RESUMEN
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.
Asunto(s)
Antiinflamatorios no Esteroideos/aislamiento & purificación , Antiinflamatorios no Esteroideos/farmacología , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/farmacología , Quimiotaxis de Leucocito/efectos de los fármacos , Staphylococcus aureus/inmunología , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/genética , Secuencia de Bases , Complemento C5a/farmacología , ADN Bacteriano/genética , Genes Bacterianos , Humanos , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Especificidad de la Especie , Staphylococcus aureus/genéticaRESUMEN
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.
Asunto(s)
Receptores de Lipopolisacáridos/inmunología , Lipopolisacáridos/inmunología , Monocitos/inmunología , Anticuerpos Monoclonales/inmunología , Cinética , Monocitos/metabolismo , ARN Mensajero/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
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.
Asunto(s)
Rodamiento de Leucocito/efectos de los fármacos , Glicoproteínas de Membrana/metabolismo , Neutrófilos/efectos de los fármacos , Neutrófilos/fisiología , Selectina-P/metabolismo , Staphylococcus aureus/patogenicidad , Superantígenos/metabolismo , Superantígenos/toxicidad , Animales , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/toxicidad , Secuencia de Bases , Unión Competitiva , Células CHO , Células Cultivadas , Clonación Molecular , Cricetinae , Cricetulus , ADN Bacteriano/genética , Exotoxinas/metabolismo , Exotoxinas/toxicidad , Humanos , Técnicas In Vitro , Rodamiento de Leucocito/fisiología , Unión Proteica , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/toxicidad , Staphylococcus aureus/genética , Staphylococcus aureus/inmunología , Superantígenos/genéticaRESUMEN
Chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) is excreted by the majority of S. aureus strains and is a potent inhibitor of C5a- and formylated peptide-mediated chemotaxis of neutrophils and monocytes. Recently, we reported that CHIPS binds to the C5a receptor (C5aR) and the formylated peptide receptor, thereby blocking activation by C5a and formylated peptides, respectively. The anaphylatoxin C5a plays an important role in host immunity and pathological inflammatory processes. For C5a a two-site binding model is proposed in which C5a initially binds the C5aR N terminus, followed by interaction of the C5a C-terminal tail with an effector domain on the receptor. We have shown here that CHIPS does not affect activation of the C5aR by a peptide mimic of the C5a C terminus. Moreover, CHIPS was found to bind human embryonic kidney 293 cells expressing only the C5aR N terminus. Deletion and mutation experiments within this C5aR N-terminal expression system revealed that the binding site of CHIPS is contained in a short stretch of 9 amino acids (amino acids 10-18), of which the aspartic acid residues at positions 10, 15, and 18 plus the glycine at position 12 are crucial. Binding studies with C5aR/C3aR and C5aR/IL8RA chimeras confirmed that CHIPS binds only to the C5aR N terminus without involvement of its extracellular loops. CHIPS may provide new strategies to block the C5aR, which may lead to the development of new C5aR antagonists.
Asunto(s)
Proteínas Bacterianas/metabolismo , Receptor de Anafilatoxina C5a/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Complemento C5a/metabolismo , Cartilla de ADN , Vectores Genéticos , Humanos , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Receptor de Anafilatoxina C5a/química , Receptor de Anafilatoxina C5a/genéticaRESUMEN
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.