Complement Split Product C5a Mediates the Lipopolysaccharide‐Induced Mobilization of Cfu‐S and Haemopoietic Progenitor Cells, But Not the Mobilization Induced By Proteolytic Enzymes

Abstract. Intravenous (i.v.) injection of mice with lipopolysaccharide (LPS), and the proteolytic enzymes trypsin and proteinase, mobilizes pluripotent haemopoietic stem cells (CFU‐s) as well as granulocyte‐macrophage progenitor cells (GM‐CFU) and the early progenitors of the erythroid lineage (E‐BFU) from the haemopoietic tissues into the peripheral blood. We investigated the involvement of the complement (C) system in this process. It appeared that the early mobilization induced by LPS and other activators of the alternative complement pathway, such as Listeria monocytogenes (Lm) and zymosan, but not that induced by the proteolytic enzymes, was absent in C5‐deficient mice. the mobilization by C activators in these mice could be restored by injection of C5‐sufficient serum, suggesting a critical role for C5. The manner in which C5 was involved in the C activation‐mediated stem cell mobilization was studied using a serum transfer system. C5‐sufficient serum, activated in vitro by incubation with Lm and subsequently liberated from the bacteria, caused mobilization in both C5‐sufficient and C5‐deficient mice. C5‐deficient serum was not able to do so. the resistance of the mobilizing principle to heat treatment (56°C, 30 min) strongly suggests that it is identical with the C5 split product C5a, or an in vivo derivative of C5a. This conclusion was reinforced by the observation that a single injection of purified rat C5a into C5‐deficient mice also induced mobilization of CFU‐s. Copyrigh

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

Do not discard Staphylococcus aureus WTA as a vaccine antigen

NWO91713303Bio-organic Synthesi

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 α-helix (residues 38–51) packed onto a four-stranded anti-parallel β-sheet. Strands β2 and β3 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 α-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

Supplementary Material for: Effective Neutrophil Phagocytosis of <b><i>Aspergillus</i></b> <b><i>fumigatus</i></b> Is Mediated by Classical Pathway Complement Activation

<i>Aspergillus fumigatus</i> is an important airborne fungal pathogen and a major cause of invasive fungal infections. Susceptible individuals become infected via the inhalation of dormant conidia. If the immune system fails to clear these conidia, they will swell, germinate and grow into large hyphal structures. Neutrophils are essential effector cells for controlling <i>A. fumigatus</i> infection. In general, opsonization of microbial particles is crucial for efficient phagocytosis and killing by neutrophils. Although the antibodies present in human serum do bind to all fungal morphotypes, we observed no direct antibody-mediated phagocytosis of <i>A. fumigatus</i>. We show that opsonization, phagocytosis and killing by neutrophils of <i>A. fumigatus</i> is complement-dependent. Using human sera depleted of key complement components, we investigated the contribution of the different complement initiation pathways in complement activation on the fungal surface. We describe the classical complement pathway as the main initiator of complement activation on <i>A. fumigatus</i> swollen conidia and germ tubes. Antibodies play an important role in complement activation and efficient innate recognition, phagocytosis and killing of <i>A. fumigatus</i> by neutrophils

Pseudomonas evades immune recognition of flagellin in both mammals and plants

The building blocks of bacterial flagella, flagellin monomers, are potent stimulators of host innate immune systems. Recognition of flagellin monomers occurs by flagellin-specific pattern-recognition receptors, such as Toll-like receptor 5 (TLR5) in mammals and flagellin-sensitive 2 (FLS2) in plants. Activation of these immune systems via flagellin leads eventually to elimination of the bacterium from the host. In order to prevent immune activation and thus favor survival in the host, bacteria secrete many proteins that hamper such recognition. In our search for Toll like receptor (TLR) antagonists, we screened bacterial supernatants and identified alkaline protease (AprA) of Pseudomonas aeruginosa as a TLR5 signaling inhibitor as evidenced by a marked reduction in IL-8 production and NF-κB activation. AprA effectively degrades the TLR5 ligand monomeric flagellin, while polymeric flagellin (involved in bacterial motility) and TLR5 itself resist degradation. The natural occurring alkaline protease inhibitor AprI of P. aeruginosa blocked flagellin degradation by AprA. P. aeruginosa aprA mutants induced an over 100-fold enhanced activation of TLR5 signaling, because they fail to degrade excess monomeric flagellin in their environment. Interestingly, AprA also prevents flagellin-mediated immune responses (such as growth inhibition and callose deposition) in Arabidopsis thaliana plants. This was due to decreased activation of the receptor FLS2 and clearly demonstrated by delayed stomatal closure with live bacteria in plants. Thus, by degrading the ligand for TLR5 and FLS2, P. aeruginosa escapes recognition by the innate immune systems of both mammals and plants

Staphylococcus aureus Staphopain A inhibits CXCR2-dependent neutrophil activation and chemotaxis

The CXC chemokine receptor 2 (CXCR2) on neutrophils, which recognizes chemokines produced at the site of infection, plays an important role in antimicrobial host defenses such as neutrophil activation and chemotaxis. Staphylococcus aureus is a successful human pathogen secreting a number of proteolytic enzymes, but their influence on the host immune system is not well understood. Here, we identify the cysteine protease Staphopain A as a chemokine receptor blocker. Neutrophils treated with Staphopain A are unresponsive to activation by all unique CXCR2 chemokines due to cleavage of the N-terminal domain, which can be neutralized by specific protease inhibitors. Moreover, Staphopain A inhibits neutrophil migration towards CXCR2 chemokines. By comparing a methicillin-resistant S. aureus (MRSA) strain with an isogenic Staphopain A mutant, we demonstrate that Staphopain A is the only secreted protease with activity towards CXCR2. Although the inability to cleave murine CXCR2 limits in-vivo studies, our data indicate that Staphopain A is an important immunomodulatory protein that blocks neutrophil recruitment by specific cleavage of the N-terminal domain of human CXCR2

Supplementary Material for: The TLR2 Antagonist Staphylococcal Superantigen-Like Protein 3 Acts as a Virulence Factor to Promote Bacterial Pathogenicity in vivo

Toll-like receptor (TLR) signaling is important in the initiation of immune responses and subsequent instigation of adaptive immunity. TLR2 recognizes bacterial lipoproteins and plays a central role in the host defense against bacterial infections, including those caused by <i>Staphylococcus aureus</i>. Many studies have demonstrated the importance of TLR2 in murine <i>S. aureus</i> infection. <i>S. aureus</i> evades TLR2 activation by secreting two proteins, staphylococcal superantigen-like protein 3 (SSL3) and 4 (SSL4). In this study, we demonstrate that antibodies against SSL3 and SSL4 are found in healthy individuals, indicating that humans are exposed to these proteins during <i>S. aureus</i> colonization or infection. To investigate the TLR2-antagonistic properties of SSL3 and SSL4, we compared the infection with wild-type and SSL3/4 knockout <i>S. aureus</i> strains in an intravenous murine infection model. Direct evaluation of the contribution of SSL3/4 to infection pathogenesis was hindered by the fact that the SSLs were not expressed in the murine system. To circumvent this limitation, an SSL3-overproducing strain (pLukM-SSL3) was generated, resulting in constitutive expression of SSL3. pLukM-SSL3 exhibited increased virulence compared to the parental strain in a murine model that was found to be TLR2 dependent. Altogether, these data indicate that SSL3 contributes to <i>S. aureus</i> virulence in vivo