, SSL5 bound to PSGL-1/Ig fusion protein immobilized on a biosensor chip. SSL5 affected binding of soluble P-selectin/Fc chimera, the principle ligand of PSGL-1, to CHO-PSGL-1 cells and inhibited adhesion of neutrophils to immobilized P-selectin under static conditions. Under flow conditions SSL5 strongly decreased neutrophil rolling on immobilized P-selectin/Fc and activated human endothelial cells. In conclusion, SSL5 interferes with the interaction between PSGL-1 and P-selectin, suggesting that S aureus uses SSL5 to prevent neutrophil extravasation toward the site of infection. This makes SSL5 a potential lead for the development of new anti-inflammatory compounds for disorders characterized by excessive recruitment of leukocytes.

Animals↗

PubMed2006

A new staphylococcal anti-inflammatory protein that antagonizes the formyl peptide receptor-like 1.

Bacteria have developed mechanisms to escape the first line of host defense, which is constituted by the recruitment of phagocytes to the sites of bacterial invasion. We previously described the chemotaxis inhibitory protein of Staphylococcus aureus, a protein that blocks the activation of neutrophils via the formyl peptide receptor (FPR) and C5aR. We now describe a new protein from S. aureus that impaired the neutrophil responses to FPR-like1 (FPRL1) agonists. FPRL1 inhibitory protein (FLIPr) inhibited the calcium mobilization in neutrophils stimulated with MMK-1, WKYMVM, prion-protein fragment PrP(106-126), and amyloid beta(1-42). Stimulation with low concentrations of fMLP was partly inhibited. Directed migration was also completely prevented toward MMK-1 and partly toward fMLP. Fluorescence-labeled FLIPr efficiently bound to neutrophils, monocytes, B cells, and NK cells. HEK293 cells transfected with human C5aR, FPR, FPRL1, and FPRL2 clearly showed that FLIPr directly bound to FPRL1 and, at higher concentrations, also to FPR but not to C5aR and FPRL2. FLIPr can reveal unknown inflammatory ligands crucial during S. aureus infections. As a novel described FPRL1 antagonist, it might lead to the development of therapeutic agents in FPRL1-mediated inflammatory components of diseases such as systemic amyloidosis, Alzheimer's, and prion disease.

Anti-Inflammatory Agents↗

PubMed2006

Bacterial complement evasion.

The human complement system is elemental to recognize bacteria, opsonize them for handling by phagocytes, or kill them by direct lysis. However, successful bacterial pathogens have in turn evolved ingenious strategies to overcome this part of the immune system. In this review we discuss the different stages of complement activation sequentially and illustrate the immune evasion strategies that various bacteria have developed to evade each subsequent step. The focus is on bacterial proteins, either surface-bound or excreted, that block complement activation. The underlying molecular mechanism of action and the possible role in pathophysiology of bacterial infections are discussed.

Animals↗

PubMed2006

Early expression of SCIN and CHIPS drives instant immune evasion by Staphylococcus aureus.

Chemotaxis inhibitory protein of staphylococci (CHIPS) and Staphylococcal complement inhibitor (SCIN) are small, excreted molecules that play a crucial role in the staphylococcal defence against the human innate immune system. Here we show that they both counteract crucial acute responses of our immune system such as complement activation, neutrophil chemotaxis and neutrophil activation. By studying gene expression via promoter-green fluorescent protein fusions, Northern blots and protein expression analyses, we show that SCIN and CHIPS are produced during the early (exponential) growth stages. Although the SCIN and CHIPS genes are expressed simultaneously, they are differently regulated by various Staphylococcus aureus regulatory loci. However, the sae locus is crucial for upregulation of both SCIN and CHIPS. This is the first study that presents the expression of two extracellular S. aureus proteins early during growth. Because SCIN and CHIPS are both efficient modulators of neutrophil chemotaxis, phagocytosis and killing, their early expression is necessary for efficient modulation of the early immune response.

Antigens, Bacterial↗

PubMed2006

Clumping factor A of Staphylococcus aureus inhibits phagocytosis by human polymorphonuclear leucocytes.

Staphylococcus aureus is a major cause of nosocomial and community-acquired infection. It expresses several factors that promote avoidance of phagocytosis by polymorphonuclear leucocytes. Clumping factor A (ClfA) is a fibrinogen-binding surface protein of S. aureus that is an important virulence factor in several infection models. This study investigated whether ClfA is an antiphagocytic factor, and whether its antiphagocytic properties were based on its ability to bind fibrinogen. In S. aureus, ClfA was shown to be of equal importance to protein A, the antiphagocytic properties of which are well established. ClfA expressed in a surrogate Gram-positive host was also found to be antiphagocytic. A ClfA mutant that was unable to bind fibrinogen had a similar antiphagocytic effect to native ClfA in the absence of fibrinogen. ClfA inhibited phagocytosis in the absence of fibrinogen, and showed enhanced inhibition in the presence of fibrinogen.

Coagulase↗

PubMed2006

The innate immune modulators staphylococcal complement inhibitor and chemotaxis inhibitory protein of Staphylococcus aureus are located on beta-hemolysin-converting bacteriophages.

Two newly discovered immune modulators, chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) and staphylococcal complement inhibitor (SCIN), cluster on the conserved 3' end of beta-hemolysin (hlb)-converting bacteriophages (betaC-phis). Since these betaC-phis also carry the genes for the immune evasion molecules staphylokinase (sak) and enterotoxin A (sea), this 8-kb region at the 3' end of betaC-phi represents an innate immune evasion cluster (IEC). By PCR and Southern analyses of 85 clinical Staphylococcus aureus strains and 5 classical laboratory strains, we show that 90% of S. aureus strains carry a betaC-phi with an IEC. Seven IEC variants were discovered, carrying different combinations of chp, sak, or sea (or sep), always in the same 5'-to-3' orientation and on the 3' end of a betaC-phi. From most IEC variants we could isolate active bacteriophages by mitomycin C treatment, of which lysogens were generated in S. aureus R5 (broad phage host). All IEC-carrying bacteriophages integrated into hlb, as was measured by Southern blotting of R5 lysogens. Large quantities of the different bacteriophages were obtained by mitomycin C treatment of the lysogens, and bacteriophages were collected and used to reinfect all lysogenic R5 strains. In total, five lytic families were found. Furthermore, phage DNA was isolated and digested with EcoR1, revealing that one IEC variant can be found on different betaI-phis. In conclusion, the four human-specific innate immune modulators SCIN, CHIPS, SAK, and SEA form an IEC that is easily transferred among S. aureus strains by a diverse group of beta-hemolysin-converting bacteriophages.

3' Flanking Region↗

PubMed2005

Staphylococcal innate immune evasion.

Upon entering the human body, bacteria are confronted with the sophisticated innate defense mechanisms of the human host. From work in recent years it has become obvious that a new and growing family of small and excreted proteins can counteract the antibacterial effects of innate immunity. These highly selective proteins pick out crucial elements of our immune system and inhibit their function. In Staphylococcus aureus these proteins act on specific cellular receptors, on antimicrobial peptides and especially on the complement system. The combined action of this growing group of essential virulence factors ascertains efficient innate immune evasion.

Humans↗

PubMed2005

The structure of the C5a receptor-blocking domain of chemotaxis inhibitory protein of Staphylococcus aureus is related to a group of immune evasive molecules.

The chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) is a 121 residue excreted virulence factor. It acts by binding the C5a- (C5aR) and formylated peptide receptor (FPR) and thereby blocks specific phagocyte responses. Here, we report the solution structure of a CHIPS fragment consisting of residues 31-121 (CHIPS31-121). CHIPS31-121 has the same activity in blocking the C5aR compared to full-length CHIPS, but completely lacks FPR antagonism. CHIPS31-121 has a compact fold comprising an alpha-helix (residues 38-51) packed onto a four-stranded anti-parallel beta-sheet. Strands beta2 and beta3 are joined by a long loop with a relatively well-defined conformation. Comparison of CHIPS31-121 with known structures reveals striking homology with the C-terminal domain of staphylococcal superantigen-like proteins (SSLs) 5 and 7, and the staphyloccocal and streptococcal superantigens TSST-1 and SPE-C. Also, the recently reported structures of several domains of the staphylococcal extracellullar adherence protein (EAP) show a high degree of structural similarity with CHIPS. Most of the conserved residues in CHIPS and its structural homologues are present in the alpha-helix. A conserved arginine residue (R46 in CHIPS) appears to be involved in preservation of the structure. Site-directed mutagenesis of all positively charged residues in CHIPS31-121 reveals a major involvement of arginine 44 and lysine 95 in C5aR antagonism. The structure of CHIPS31-121 will be vital in the further unraveling of its precise mechanism of action. Its structural homology to S.aureus SSLs, superantigens, and EAP might help the design of future experiments towards an understanding of the relationship between structure and function of these proteins.

Amino Acid Sequence↗

PubMed2005

Immune evasion by a staphylococcal complement inhibitor that acts on C3 convertases.

The complement system is pivotal in host defense but also contributes to tissue injury in several diseases. The assembly of C3 convertases (C4b2a and C3bBb) is a prerequisite for complement activation. The convertases catalyze C3b deposition on activator surfaces. Here we describe the identification of staphylococcal complement inhibitor, an excreted 9.8-kilodalton protein that blocks human complement by specific interaction with C4b2a and C3bBb. Staphylococcal complement inhibitor bound and stabilized C3 convertases, interfering with additional C3b deposition through the classical, lectin and alternative complement pathways. This led to a substantial decrease in phagocytosis and killing of Staphylococcus aureus by human neutrophils. As a highly active and small soluble protein that acts exclusively on surfaces, staphylococcal complement inhibitor may represent a promising anti-inflammatory molecule.

Bacterial Proteins↗

PubMed2004

N-terminal residues of the chemotaxis inhibitory protein of Staphylococcus aureus are essential for blocking formylated peptide receptor but not C5a receptor.

Staphylococcus aureus excretes a factor that specifically and simultaneously acts on the C5aR and the formylated peptide receptor (FPR). This chemotaxis inhibitory protein of S. aureus (CHIPS) blocks C5a- and fMLP-induced phagocyte activation and chemotaxis. Monoclonal anti-CHIPS Abs inhibit CHIPS activity against one receptor completely without affecting the other receptor, indicating that two distinct sites are responsible for both actions. A CHIPS-derived N-terminal 6 aa peptide is capable of mimicking the anti-FPR properties of CHIPS but has no effect on the C5aR. Synthetic peptides in which the first 6 aa are substituted individually for all other naturally occurring amino acids show that the first and third residue play an important role in blocking the FPR. Using an Escherichia coli expression system, we created mutant CHIPS proteins in which these amino acids are substituted. These mutant proteins have impaired or absent FPR- but still an intact C5aR-blocking activity, indicating that the loss of the FPR-blocking activity is not caused by any structural impairment. This identifies the first and third amino acid, both a phenylalanine, to be essential for CHIPS blocking the fMLP-induced activation of phagocytes. The unique properties of CHIPS to specifically inhibit the FPR with high affinity (kd=35.4 +/- 7.7 nM) could be an important new tool to further stimulate the fundamental research on the mechanisms underlying the FPR and its role in disease processes.

Amino Acid Sequence↗

PubMed2004

Chemotaxis inhibitory protein of Staphylococcus aureus binds specifically to the C5a and formylated peptide receptor.

Chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) is an exoprotein produced by several strains of S. aureus, and a potent inhibitor of neutrophil and monocyte chemotaxis toward C5a and formylated peptides like fMLP. These chemoattractants act on their target cells by binding and activating the C5aR and formylated peptide receptor (FPR), respectively. In the present report, we examined the mechanism by which CHIPS affects both of these receptors. We showed that CHIPS blocked binding of anti-C5aR mAb and formylated peptide to human neutrophils as efficiently at temperatures of 0 and 37 degrees C, implying that it is independent of signal transducing systems. This was confirmed by showing that CHIPS acts completely independently of ATP. Additionally, CHIPS was not internalized upon binding to neutrophils. Furthermore, we showed that CHIPS binds specifically to the C5aR and FPR expressed on U937 cells. This binding was functional in blocking C5a- and fMLP-induced calcium mobilization in these cell lines. These results suggest that CHIPS binds directly to the C5aR and FPR, thereby preventing the natural ligands from activating these receptors. The apparent K(d) values of CHIPS for the C5aR and FPR were 1.1 +/- 0.2 nM and 35.4 +/- 7.7 nM, respectively. Moreover, after screening a wide variety of other G protein-coupled receptors, CHIPS was found to affect exclusively the C5aR and FPR. This selectivity and high-affinity binding with potent antagonistic effects makes CHIPS a promising lead for the development of new anti-inflammatory compounds for diseases in which damage by neutrophils plays a key role.

Bacterial Proteins↗

PubMed2004

Spare CD14 molecules on human monocytes enhance the sensitivity for low LPS concentrations.

Human monocytes express on their plasma membrane relatively large number of CD14 molecules, known to play a crucial role in the lipopolisaccharide (LPS)-mediated cellular activation. Indirect data (J. Biol. Chem. 270 (1995) 9904) suggest that not all of these CD14 molecules participate in LPS-signaling, but the importance of these spare receptors and the exact number of CD14 involved in activation upon different LPS-stimuli is not known. Using different concentrations of a blocking anti-CD14 monoclonal antibody (mAb 60bca) we created monocytes with graded amounts of CD14. The exact number of occupied and free receptors was quantitated by flow cytometry and special mAb-labeled standard beads. The number of free CD14 molecules per monocyte in the presence of 10, 3.33, 0.73, 0.25 and 0.041 microg/ml mAb was 0, 13,100, 49,300, 97,700 and 165,900. Stimulation of these partially blocked monocytes with 0.1, 1, 10 and 100 ng/ml ReLPS in the presence of 3% human serum revealed that already 13,100 and 97,700 CD14 molecules provided a maximal Tumor necrosis factor alpha (TNFalpha) mRNA response using 100 and 10 ng/ml ReLPS, while the activation totally depended on the number of available CD14 molecules in the case of 1 and 0.1 ng/ml ReLPS. Our data imply that the number of CD14 molecules available for LPS-binding influence the cellular response. In the presence of higher concentrations of LPS only fractions of CD14 participate in the cell activation, while the presence of the spare receptors enhance the sensitivity against lower LPS amounts.

Antibodies, Monoclonal↗

PubMed2004

Chemotaxis inhibitory protein of Staphylococcus aureus, a bacterial antiinflammatory agent.

Leukocyte migration is a key event both in host defense against invading pathogens as well as in inflammation. Bacteria generate chemoattractants primarily by excretion (formylated peptides), complement activation (C5a), and subsequently through activation of leukocytes (e.g., leukotriene B4, platelet-activating factor, and interleukin 8). Here we describe a new protein secreted by Staphylococcus aureus that specifically impairs the response of neutrophils and monocytes to formylated peptides and C5a. This chemotaxis inhibitory protein of S. aureus (CHIPS) is a 14.1-kD protein encoded on a bacteriophage and is found in >60% of clinical isolates. CHIPS reduces the neutrophil recruitment toward C5a in a mouse peritonitis model, even though its activity is much more potent on human than on mouse cells. These findings suggest a new immune escape mechanism of S. aureus and put forward CHIPS as a potential new antiinflammatory therapeutic compound.

Amino Acid Sequence↗

PubMed2003

Lipoteichoic acid from Staphylococcus aureus is a potent stimulus for neutrophil recruitment.

Lipoteichoic acid (LTA) is a major immunostimulatory principle of Gram-positive bacteria. Intranasal application of LTA from S. aureus to mice resulted in greatly increased neutrophil and macrophage counts in the bronchoalveolar lavage as well as increased levels of the chemokine KC. The potential of highly pure, bioactive LTA from S. aureus to induce neutrophil recruitment and activation was investigated further in the human system. Although neutrophils expressed the key known receptors, CD14, TLR2 and TLR6, LTA did not induce or prime neutrophils for oxidative burst, or release of chemokines, bactericidal permeability-increasing protein or myeloperoxidase. However, LTA induced a strong release of the chemoattractants LTB4, IL-8, C5a, MCP-1 and the colony-stimulating factor G-CSF in whole blood comparable to stimulation with the same concentration of LPS (S. abortus equi). Further, the cytokine and chemoattractant pattern induced by LTA correlated well with that induced by live S. aureus of the same strain. LTA does not appear to activate neutrophils directly, but is a strong stimulus for the recruitment of phagocytes to the site of infection.

Animals↗

PubMed2003

Lipoprotein metabolism in patients with severe sepsis.

OBJECTIVE: Lipoproteins have been implicated to play a role in innate immunity. Changes in lipoprotein levels have been reported in a variety of inflammatory disorders. Not much is known about lipoprotein metabolism in patients with severe sepsis. We conducted an ancillary study in a multiple-center phase III sepsis trial to investigate the dynamics of plasma lipoproteins in patients with severe sepsis. DESIGN: Prospective analysis in patients meeting criteria for severe sepsis as part of a multiple-center sepsis study (KyberSept) with antithrombin III (Kybernin P). SETTING: University hospital intensive care unit. PATIENTS: Seventeen patients were included in the study. INTERVENTIONS: Randomized patients received a loading dose of 6000 IU of antithrombin III (Kybernin P) or placebo followed by a 96-hr continuous infusion of 250 IU/hr antithrombin III (Kybernin P) or placebo. In each patient, serial blood samples for total cholesterol, lipoprotein cholesterol, triglycerides, apolipoprotein A-1, apolipoprotein B, and C-reactive protein determination as well as clinical data were collected over 28 days. MEASUREMENTS AND MAIN RESULTS: Plasma cholesterol levels rapidly decreased from 2.67 +/- 2.02 mmol/L on day 0 to a nadir of 1.41 +/- 0.70 mmol/L on day 3, followed by a slow increase to 4.18 +/- 1.94 mmol/L on day 28. High-density lipoprotein (HDL) cholesterol concentrations decreased rapidly from 0.84 +/- 0.92 mmol/L to a nadir of 0.42 +/- 0.35 mmol/L on day 3, to show a slow increase during the following 4 wks to 0.84 +/- 0.42 mmol/L. The low-density lipoprotein (LDL) cholesterol concentrations were already low (0.94 +/- 0.81 mmol/L) at study entry, to show a progressive increase to subnormal values (2.01 +/- 0.94 mmol/L) at 4 wks. Nadir and recovery lipoprotein concentrations were significantly different (paired Student's t-test, p <.05). A significant correlation was found between HDL cholesterol and apolipoprotein A-1 (r =.714, p <.05) and between LDL cholesterol and apolipoprotein B (r =.733, p <.05). There was no statistical difference in lipoprotein concentrations either between survivors and nonsurvivors or between patients receiving antithrombin III or placebo. Serum amyloid A was a major apoprotein (45%) in HDL at the start of the sepsis and was slowly replaced by apolipoprotein A-1 during recovery. A positive correlation was found between plasma C-reactive protein concentrations and serum amyloid A concentrations in HDL (r =.684, p <.05). No other relevant correlations were found between inflammatory and lipoprotein parameters. CONCLUSIONS: In patients with severe sepsis, lipoprotein concentrations rapidly change and can be reduced to 50% of recovery concentrations. The pattern of early rapid decline is found primarily in the HDL and a slow recovery in both HDL and LDL fractions. The correlation between apolipoprotein and lipoprotein cholesterol concentrations suggests a decline in lipoprotein particles. During severe sepsis, HDL is shifted to acute phase HDL, which is enriched in serum amyloid A and depleted of cholesterol and apolipoprotein A-1. Lipoprotein concentrations are unable to discriminate between survivors and nonsurvivors.

APACHE↗