RESUMO
IgG molecules are crucial for the human immune response against bacterial infections. IgGs can trigger phagocytosis by innate immune cells, like neutrophils. To do so, IgGs should bind to the bacterial surface via their variable Fab regions and interact with Fcγ receptors and complement C1 via the constant Fc domain. C1 binding to IgG-labeled bacteria activates the complement cascade, which results in bacterial decoration with C3-derived molecules that are recognized by complement receptors on neutrophils. Next to FcγRs and complement receptors on the membrane, neutrophils also express the intracellular neonatal Fc receptor (FcRn). We previously reported that staphylococcal protein A (SpA), a key immune-evasion protein of Staphylococcus aureus, potently blocks IgG-mediated complement activation and killing of S. aureus by interfering with IgG hexamer formation. SpA is also known to block IgG-mediated phagocytosis in absence of complement, but the mechanism behind it remains unclear. In this study, we demonstrate that SpA blocks IgG-mediated phagocytosis and killing of S. aureus and that it inhibits the interaction of IgGs with FcγRs (FcγRIIa and FcγRIIIb, but not FcγRI) and FcRn. Furthermore, our data show that multiple SpA domains are needed to effectively block IgG1-mediated phagocytosis. This provides a rationale for the fact that SpA from S. aureus contains four to five repeats. Taken together, our study elucidates the molecular mechanism by which SpA blocks IgG-mediated phagocytosis and supports the idea that in addition to FcγRs, the intracellular FcRn is also prevented from binding IgG by SpA.
Assuntos
Imunoglobulina G , Fagocitose , Receptores de IgG , Proteína Estafilocócica A , Staphylococcus aureus , Complemento C1 , Humanos , Imunoglobulina G/imunologia , Receptores de Complemento , Receptores de IgG/metabolismo , Proteína Estafilocócica A/metabolismoRESUMO
Immunoglobulin (Ig) G molecules are essential players in the human immune response against bacterial infections. An important effector of IgG-dependent immunity is the induction of complement activation, a reaction that triggers a variety of responses that help kill bacteria. Antibody-dependent complement activation is promoted by the organization of target-bound IgGs into hexamers that are held together via noncovalent Fc-Fc interactions. Here we show that staphylococcal protein A (SpA), an important virulence factor and vaccine candidate of Staphylococcus aureus, effectively blocks IgG hexamerization and subsequent complement activation. Using native mass spectrometry and high-speed atomic force microscopy, we demonstrate that SpA blocks IgG hexamerization through competitive binding to the Fc-Fc interaction interface on IgG monomers. In concordance, we show that SpA interferes with the formation of (IgG)6:C1q complexes and prevents downstream complement activation on the surface of S. aureus. Finally, we demonstrate that IgG3 antibodies against S. aureus can potently induce complement activation and opsonophagocytic killing even in the presence of SpA. Together, our findings identify SpA as an immune evasion protein that specifically blocks IgG hexamerization.
Assuntos
Ativação do Complemento , Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoglobulina G/metabolismo , Multimerização Proteica , Proteína Estafilocócica A/metabolismo , Sítios de Ligação , Células Cultivadas , Humanos , Fagócitos/imunologia , Fagocitose , Ligação Proteica , Staphylococcus aureus/imunologiaRESUMO
Complement is an important effector mechanism for antibody-mediated clearance of infections and tumor cells. Upon binding to target cells, the antibody's constant (Fc) domain recruits complement component C1 to initiate a proteolytic cascade that generates lytic pores and stimulates phagocytosis. The C1 complex (C1qr2s2) consists of the large recognition protein C1q and a heterotetramer of proteases C1r and C1s (C1r2s2). While interactions between C1 and IgG-Fc are believed to be mediated by the globular heads of C1q, we here find that C1r2s2 proteases affect the capacity of C1q to form an avid complex with surface-bound IgG molecules (on various 2,4-dinitrophenol [DNP]-coated surfaces and pathogenic Staphylococcus aureus). The extent to which C1r2s2 contributes to C1q-IgG stability strongly differs between human IgG subclasses. Using antibody engineering of monoclonal IgG, we reveal that hexamer-enhancing mutations improve C1q-IgG stability, both in the absence and presence of C1r2s2 In addition, hexamer-enhanced IgGs targeting S. aureus mediate improved complement-dependent phagocytosis by human neutrophils. Altogether, these molecular insights into complement binding to surface-bound IgGs could be important for optimal design of antibody therapies.
Assuntos
Membrana Celular/metabolismo , Complemento C1q/metabolismo , Complemento C1r/metabolismo , Complemento C1s/metabolismo , Imunoglobulina G/metabolismo , Ativação do Complemento , Humanos , Microscopia de Força Atômica , Mutação/genética , Fagocitose , Ligação Proteica , Multimerização Proteica , Estabilidade Proteica , Staphylococcus aureus/imunologiaRESUMO
Neutrophils are critical to the generation of effective immune responses and for killing invading microbes. Paired immune receptors provide important mechanisms to modulate neutrophil activation thresholds and effector functions. Expression of the leukocyte Ig-like receptor (LILR)A6 (ILT8/CD85b) and LILRB3 (ILT5/CD85a) paired-receptor system on human neutrophils has remained unclear because of the lack of specific molecular tools. Additionally, there is little known of their possible functions in neutrophil biology. The objective of this study was to characterize expression of LILRA6/LILRB3 receptors during human neutrophil differentiation and activation, and to assess their roles in modulating Fc receptor-mediated effector functions. LILRB3, but not LILRA6, was detected in human neutrophil lysates following immunoprecipitation by mass spectrometry. We demonstrate high LILRB3 expression on the surface of resting neutrophils and release from the surface following neutrophil activation. Surface expression was recapitulated in a human PLB-985 cell model of neutrophil-like differentiation. Continuous ligation of LILRB3 inhibited key IgA-mediated effector functions, including production of reactive oxygen species, phagocytic uptake, and microbial killing. This suggests that LILRB3 provides an important checkpoint to control human neutrophil activation and their antimicrobial effector functions during resting and early-activation stages of the neutrophil life cycle.
Assuntos
Antígenos CD/metabolismo , Neutrófilos/imunologia , Receptores Fc/metabolismo , Receptores Imunológicos/metabolismo , Infecções Estafilocócicas/imunologia , Antígenos CD/genética , Antígenos CD/isolamento & purificação , Diferenciação Celular/imunologia , Linhagem Celular , Regulação para Baixo/imunologia , Humanos , Ativação de Neutrófilo , Neutrófilos/metabolismo , Fagocitose , Cultura Primária de Células , Espécies Reativas de Oxigênio/metabolismo , Receptores Imunológicos/genética , Receptores Imunológicos/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Infecções Estafilocócicas/microbiologia , Staphylococcus capitis/imunologiaRESUMO
Staphylococcus aureus Panton-Valentine leukocidin is a pore-forming toxin targeting the human C5a receptor (hC5aR), enabling this pathogen to battle the immune response by destroying phagocytes through targeted lysis. The mechanisms that contribute to rapid cell lysis are largely unexplored. Here, we show that cell lysis may be enabled by a process of toxins targeting receptor clusters and present indirect evidence for receptor "recycling" that allows multiple toxin pores to be formed close together. With the use of live cell single-molecule super-resolution imaging, Förster resonance energy transfer and nanoscale total internal reflection fluorescence colocalization microscopy, we visualized toxin pore formation in the presence of its natural docking ligand. We demonstrate disassociation of hC5aR from toxin complexes and simultaneous binding of new ligands. This effect may free mobile receptors to amplify hyperinflammatory reactions in early stages of microbial infections and have implications for several other similar bicomponent toxins and the design of new antibiotics.-Haapasalo, K., Wollman, A. J. M., de Haas, C. J. C., van Kessel, K. P. M., van Strijp, J. A. G., Leake, M. C. Staphylococcus aureus toxin LukSF dissociates from its membrane receptor target to enable renewed ligand sequestration.
Assuntos
Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Exotoxinas/metabolismo , Leucocidinas/metabolismo , Receptores de Superfície Celular/metabolismo , Infecções Estafilocócicas/metabolismo , Staphylococcus aureus/metabolismo , Linhagem Celular , Humanos , Ligantes , Fagócitos , Receptor da Anafilatoxina C5a/metabolismoRESUMO
Staphylococcus aureus is highly adapted to its host and has evolved many strategies to resist opsonization and phagocytosis. Even after uptake by neutrophils, S. aureus shows resistance to killing, which suggests the presence of phagosomal immune evasion molecules. With the aid of secretome phage display, we identified a highly conserved protein that specifically binds and inhibits human myeloperoxidase (MPO), a major player in the oxidative defense of neutrophils. We have named this protein "staphylococcal peroxidase inhibitor" (SPIN). To gain insight into inhibition of MPO by SPIN, we solved the cocrystal structure of SPIN bound to a recombinant form of human MPO at 2.4-Å resolution. This structure reveals that SPIN acts as a molecular plug that prevents H2O2 substrate access to the MPO active site. In subsequent experiments, we observed that SPIN expression increases inside the neutrophil phagosome, where MPO is located, compared with outside the neutrophil. Moreover, bacteria with a deleted gene encoding SPIN showed decreased survival compared with WT bacteria after phagocytosis by neutrophils. Taken together, our results demonstrate that S. aureus secretes a unique proteinaceous MPO inhibitor to enhance survival by interfering with MPO-mediated killing.
Assuntos
Peroxidase/antagonistas & inibidores , Proteínas de Bactérias , Regulação Bacteriana da Expressão Gênica , Humanos , Modelos Moleculares , Neutrófilos/fisiologia , Fagocitose , Ligação Proteica , Conformação Proteica , Staphylococcus aureus/metabolismo , Regulação para CimaRESUMO
The heme-containing enzyme myeloperoxidase (MPO) is critical for optimal antimicrobial activity of human neutrophils. We recently discovered that the bacterium Staphylococcus aureus expresses a novel immune evasion protein, called SPIN, that binds tightly to MPO, inhibits MPO activity, and contributes to bacterial survival following phagocytosis. A co-crystal structure of SPIN bound to MPO suggested that SPIN blocks substrate access to the catalytic heme by inserting an N-terminal ß-hairpin into the MPO active-site channel. Here, we describe a series of experiments that more completely define the structure/function relationships of SPIN. Whereas the SPIN N terminus adopts a ß-hairpin confirmation upon binding to MPO, the solution NMR studies presented here are consistent with this region of SPIN being dynamically structured in the unbound state. Curiously, whereas the N-terminal ß-hairpin of SPIN accounts for â¼55% of the buried surface area in the SPIN-MPO complex, its deletion did not significantly change the affinity of SPIN for MPO but did eliminate the ability of SPIN to inhibit MPO. The flexible nature of the SPIN N terminus rendered it susceptible to proteolytic degradation by a series of chymotrypsin-like proteases found within neutrophil granules, thereby abrogating SPIN activity. Degradation of SPIN was prevented by the S. aureus immune evasion protein Eap, which acts as a selective inhibitor of neutrophil serine proteases. Together, these studies provide insight into MPO inhibition by SPIN and suggest possible functional synergy between two distinct classes of S. aureus immune evasion proteins.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Peroxidase/química , Peroxidase/metabolismo , Infecções Estafilocócicas/enzimologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/metabolismo , Motivos de Aminoácidos , Proteínas de Bactérias/genética , Cristalografia por Raios X , Humanos , Espectroscopia de Ressonância Magnética , Peroxidase/genética , Ligação Proteica , Staphylococcus aureus/química , Staphylococcus aureus/genéticaRESUMO
We implement a general imaging method by measuring the complex degree of coherence using linear optics and photon number resolving detectors. In the absence of collective or entanglement-assisted measurements, our method is optimal over a large range of practically relevant values of the complex degree of coherence. We measure the size and position of a small distant source of pseudothermal light, and show that our method outperforms the traditional imaging method by an order of magnitude in precision. Finally, we show that a lack of photon-number resolution in the detectors has only a modest detrimental effect on measurement precision and simulate imaging using the new and traditional methods with an array of detectors, showing that the new method improves both image clarity and contrast.
RESUMO
Staphylococcal superantigen-like (SSL) proteins, one of the major virulence factor families produced by Staphylococcus aureus, were previously demonstrated to be immune evasion molecules that interfere with a variety of innate immune defences. However, in contrast to characterised SSLs, which inhibit immune functions, we show that SSL13 is a strong activator of neutrophils via the formyl peptide receptor 2 (FPR2). Moreover, our data show that SSL13 acts as a chemoattractant and induces degranulation and oxidative burst in neutrophils. As with many other staphylococcal immune evasion proteins, SSL13 shows a high degree of human specificity. SSL13 is not able to efficiently activate mouse neutrophils, hampering in vivo experiments. In conclusion, SSL13 is a neutrophil chemoattractant and activator that acts via FPR2. Therefore, SSL13 is a unique SSL member that does not belong to the immune evasion class but is a pathogen alarming molecule. Our study provides a new concept of SSLs; SSLs not only inhibit host immune processes but also recruit human neutrophils to the site of infection. This new insight allows us to better understand complex interactions between host and S. aureus pathological processes.
Assuntos
Proteínas de Bactérias/metabolismo , Neutrófilos/microbiologia , Receptores de Formil Peptídeo/metabolismo , Receptores de Lipoxinas/metabolismo , Staphylococcus aureus/patogenicidade , Fatores de Virulência/metabolismo , Animais , Proteínas de Bactérias/genética , Degranulação Celular , Fatores Quimiotáticos/metabolismo , Feminino , Células HL-60 , Humanos , Evasão da Resposta Imune , Camundongos Endogâmicos , Ativação de Neutrófilo , Neutrófilos/fisiologia , Peritonite/metabolismo , Peritonite/microbiologia , Explosão Respiratória , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/patologiaRESUMO
We report strongly nonreciprocal behavior for quantum dot exciton spins coupled to nanophotonic waveguides under resonant laser excitation. A clear dependence of the transmission spectrum on the propagation direction is found for a chirally coupled quantum dot, with spin up and spin down exciton spins coupling to the left and right propagation directions, respectively. The reflection signal shows an opposite trend to the transmission, which a numerical model indicates is due to direction-selective saturation of the quantum dot. The chiral spin-photon interface we demonstrate breaks reciprocity of the system and opens the way to spin-based quantum optical components such as optical diodes and circulators in a chip-based solid-state environment.
RESUMO
Staphylococcus aureus and related species are highly adapted to their hosts and have evolved numerous strategies to evade the immune system. S. aureus shows resistance to killing following uptake into the phagosome, which suggests that the bacterium evades intracellular killing mechanisms used by neutrophils. We recently discovered an S. aureus protein (SPIN for Staphylococcal Peroxidase INhibitor) that binds to and inhibits myeloperoxidase (MPO), a major player in the oxidative defense of neutrophils. To allow for comparative studies between multiple SPIN sequences, we identified a panel of homologs from species closely related to S. aureus. Characterization of these proteins revealed that SPIN molecules from S. agnetis, S. delphini, S. schleiferi, and S. intermedius all bind human MPO with nanomolar affinities, and that those from S. delphini, S. schleiferi, and S. intermedius inhibit human MPO in a dose-dependent manner. A 2.4â¯Å resolution co-crystal structure of SPIN-delphini bound to recombinant human MPO allowed us to identify conserved structural features of SPIN proteins, and to propose sequence-dependent physical explanations for why SPIN-aureus binds human MPO with higher affinity than SPIN-delphini. Together, these studies expand our understanding of MPO binding and inhibition by a recently identified component of the staphylococcal innate immune evasion arsenal.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Peroxidase/antagonistas & inibidores , Staphylococcus/química , Sequência de Aminoácidos , Inibidores Enzimáticos/metabolismo , Humanos , Modelos Moleculares , Peroxidase/química , Peroxidase/metabolismo , Conformação ProteicaRESUMO
Toll-like receptors (TLRs) are crucial in innate recognition of invading micro-organisms and their subsequent clearance. Bacteria are not passive bystanders and have evolved complex evasion mechanisms. Staphylococcus aureus secretes a potent TLR2 antagonist, staphylococcal superantigen-like protein 3 (SSL3), which prevents receptor stimulation by pathogen-associated lipopeptides. Here, we present crystal structures of SSL3 and its complex with TLR2. The structure reveals that formation of the specific inhibitory complex is predominantly mediated by hydrophobic contacts between SSL3 and TLR2 and does not involve interaction of TLR2-glycans with the conserved Lewis(X) binding site of SSL3. In the complex, SSL3 partially covers the entrance to the lipopeptide binding pocket in TLR2, reducing its size by â¼50%. We show that this is sufficient to inhibit binding of agonist Pam2CSK4 effectively, yet allows SSL3 to bind to an already formed TLR2-Pam2CSK4 complex. The binding site of SSL3 overlaps those of TLR2 dimerization partners TLR1 and TLR6 extensively. Combined, our data reveal a robust dual mechanism in which SSL3 interferes with TLR2 activation at two stages: by binding to TLR2, it blocks ligand binding and thus inhibits activation. Second, by interacting with an already formed TLR2-lipopeptide complex, it prevents TLR heterodimerization and downstream signaling.
Assuntos
Endotoxinas/fisiologia , Staphylococcus aureus/fisiologia , Receptor 2 Toll-Like/antagonistas & inibidores , Dimerização , Endotoxinas/química , Endotoxinas/genética , Estrutura Molecular , Mutagênese , Ligação Proteica , Receptor 2 Toll-Like/químicaRESUMO
BACKGROUND: The enhanced liver fibrosis (ELF) test has been introduced to screen, diagnose and/or monitor liver conditions in large groups of patients with liver diseases. It has not been used in inflammatory skin or joint diseases. OBJECTIVES: To evaluate the distribution of the ELF test, apply existing cut-offs for hepatic patients and healthy controls, and compare it with the procollagen-3 N-terminal peptide (P3NP) test in patients with psoriasis (PSO), psoriatic arthritis (PsA) and rheumatoid arthritis (RA), and controls. METHODS: In total, 531 patients were included. Demographic, lifestyle and disease-specific data were collected. ELF and P3NP tests were performed. RESULTS: Prevalence of an increased ELF score (> 11) and P3NP was highest in patients with RA (7·7% and 6·1%, respectively) followed by patients with PSO (1·7% and 5·2%, respectively) and PsA (0·7% and 1·3%, respectively). Mean ± SD ELF scores for PSO, PsA and RA were, respectively, 9·09 ± 0·86, 8·96 ± 0·76 and 9·55 ± 1·04. All subgroups with moderate-to-severe disease severity had higher (> 9·8) ELF scores (PSO 27·0% vs. 18·3%; PsA 19·2% vs. 12%; RA 45·8% vs. 30·5%) and P3NP values. Distribution of the ELF score was smaller than the P3NP value [mean ± SD: 9·15 ± 0·92 (range 6·53-13·05) vs. 8·37 ± 4·30 (range 0·53-63·88)]. CONCLUSIONS: ELF score and P3NP are elevated in PSO, PsA and RA. ELF may be superior to P3NP alone, but further research should be done to validate the ELF test in determining susceptibility for developing liver fibrosis in PSO, PsA and RA.
Assuntos
Artrite Reumatoide/complicações , Cirrose Hepática/diagnóstico , Fragmentos de Peptídeos/metabolismo , Pró-Colágeno/metabolismo , Psoríase/complicações , Idoso , Artrite Psoriásica/complicações , Biomarcadores/metabolismo , Estudos Transversais , Feminino , Humanos , Testes de Função Hepática , Masculino , Pessoa de Meia-IdadeRESUMO
Staphylococcus aureus is well adapted to the human host. Evasion of the host phagocyte response is critical for successful infection. The staphylococcal bicomponent pore-forming toxins Panton-Valentine leukocidin LukSF-PV (PVL) and γ-hemolysin CB (HlgCB) target human phagocytes through interaction with the complement receptors C5aR1 and C5aR2. Currently, the apparent redundancy of both toxins cannot be adequately addressed in experimental models of infection because mice are resistant to PVL and HlgCB. The molecular basis for species specificity of the two toxins in animal models is not completely understood. We show that PVL and HlgCB feature distinct activity toward neutrophils of different mammalian species, where activity of PVL is found to be restricted to fewer species than that of HlgCB. Overexpression of various mammalian C5a receptors in HEK cells confirms that cytotoxicity toward neutrophils is driven by species-specific interactions of the toxins with C5aR1. By taking advantage of the species-specific engagement of the toxins with their receptors, we demonstrate that PVL and HlgCB differentially interact with human C5aR1 and C5aR2. In addition, binding studies illustrate that different parts of the receptor are involved in the initial binding of the toxin and the subsequent formation of lytic pores. These findings allow a better understanding of the molecular mechanism of pore formation. Finally, we show that the toxicity of PVL, but not of HlgCB, is neutralized by various C5aR1 antagonists. This study offers directions for the development of improved preclinical models for infection, as well as for the design of drugs antagonizing leukocidin toxicity.
Assuntos
Proteínas de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Exotoxinas/imunologia , Proteínas Hemolisinas/imunologia , Leucocidinas/imunologia , Receptor da Anafilatoxina C5a/imunologia , Receptores de Quimiocinas/imunologia , Sequência de Aminoácidos , Animais , Bovinos , Linhagem Celular , Células HEK293 , Humanos , Evasão da Resposta Imune/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Neutrófilos/imunologia , Fagócitos/imunologia , Ligação Proteica , Estrutura Terciária de Proteína , Receptor da Anafilatoxina C5a/antagonistas & inibidores , Receptores de Quimiocinas/antagonistas & inibidores , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/patogenicidadeRESUMO
Neutrophils are indispensable for clearing infections with the prominent human pathogen Staphylococcus aureus. Here, we report that S. aureus secretes a family of proteins that potently inhibits the activity of neutrophil serine proteases (NSPs): neutrophil elastase (NE), proteinase 3, and cathepsin G. The NSPs, but not related serine proteases, are specifically blocked by the extracellular adherence protein (Eap) and the functionally orphan Eap homologs EapH1 and EapH2, with inhibitory-constant values in the low-nanomolar range. Eap proteins are together essential for NSP inhibition by S. aureus in vitro and promote staphylococcal infection in vivo. The crystal structure of the EapH1/NE complex showed that Eap molecules constitute a unique class of noncovalent protease inhibitors that occlude the catalytic cleft of NSPs. These findings increase our insights into the complex pathogenesis of S. aureus infections and create opportunities to design novel treatment strategies for inflammatory conditions related to excessive NSP activity.
Assuntos
Neutrófilos/metabolismo , Inibidores de Serina Proteinase/metabolismo , Staphylococcus aureus/metabolismo , Animais , Aderência Bacteriana , Proteínas de Bactérias/metabolismo , Biocatálise , Espaço Extracelular/metabolismo , Feminino , Humanos , Elastase de Leucócito/antagonistas & inibidores , Elastase de Leucócito/metabolismo , Camundongos Endogâmicos C57BL , Modelos Moleculares , Infecções Estafilocócicas/patologiaRESUMO
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.
Assuntos
Proteínas de Bactérias/imunologia , Cisteína Endopeptidases/imunologia , Neutrófilos/imunologia , Receptores de Interleucina-8B/imunologia , Animais , Células Cultivadas , Quimiotaxia de Leucócito/imunologia , Feminino , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Ativação de Neutrófilo/imunologia , Infiltração de Neutrófilos/imunologia , Receptores de Interleucina-8B/antagonistas & inibidores , Células U937RESUMO
Staphylococcus aureus has developed many mechanisms to escape from human immune responses. To resist phagocytic clearance, S. aureus expresses a polysaccharide capsule, which effectively masks the bacterial surface and surface-associated proteins, such as opsonins, from recognition by phagocytic cells. Additionally, secretion of the extracellular fibrinogen binding protein (Efb) potently blocks phagocytic uptake of the pathogen. Efb creates a fibrinogen shield surrounding the bacteria by simultaneously binding complement C3b and fibrinogen at the bacterial surface. By means of neutrophil phagocytosis assays with fluorescently labelled encapsulated serotype 5 (CP5) and serotype 8 (CP8) strains we compare the immune-modulating function of these shielding mechanisms. The data indicate that, in highly encapsulated S. aureus strains, the polysaccharide capsule is able to prevent phagocytic uptake at plasma concentrations <10 %, but loses its protective ability at higher concentrations of plasma. Interestingly, Efb shows a strong inhibitory effect on both capsule-negative and encapsulated strains at all tested plasma concentrations. Furthermore, the results suggest that both shielding mechanisms can exist simultaneously and collaborate to provide optimal protection against phagocytosis at a broad range of plasma concentrations. As opsonizing antibodies will be shielded from recognition by either mechanism, incorporating both capsular polysaccharides and Efb in future vaccines could be of great importance.
Assuntos
Cápsulas Bacterianas/metabolismo , Proteínas de Bactérias/metabolismo , Fibrinogênio/metabolismo , Neutrófilos/imunologia , Fagocitose/imunologia , Polissacarídeos Bacterianos/metabolismo , Staphylococcus aureus/imunologia , Anticorpos Antibacterianos/imunologia , Complemento C3b/metabolismo , Corantes Fluorescentes , Humanos , Imunomodulação/imunologia , Microscopia Confocal , Proteínas Opsonizantes/metabolismo , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/patologia , Staphylococcus aureus/metabolismoRESUMO
Matrix metalloproteinases (MMPs) are endopeptidases that degrade components of the extracellular matrix, but also modulate inflammation. During bacterial infections, MMPs are important in the recruitment and migration of inflammatory cells. Besides facilitating cell migration by degrading extracellular matrix components, they potentiate the action of several inflammatory molecules, including cytokines, chemokines, and antimicrobial peptides. Staphylococcus aureus secretes an arsenal of immune evasion molecules that interfere with immune cell functioning and hamper proper immune responses. An earlier study identified staphylococcal superantigen-like protein 5 (SSL5) as an MMP9 inhibitor. Since multiple MMPs are involved in neutrophil recruitment, we set up an in-depth search for additional MMP inhibitors by testing a panel of over 70 secreted staphylococcal proteins on the inhibition of the two main neutrophil MMPs: MMP8 (neutrophil collagenase) and MMP9 (neutrophil gelatinase B). We identified SSL1 and SSL5 as potent inhibitors of both neutrophil MMPs and show that they are actually broad range MMP inhibitors. SSL1 and SSL5 prevent MMP-induced cleavage and potentiation of IL-8 and inhibit the migration of neutrophils through collagen. Thus, through MMP-inhibition, SSL1 and SSL5 interfere with neutrophil activation, chemotaxis, and migration, all vital neutrophil functions in bacterial clearance. Studies on MMP-SSL interactions can have therapeutic potential and SSL based derivatives might prove useful in treatment of cancer and destructive inflammatory diseases.
Assuntos
Proteínas de Bactérias/metabolismo , Metaloproteinases da Matriz/metabolismo , Staphylococcus aureus/metabolismo , Proteínas de Bactérias/farmacologia , Movimento Celular/efeitos dos fármacos , Quimiotaxia , Ensaio de Imunoadsorção Enzimática , Humanos , Imunidade Inata/efeitos dos fármacos , Inibidores de Metaloproteinases de Matriz/farmacologia , Metaloproteinases da Matriz/química , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Ligação Proteica , Células U937RESUMO
Upon contact with human plasma, bacteria are rapidly recognized by the complement system that labels their surface for uptake and clearance by phagocytic cells. Staphylococcus aureus secretes the 16 kD Extracellular fibrinogen binding protein (Efb) that binds two different plasma proteins using separate domains: the Efb N-terminus binds to fibrinogen, while the C-terminus binds complement C3. In this study, we show that Efb blocks phagocytosis of S. aureus by human neutrophils. In vitro, we demonstrate that Efb blocks phagocytosis in plasma and in human whole blood. Using a mouse peritonitis model we show that Efb effectively blocks phagocytosis in vivo, either as a purified protein or when produced endogenously by S. aureus. Mutational analysis revealed that Efb requires both its fibrinogen and complement binding residues for phagocytic escape. Using confocal and transmission electron microscopy we show that Efb attracts fibrinogen to the surface of complement-labeled S. aureus generating a 'capsule'-like shield. This thick layer of fibrinogen shields both surface-bound C3b and antibodies from recognition by phagocytic receptors. This information is critical for future vaccination attempts, since opsonizing antibodies may not function in the presence of Efb. Altogether we discover that Efb from S. aureus uniquely escapes phagocytosis by forming a bridge between a complement and coagulation protein.