RESUMO
The respiratory pathogen Moraxella catarrhalis is a human-specific commensal that frequently causes acute otitis media in children and stimulates acute exacerbations in chronic obstructive pulmonary disease patients. The exact molecular mechanisms defining host-pathogen interactions promoting pathogenesis are not clearly understood. Limited knowledge hampers vaccine and immunotherapeutic development required to treat this emerging pathogen. In this study, we reveal in detail a novel antibacterial role displayed by short leucine-rich proteoglycans (SLRPs) in concert with complement. We show that fibromodulin (FMOD), osteoadherin (OSAD), and biglycan (BGN) but not decorin (DCN) enhance serum killing of M. catarrhalis. Our results suggest that M. catarrhalis binding to SLRPs is a conserved feature, as the overwhelming majority of clinical and laboratory strains bound all four SLRPs. Furthermore, we resolve the binding mechanism responsible for this interaction and highlight the role of the ubiquitous surface protein (Usp) A2/A2H in mediating binding to host SLRPs. A conserved immune evasive strategy used by M. catarrhalis and other pathogens is the surface acquisition of host complement inhibitors such as C4b-binding protein (C4BP). We observed that FMOD, OSAD, and BGN competitively inhibit binding of C4BP to the surface of M. catarrhalis, resulting in increased C3b/iC3b deposition, membrane attack complex (MAC) formation, and subsequently decreased bacterial survival. Furthermore, both OSAD and BGN promote enhanced neutrophil killing in vitro, both in a complement-dependent and independent fashion. In summary, our results illustrate that SLRPs, FMOD, OSAD, and BGN portray complement-modulating activity enhancing M. catarrhalis killing, defining a new antibacterial role supplied by SLRPs.
Assuntos
Ativação do Complemento/imunologia , Interações Hospedeiro-Patógeno/imunologia , Moraxella catarrhalis/imunologia , Infecções por Moraxellaceae/imunologia , Proteoglicanas/imunologia , Humanos , LeucinaRESUMO
Streptococcus pyogenes is an exclusively human pathogen that can provoke mild skin and throat infections but can also cause fatal septicemia. This gram-positive bacterium has developed several strategies to evade the human immune system, enabling S. pyogenes to survive in the host. These strategies include recruiting several human plasma proteins, such as the complement inhibitor, C4b-binding protein (C4BP), and human (hu)-IgG through its Fc region to the bacterial surface to evade immune recognition. We identified a novel virulence mechanism whereby IgG-enhanced binding of C4BP to five of 12 tested S. pyogenes strains expressed diverse M proteins that are important surface-expressed virulence factors. Importantly, all strains that bound C4BP in the absence of IgG bound more C4BP when IgG was present. Further studies with an M1 strain that additionally expressed protein H, also a member of the M protein family, revealed that binding of hu-IgG Fc to protein H increased the affinity of protein H for C4BP. Increased C4BP binding accentuated complement downregulation, resulting in diminished bacterial killing. Accordingly, mortality from S. pyogenes infection in hu-C4BP transgenic mice was increased when hu-IgG or its Fc portion alone was administered concomitantly. Electron microscopy analysis of human tissue samples with necrotizing fasciitis confirmed increased C4BP binding to S. pyogenes when IgG was present. Our findings provide evidence of a paradoxical function of hu-IgG bound through Fc to diverse S. pyogenes isolates that increases their virulence and may counteract the beneficial effects of IgG opsonization.
Assuntos
Proteínas do Sistema Complemento/imunologia , Imunoglobulina G/imunologia , Streptococcus pyogenes/imunologia , Virulência/imunologia , Animais , Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas de Transporte/imunologia , Proteína de Ligação ao Complemento C4b/imunologia , Inativadores do Complemento/imunologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Fagocitose/imunologia , Ligação Proteica/imunologia , Infecções Estreptocócicas/imunologia , Fatores de Virulência/imunologiaRESUMO
Respiratory tract infections are one of the leading causes of mortality worldwide urging better understanding of interactions between pathogens causing these infections and the host. Here we report that an extracellular matrix component proline/arginine-rich end leucine-rich repeat protein (PRELP) is a novel antibacterial component of innate immunity. We detected the presence of PRELP in human bronchoalveolar lavage fluid and showed that PRELP can be found in alveolar fluid, resident macrophages/monocytes, myofibroblasts, and the adventitia of blood vessels in lung tissue. PRELP specifically binds respiratory tract pathogens Moraxella catarrhalis, Haemophilus influenzae, and Streptococcus pneumoniae, but not other bacterial pathogens tested. We focused our study on M. catarrhalis and found that PRELP binds the majority of clinical isolates of M. catarrhalis (n = 49) through interaction with the ubiquitous surface protein A2/A2H. M. catarrhalis usually resists complement-mediated serum killing by recruiting to its surface a complement inhibitor C4b-binding protein, which is also a ligand for PRELP. We found that PRELP competitively inhibits binding of C4b-binding protein to bacteria, which enhances membrane attack complex formation on M. catarrhalis and thus leads to increased serum sensitivity. Furthermore, PRELP enhances phagocytic killing of serum-opsonized M. catarrhalis by human neutrophils in vitro. Moreover, PRELP reduces Moraxella adherence to and invasion of human lung epithelial A549 cells. Taken together, PRELP enhances host innate immunity against M. catarrhalis through increasing complement-mediated attack, improving phagocytic killing activity of neutrophils, and preventing bacterial adherence to lung epithelial cells.
Assuntos
Proteínas da Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Macrófagos/imunologia , Moraxella catarrhalis/imunologia , Infecções por Moraxellaceae/imunologia , Miofibroblastos/imunologia , Mucosa Respiratória/imunologia , Infecções Respiratórias/imunologia , Citotoxicidade Celular Dependente de Anticorpos , Aderência Bacteriana , Linhagem Celular , Inativadores do Complemento/antagonistas & inibidores , Inativadores do Complemento/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Evasão da Resposta Imune , Imunidade Inata , Fagocitose , Mucosa Respiratória/patologiaRESUMO
Bacteria can cause life-threatening infections, such as pneumonia, meningitis, or sepsis. Antibiotic therapy is a mainstay of treatment, although antimicrobial resistance has drastically increased over the years. Unfortunately, safe and effective vaccines against most pathogens have not yet been approved, and thus developing alternative treatments is important. We analyzed the efficiency of factor H (FH)6-7/Fc, a novel antibacterial immunotherapeutic protein against the Gram-positive bacterium Streptococcus pyogenes This protein is composed of two domains of complement inhibitor human FH (FH complement control protein modules 6 and 7) that bind to S. pyogenes, linked to the Fc region of IgG (FH6-7/Fc). FH6-7/Fc has previously been shown to enhance complement-dependent killing of, and facilitate bacterial clearance in, animal models of the Gram-negative pathogens Haemophilus influenzae and Neisseria meningitidis We hypothesized that activation of complement by FH6-7/Fc on the surface of Gram-positive bacteria such as S. pyogenes will enable professional phagocytes to eliminate the pathogen. We found that FH6-7/Fc alleviated S. pyogenes-induced sepsis in a transgenic mouse model expressing human FH (S. pyogenes binds FH in a human-specific manner). Furthermore, FH6-7/Fc, which binds to protein H and selected M proteins, displaced FH from the bacterial surface, enhanced alternative pathway activation, and reduced bacterial blood burden by opsonophagocytosis in a C3-dependent manner in an ex vivo human whole-blood model. In conclusion, FH-Fc chimeric proteins could serve as adjunctive treatments against multidrug-resistant bacterial infections.
Assuntos
Fator H do Complemento/uso terapêutico , Imunoterapia/métodos , Proteínas Recombinantes de Fusão/uso terapêutico , Sepse/terapia , Vacinas Antiestafilocócicas/imunologia , Infecções Estreptocócicas/terapia , Streptococcus pyogenes/imunologia , Animais , Antibacterianos/uso terapêutico , Células Cultivadas , Complemento C3/metabolismo , C3 Convertase da Via Alternativa do Complemento , Fator H do Complemento/genética , Resistência a Múltiplos Medicamentos , Humanos , Camundongos , Camundongos Transgênicos , Fagocitose , Proteínas Recombinantes de Fusão/genética , Sepse/imunologia , Infecções Estreptocócicas/imunologiaRESUMO
Moraxella catarrhalis is a respiratory tract pathogen commonly causing otitis media in children and acute exacerbations in patients suffering from chronic obstructive pulmonary disease. Cartilage oligomeric matrix protein (COMP) functions as a structural component in cartilage, as well as a regulator of complement activity. Importantly, COMP is detected in resident macrophages and monocytes, alveolar fluid, and the endothelium of blood vessels in lung tissue. We show that the majority of clinical isolates of M. catarrhalis (n = 49), but not other tested bacterial pathogens, bind large amounts of COMP. COMP interacts directly with the ubiquitous surface protein A2 of M. catarrhalis. Binding of COMP correlates with survival of M. catarrhalis in human serum by inhibiting bactericidal activity of the complement membrane attack complex. Moreover, COMP inhibits phagocytic killing of M. catarrhalis by human neutrophils. We further observed that COMP reduces bacterial adhesion and uptake by human lung epithelial cells, thus protecting M. catarrhalis from intracellular killing by epithelial cells. Taken together, our findings uncover a novel mechanism that M. catarrhalis uses to evade host innate immunity.
Assuntos
Proteína de Matriz Oligomérica de Cartilagem/imunologia , Evasão da Resposta Imune/imunologia , Imunidade Inata/imunologia , Moraxella catarrhalis/imunologia , Infecções por Moraxellaceae/imunologia , Aderência Bacteriana/imunologia , Proteína de Matriz Oligomérica de Cartilagem/metabolismo , Linhagem Celular , Citometria de Fluxo , Humanos , Moraxella catarrhalis/metabolismo , Infecções por Moraxellaceae/metabolismoRESUMO
Periodontal disease is one of the most common inflammatory infectious diseases worldwide and it is associated with other syndromes, such as cardiovascular disease or rheumatoid arthritis. Recent advances in sequencing allowed for identification of novel periodontopathogens such as Gram-positive Filifactor alocis, but its virulence mechanisms remain largely unknown. We confirmed that F. alocis is a prevalent species in periodontitis patients, and we also observed strong correlation of this bacterium with clinical parameters, highlighting its role in the pathogenesis of the disease. Further, we found that preincubation of human serum with F. alocis resulted in abolished bactericidal activity and that F. alocis was surviving readily in full blood. We demonstrated that one of the key contributors to F. alocis complement resistance is a unique protein, FACIN (F. alocis complement inhibitor), which binds to C3, resulting in suppression of all complement pathways. Interestingly, FACIN is a nonclassical cell surface protein, a cytosolic enzyme acetylornithine transaminase, for which we now identified a moonlighting function. FACIN binds to C3 alone, but more importantly it also captures activated complement factor 3 within the complex with factor B, thereby locking in the convertase in an inactive state. Because of the indispensable role of alternative pathway convertase in amplifying complement cascades, its inhibition by FACIN results in a very potent downregulation of activated complement factor 3 opsonization on the pathogen surface, accompanied by reduction of downstream C5 cleavage.
Assuntos
Bactérias Anaeróbias/enzimologia , Bactérias Anaeróbias/imunologia , Complemento C3/antagonistas & inibidores , Complemento C3/metabolismo , Transaminases/metabolismo , Ativação do Complemento , Complemento C3/imunologia , HumanosRESUMO
BACKGROUND: Neutrophils are traditionally considered transcriptionally inactive. Compared to other immune cells, little is known about their transcriptional profile during interaction with pathogens. METHODS: We analyzed the meta-transcriptome of the neutrophil-Candida albicans interplay and the transcriptome of C. albicans challenged with neutrophil extracellular traps (NETs) by RNA-Seq, considering yeast and hypha individually in each approach. RESULTS: The neutrophil response to C. albicans yeast and hyphae was dominated by a morphotype-independent core response. However, 11 % of all differentially expressed genes were regulated in a specific manner when neutrophils encountered the hyphal form of C. albicans. While involving genes for transcriptional regulators, receptors, and cytokines, the neutrophil core response lacked typical antimicrobial effectors genes. Genes of the NOD-like receptor pathway, including NLRP3, were enriched. Neutrophil- and NET-provoked responses in C. albicans differed. At the same time, the Candida transcriptome upon neutrophil encounter and upon NET challenge included genes from various metabolic processes and indicate a mutual role of the regulators Tup1p, Efg1p, Hap43p, and Cap1p. Upon challenge with neutrophils and NETs, the overall Candida response was partially morphotype-specific. Yet again, actual oppositional regulation in yeasts and hyphae was only detected for the arginine metabolism in neutrophil-infecting C. albicans. CONCLUSIONS: Taken together, our study provides a comprehensive and quantitative transcript profile of the neutrophil-C. albicans interaction. By considering the two major appearances of both, neutrophils and C. albicans, our study reveals yet undescribed insights into this medically relevant encounter. Hence, our findings will facilitate future research and potentially inspire novel therapy developments.
Assuntos
Candida albicans/genética , Candida albicans/imunologia , Perfilação da Expressão Gênica , Neutrófilos/metabolismo , Neutrófilos/microbiologia , Arginina/metabolismo , Candida albicans/fisiologia , Citocinas/genética , Citoesqueleto/metabolismo , Hifas/genética , Neutrófilos/citologia , Neutrófilos/imunologia , Transdução de Sinais/genética , Estresse Fisiológico/genética , Açúcares/metabolismoRESUMO
Streptococcus pyogenes, also known as Group A Streptococcus (GAS), is an important human bacterial pathogen that can cause invasive infections. Once it colonizes its exclusively human host, GAS needs to surmount numerous innate immune defense mechanisms, including opsonization by complement and consequent phagocytosis. Several strains of GAS bind to human-specific complement inhibitors, C4b-binding protein (C4BP) and/or Factor H (FH), to curtail complement C3 (a critical opsonin) deposition. This results in diminished activation of phagocytes and clearance of GAS that may lead to the host being unable to limit the infection. Herein we describe the course of GAS infection in three human complement inhibitor transgenic (tg) mouse models that examined each inhibitor (human C4BP or FH) alone, or the two inhibitors together (C4BPxFH or 'double' tg). GAS infection with strains that bound C4BP and FH resulted in enhanced mortality in each of the three transgenic mouse models compared to infection in wild type mice. In addition, GAS manifested increased virulence in C4BPxFH mice: higher organism burdens and greater elevations of pro-inflammatory cytokines and they died earlier than single transgenic or wt controls. The effects of hu-C4BP and hu-FH were specific for GAS strains that bound these inhibitors because strains that did not bind the inhibitors showed reduced virulence in the 'double' tg mice compared to strains that did bind; mortality was also similar in wild-type and C4BPxFH mice infected by non-binding GAS. Our findings emphasize the importance of binding of complement inhibitors to GAS that results in impaired opsonization and phagocytic killing, which translates to enhanced virulence in a humanized whole animal model. This novel hu-C4BPxFH tg model may prove invaluable in studies of GAS pathogenesis and for developing vaccines and therapeutics that rely on human complement activation for efficacy.
Assuntos
Antígenos de Bactérias/imunologia , Inativadores do Complemento/imunologia , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/patogenicidade , Animais , Proteínas da Membrana Bacteriana Externa/metabolismo , Ativação do Complemento , Humanos , Camundongos , Infecções Estreptocócicas/imunologia , VirulênciaRESUMO
Polynuclear manganese compounds have garnered interest as mimics and models of the water oxidizing complex (WOC) in photosystem II and as single molecule magnets. Molecular systems in which composition can be correlated to physical phenomena, such as magnetic exchange interactions, remain few primarily because of synthetic limitations. Here, we report the synthesis of a family of trimanganese(II) complexes of the type Mn3X3L (X = Cl-, H-, and MeO-) where L3- is a tris(ß-diketiminate) cyclophane. The tri(chloride) complex (2) is structurally similar to the reported tri(bromide) complex (1) with the Mn3X3 core having a ladder-like arrangement of alternating M-X rungs, whereas the tri(µ-hydride) (3) and tri(µ-methoxide) (4) complexes contain planar hexagonal cores. The hydride and methoxide complexes are synthesized in good yield (48% and 56%) starting with the bromide complex employing a metathesis-like strategy. Compounds 2-4 were characterized by combustion analysis, X-ray crystallography, X-band EPR spectroscopy, SQUID magnetometry, and infrared and UV-visible spectroscopy. Magnetic susceptibility measurements indicate that the Mn3 clusters in 2-4 are antiferromagnetically coupled, and the spin ground state of the compounds (S = 3/2 (1, 2) or S = 1/2 (3, 4)) is correlated to the identity of the bridging ligand and structural arrangement of the Mn3X3 core (X = Br, Cl, H, OCH3). Electrochemical experiments on isobutyronitrile solutions of 3 and 4 display broad irreversible oxidations centered at 0.30 V.
RESUMO
The designed [3M-3(µ-H)] clusters (M = Fe(II), Co(II)) Fe3H3L (1-H) and Co3H3L (2-H) [where L(3-) is a tris(ß-diketiminate) cyclophane] were synthesized by treating the corresponding M3Br3L complexes with KBEt3H. From single-crystal X-ray analysis, the hydride ligands are sterically protected by the cyclophane ligand, and these complexes selectively react with CO2 over other unsaturated substrates (e.g., CS2, Me3SiCCH, C2H2, and CH3CN). The reaction of 1-H or 2-H with CO2 at room temperature yielded Fe3(OCHO)(H)2L (1-CO2) or Co3(OCHO)(H)2L (2-CO2), respectively, which evidence the differential reactivity of the hydride ligands within these complexes. The analogous reactions at elevated temperatures revealed a distinct difference in the reactivity pattern for 2-H as compared to 1-H; Fe3(OCHO)3L (1-3CO2) was generated from 1-H, while 2-H afforded only 2-CO2.
RESUMO
Using a simple metathesis approach, the triiron(II) tribromide complex Fe3Br3L (1) reacts with tetrabutylammonium azide to afford the monoazide dibromide analogue Fe3(Br)2(N3)L (2) in high yield. The inclusion of azide was confirmed by IR spectroscopy with a ν(N3) = 2082 cm(-1) as well as combustion analysis and X-ray crystallography. Heating 2 in the solid state results in the complete loss of the azide vibration in the IR spectra and the isolation of the olive-green mononitride complex Fe3(Br)2(N)L (3). Solution magnetic susceptibility measurements support that the trimetallic core within 2 is oxidized upon generation of 3 (5.07 vs 3.09 µB). Absorption maxima in the UV-visible-near-IR (NIR) spectra of 2 and 3 support the azide-to-nitride conversion, and a broad NIR absorption centered at 1117 nm is similar to that previously reported for the intervalence charge-transfer band for a mixed-valent nitridodiiron cluster. The cyclic voltammograms recorded for 3 are comparable to those of 1 with no reductive waves observed between â¼0 and -2.5 V (vs Fc/Fc(+)), whereas a reversible one-electron redox process is observed for Fe3(NH2)3L (4). These results suggest that intercluster cooperativity is unlikely to predominate the dinitrogen reduction mechanism when 1 is treated with KC8 under N2.
RESUMO
The reaction of [Zn3Cl3L], in which L(3-) is a tris(ß-diketiminate) cyclophane, with K(sBu)3BH afforded [Zn3(µ-H)3L] (2), as confirmed by NMR spectroscopy, NOESY, and X-ray crystallography. The complex 2 was air-stable and unreactive towards water, methanol, and other substrates (e.g., nitriles) at room temperature over 24â h but reacted with CO2 (ca. 1â atm) to generate [Zn3(µ-H)2(µ-1,1-O2CH)] (3). In contrast, [Zn3(OH)3L] (4) was found to be unreactive toward CO2 over the course of several days at 90 °C.
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Streptococcus pyogenes AP1, a strain of the highly virulent M1 serotype, uses exclusively protein H to bind the complement inhibitor C4b-binding protein (C4BP). We found a strong correlation between the ability of AP1 and its isogenic mutants lacking protein H to inhibit opsonization with complement C3b and binding of C4BP. C4BP bound to immobilized protein H or AP1 bacteria retained its cofactor activity for degradation of (125)I-C4b. Furthermore, C4b deposited from serum onto AP1 bacterial surfaces was processed into C4c/C4d fragments, which did not occur on strains unable to bind C4BP. Recombinant C4BP mutants, which (i) lack certain CCP domains or (ii) have mutations in single aa as well as (iii) mutants with additional aa between different CCP domains were used to determine that the binding is mainly mediated by a patch of positively charged amino acid residues at the interface of domains CCP1 and CCP2. Using recombinant protein H fragments, we narrowed down the binding site to the N-terminal domain A. With a peptide microarray, we identified one single 18-amino acid-long peptide comprising residues 92-109, which specifically bound C4BP. Biacore was used to determine KD = 6 × 10(-7) M between protein H and a single subunit of C4BP. C4BP binding also correlated with elevated levels of adhesion and invasion to endothelial cells. Taken together, we identified the molecular basis of C4BP-protein H interaction and found that it is not only important for decreased opsonization but also for invasion of endothelial cells by S. pyogenes.
Assuntos
Aderência Bacteriana/fisiologia , Complemento C4b/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Streptococcus pyogenes/metabolismo , Streptococcus pyogenes/patogenicidade , Fatores de Virulência/metabolismo , Sítios de Ligação , Células Cultivadas , Complemento C4b/genética , Proteína de Ligação ao Complemento C4b , Feminino , Células Endoteliais da Veia Umbilical Humana/microbiologia , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Masculino , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Streptococcus pyogenes/genética , Ressonância de Plasmônio de Superfície , Fatores de Virulência/genéticaRESUMO
The enteropathogen Yersinia pseudotuberculosis can survive in the harsh environment of lymphoid compartments that abounds in immune cells. This capacity is dependent on the plasmid-encoded Yersinia outer proteins (Yops) that are delivered into the host cell via a mechanism involving the Yersinia type III secretion system. We show that the virulence protein YopK has a role in the mechanism by which Y. pseudotuberculosis avoids the polymorphonuclear leukocyte or neutrophil (PMN) defense. A yopK mutant, which is attenuated in the mouse infection model, where it fails to cause systemic infection, was found to colonize Peyer's patches and mesenteric lymph nodes more rapidly than the wild-type strain. Further, in mice lacking PMNs, the yopK mutant caused full disease with systemic spread and typical symptoms. Analyses of effects on PMNs revealed that both the wild-type strain and the yopK mutant inhibited internalization and reactive oxygen species production, as well as neutrophil extracellular trap formation by PMNs. However, the wild-type strain effectively avoided induction of PMN death, whereas the mutant caused a necrosis-like PMN death. Taken together, our results indicate that YopK is required for the ability of Yersinia to resist the PMN defense, which is critical for the virulence of the pathogen. We suggest a mechanism whereby YopK functions to prevent unintended Yop delivery and thereby PMN disruption, resulting in necrosis-like cell death, which would enhance the inflammatory response favoring the host.
Assuntos
Proteínas da Membrana Bacteriana Externa/imunologia , Neutrófilos/imunologia , Infecções por Yersinia pseudotuberculosis/imunologia , Yersinia pseudotuberculosis/imunologia , Animais , Morte Celular/imunologia , Feminino , Humanos , Linfonodos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Necrose/imunologia , Nódulos Linfáticos Agregados/imunologia , Espécies Reativas de Oxigênio/imunologia , Infecções por Yersinia pseudotuberculosis/sangueRESUMO
Components of the extracellular matrix (ECM), when exposed to body fluids may promote local complement activation and inflammation. Pathologic complement activation at the glomerular basement membrane and at the Bruch's membrane is implicated in renal and eye diseases, respectively. Binding of soluble complement inhibitors to the ECM, including factor H (FH), is important to prevent excessive complement activation. Since the FH-related (FHR) proteins FHR1 and FHR5 are also implicated in these diseases, our aim was to study whether these FHRs can also bind to ECM components and affect local FH activity and complement activation. Both FH and the FHRs showed variable binding to ECM components. We identified laminin, fibromodulin, osteoadherin and PRELP as ligands of FHR1 and FHR5, and found that FHR1 bound to these ECM components through its C-terminal complement control protein (CCP) domains 4-5, whereas FHR5 bound via its middle region, CCPs 3-7. Aggrecan, biglycan and decorin did not bind FH, FHR1 and FHR5. FHR5 also bound to immobilized C3b, a model of surface-deposited C3b, via CCPs 3-7. By contrast, soluble C3, C3(H2O), and the C3 fragments C3b, iC3b and C3d bound to CCPs 8-9 of FHR5. Properdin, which was previously described to bind via CCPs 1-2 to FHR5, did not bind in its physiologically occurring serum forms in our assays. FHR1 and FHR5 inhibited the binding of FH to the identified ECM proteins in a dose-dependent manner, which resulted in reduced FH cofactor activity. Moreover, both FHR1 and FHR5 enhanced alternative complement pathway activation on immobilized ECM proteins when exposed to human serum, resulting in the increased deposition of C3-fragments, factor B and C5b-9. Thus, our results identify novel ECM ligands of FH family proteins and indicate that FHR1 and FHR5 are competitive inhibitors of FH on ECM and, when bound to these ligands, they may enhance local complement activation and promote inflammation under pathological conditions.
Assuntos
Ativação do Complemento , Proteínas Inativadoras do Complemento C3b , Fator H do Complemento , Proteínas do Sistema Complemento , Proteínas Inativadoras do Complemento C3b/metabolismo , Proteínas do Sistema Complemento/metabolismo , Matriz Extracelular , Humanos , Inflamação , LigantesRESUMO
Neutrophils are the first line of defense at the site of an infection. They encounter and kill microbes intracellularly upon phagocytosis or extracellularly by degranulation of antimicrobial proteins and the release of Neutrophil Extracellular Traps (NETs). NETs were shown to ensnare and kill microbes. However, their complete protein composition and the antimicrobial mechanism are not well understood. Using a proteomic approach, we identified 24 NET-associated proteins. Quantitative analysis of these proteins and high resolution electron microscopy showed that NETs consist of modified nucleosomes and a stringent selection of other proteins. In contrast to previous results, we found several NET proteins that are cytoplasmic in unstimulated neutrophils. We demonstrated that of those proteins, the antimicrobial heterodimer calprotectin is released in NETs as the major antifungal component. Absence of calprotectin in NETs resulted in complete loss of antifungal activity in vitro. Analysis of three different Candida albicans in vivo infection models indicated that NET formation is a hitherto unrecognized route of calprotectin release. By comparing wild-type and calprotectin-deficient animals we found that calprotectin is crucial for the clearance of infection. Taken together, the present investigations confirmed the antifungal activity of calprotectin in vitro and, moreover, demonstrated that it contributes to effective host defense against C. albicans in vivo. We showed for the first time that a proportion of calprotectin is bound to NETs in vitro and in vivo.
Assuntos
Candida albicans/imunologia , Complexo Antígeno L1 Leucocitário/imunologia , Neutrófilos/imunologia , Abscesso Abdominal/imunologia , Abscesso Abdominal/microbiologia , Análise de Variância , Animais , Antifúngicos/química , Antifúngicos/metabolismo , Células Cultivadas , Estruturas Celulares/química , Estruturas Celulares/imunologia , Estruturas Celulares/ultraestrutura , Histonas/química , Histonas/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Imuno-Histoquímica , Complexo Antígeno L1 Leucocitário/química , Complexo Antígeno L1 Leucocitário/metabolismo , Pneumopatias Fúngicas/imunologia , Pneumopatias Fúngicas/microbiologia , Camundongos , Camundongos Knockout , Ativação de NeutrófiloRESUMO
Streptococcus pyogenes is a major human pathogen that causes a variety of diseases ranging from mild skin and throat infections to fatal septicemia. In severe invasive infections, S. pyogenes encounters and interacts with components of the extracellular matrix (ECM), including small leucine rich-proteoglycans (SLRPs). In this study, we report a novel antimicrobial role played by SLRPs biglycan, decorin, fibromodulin and osteoadherin, specifically in promoting the eradication of S. pyogenes in a human sepsis model of infection. SLRPs can be released from the ECM and de novo synthesized by a number of cell types. We reveal that infection of human monocytes by S. pyogenes induces the expression of decorin. Furthermore, we show that the majority of genetically distinct and clinically relevant S. pyogenes isolates interact with SLRPs resulting in decreased survival in blood killing assays. Biglycan and decorin induce TLR2 and TLR4 signaling cascades resulting in secretion of proinflammatory and chemotactic molecules and recruitment of professional phagocytes. Surprisingly, SLRP-mediated elimination of S. pyogenes occurs independently of TLR activation. Our results indicate that SLRPs act in concert with human serum, enhancing deposition of complement activation fragments and the classical activator C1q on the bacterial surface, facilitating efficient microbial eradication. Addition of the complement C3 inhibitor compstatin significantly reverses SLRP-induced blood killing, confirming active complement as a key mediator in SLRP-mediated bacterial destruction. Taken together our results add to the functional repertoire of SLRPs, expanding to encompass their role in controlling bacterial infection.
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Moraxella catarrhalis is a human-specific commensal of the respiratory tract and an opportunistic pathogen. It is one of the leading cause of otitis media in children and of acute exacerbations in patients with chronic obstructive pulmonary disease, resulting in significant morbidity and economic burden. Vaccines and new immunotherapeutic strategies to treat this emerging pathogen are needed. Complement is a key component of innate immunity that mediates the detection, response, and subsequent elimination of invading pathogens. Many pathogens including M. catarrhalis have evolved complement evasion mechanisms, which include the binding of human complement inhibitors such as C4b-binding protein (C4BP) and Factor H (FH). Inhibiting C4BP and FH acquisition by M. catarrhalis may provide a novel therapeutic avenue to treat infections. To achieve this, we created two chimeric proteins that combined the Moraxella-binding domains of C4BP and FH fused to human immunoglobulin Fcs: C4BP domains 1 and 2 and FH domains 6 and 7 fused to IgM and IgG Fc, respectively. As expected, FH6-7/IgG displaced FH from the bacterial surface while simultaneously activating complement via Fc-C1q interactions, together increasing pathogen elimination. C4BP1-2/IgM also increased serum killing of the bacteria through enhanced complement deposition, but did not displace C4BP from the surface of M. catarrhalis. These Fc fusion proteins could act as anti-infective immunotherapies. Many microbes bind the complement inhibitors C4BP and FH through the same domains as M. catarrhalis, therefore these Fc fusion proteins may be promising candidates as adjunctive therapy against many different drug-resistant pathogens.
Assuntos
Proteína de Ligação ao Complemento C4b/farmacologia , Fator H do Complemento/farmacologia , Fragmentos Fc das Imunoglobulinas/farmacologia , Moraxella catarrhalis/efeitos dos fármacos , Proteínas Recombinantes de Fusão/farmacologia , Animais , Ligação Competitiva , Atividade Bactericida do Sangue , Células CHO , Complemento C3b/análise , Complemento C3d/análise , Proteína de Ligação ao Complemento C4b/genética , Proteína de Ligação ao Complemento C4b/metabolismo , Fator H do Complemento/genética , Fator H do Complemento/metabolismo , Cricetinae , Cricetulus , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Imunoglobulina G/farmacologia , Imunoglobulina M/genética , Imunoglobulina M/metabolismo , Imunoglobulina M/farmacologia , Moraxella catarrhalis/metabolismo , Ligação Proteica , Domínios Proteicos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Neutrophils are professional phagocytes of the innate immune system that are essential to control bacterial and fungal infections. These cells engulf and kill invading microbes. Additionally, activated neutrophils are able to release neutrophil extracellular traps (NETs). These fibers consist of chromatin decorated with antimicrobial proteins to trap and kill microbes. Appropriate quantitative methods are required to understand the nature of interactions of neutrophils with pathogens. Here we present assays to measure killing mediated by phagocytosis, by NETs, by a combination of both, and by granular extract. As examples, we use Candida albicans for fungal and Shigella flexneri for bacterial pathogens.