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1.
Nat Immunol ; 10(7): 761-8, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19525969

RESUMO

Granule-mediated cytotoxicity is the main effector mechanism of cytotoxic CD8+ T cells. We report that CD8+ T cells from acid sphingomyelinase (ASMase)-deficient (ASMase-KO) mice are defective in exocytosis of cytolytic effector molecules; this defect resulted in attenuated cytotoxic activity of ASMase-KO CD8+ T cells and delayed elimination of lymphocytic choriomeningitis virus from ASMase-KO mice. Cytolytic granules of ASMase-KO and wild-type CD8+ T cells were equally loaded with granzymes and perforin, and correctly directed to the immunological synapse. In wild-type CD8+ T cells, secretory granules underwent shrinkage by 82% after fusion with the plasma membrane. In ASMase-KO CD8+ T cells, the contraction of secretory granules was markedly impaired. Thus, ASMase is required for contraction of secretory granules and expulsion of cytotoxic effector molecules.


Assuntos
Citotoxicidade Imunológica/imunologia , Vesículas Secretórias/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Linfócitos T/imunologia , Animais , Infecções por Arenaviridae/imunologia , Infecções por Arenaviridae/metabolismo , Infecções por Arenaviridae/virologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Quimiocina CCL5/metabolismo , Feminino , Granzimas/genética , Granzimas/metabolismo , Immunoblotting , Sinapses Imunológicas/imunologia , Vírus da Coriomeningite Linfocítica/fisiologia , Masculino , Camundongos , Camundongos Knockout , Microscopia de Fluorescência , Perforina/genética , Perforina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vesículas Secretórias/enzimologia , Esfingomielina Fosfodiesterase/genética , Linfócitos T/citologia , Linfócitos T/metabolismo , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo
2.
Mol Microbiol ; 99(2): 407-24, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26434356

RESUMO

Borrelia (B.) bavariensis exhibits a marked tropism for nervous tissues and frequently causes neurological manifestations in humans. The molecular mechanism by which B. bavariensis overcomes innate immunity, in particular, complement remains elusive. In contrast to other serum-resistant spirochetes, none of the B. bavariensis isolates investigated bound complement regulators of the alternative (AP) and classical pathway (CP) or proteolytically inactivated complement components. Focusing on outer surface proteins BGA66 and BGA71, we demonstrated that both molecules either inhibit AP, CP and terminal pathway (TP) activation, or block activation of the CP and TP respectively. Both molecules bind complement components C7, C8 and C9, and thereby prevent assembly of the terminal complement complex. This inhibitory activity was confirmed by the introduction of the BGA66 and BGA71 encoding genes into a serum-sensitive B. garinii strain. Transformed spirochetes producing either BGA66 or BGA71 overcome complement-mediated killing, thus indicating that both proteins independently facilitate serum resistance of B. bavariensis. The generation of C-terminally truncated proteins as well as a chimeric BGA71 protein lead to the localization of the complement-interacting binding site within the N-terminus. Collectively, our data reveal a novel immune evasion strategy of B. bavariensis that is directed against the activation of the TP.


Assuntos
Proteínas de Bactérias/imunologia , Borrelia burgdorferi/imunologia , Proteínas do Sistema Complemento/imunologia , Doença de Lyme/imunologia , Animais , Proteínas de Bactérias/genética , Borrelia burgdorferi/genética , Complexo de Ataque à Membrana do Sistema Complemento/genética , Complexo de Ataque à Membrana do Sistema Complemento/imunologia , Humanos , Doença de Lyme/microbiologia , Camundongos
3.
Immunity ; 29(5): 720-33, 2008 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-18951048

RESUMO

Granzyme A (GzmA) is considered a major proapoptotic protease. We have discovered that GzmA-induced cell death involves rapid membrane damage that depends on the synergy between micromolar concentrations of GzmA and sublytic perforin (PFN). Ironically, GzmA and GzmB, independent of their catalytic activity, both mediated this swift necrosis. Even without PFN, lower concentrations of human GzmA stimulated monocytic cells to secrete proinflammatory cytokines (interleukin-1beta [IL-1beta], TNFalpha, and IL-6) that were blocked by a caspase-1 inhibitor. Moreover, murine GzmA and GzmA(+) cytotoxic T lymphocytes (CTLs) induce IL-1beta from primary mouse macrophages, and GzmA(-/-) mice resist lipopolysaccharide-induced toxicity. Thus, the granule secretory pathway plays an unexpected role in inflammation, with GzmA acting as an endogenous modulator.


Assuntos
Granzimas/imunologia , Interleucina-1beta/imunologia , Interleucina-6/imunologia , Leucócitos Mononucleares/imunologia , Perforina/imunologia , Linfócitos T Citotóxicos/imunologia , Fator de Necrose Tumoral alfa/imunologia , Adenoviridae/imunologia , Animais , Adesão Celular , Morte Celular , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Técnicas de Silenciamento de Genes , Granzimas/metabolismo , Células HeLa , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Células Jurkat , Macrófagos/imunologia , Camundongos , Perforina/metabolismo , Linfócitos T Citotóxicos/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Células U937
4.
J Infect Dis ; 213(9): 1388-99, 2016 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-26681776

RESUMO

Acinetobacter baumannii is an emerging opportunistic pathogen, responsible for up to 10% of gram-negative, nosocomial infections. The global increase of multidrug-resistant and pan-resistant Acinetobacter isolates presents clinicians with formidable challenges. To establish a persistent infection,A. baumannii must overcome the detrimental effects of complement as the first line of defense against invading microorganisms. However, the immune evasion principles underlying serum resistance inA. baumannii remain elusive. Here, we identified a novel plasminogen-binding protein, termed CipA. Bound plasminogen, upon conversion to active plasmin, degraded fibrinogen and complement C3b and contributed to serum resistance. Furthermore, CipA directly inhibited the alternative pathway of complement in vitro, irrespective of its ability to bind plasminogen. A CipA-deficient mutant was efficiently killed by human serum and showed a defect in the penetration of endothelial monolayers, demonstrating that CipA is a novel multifunctional protein that contributes to the pathogenesis ofA. baumannii.


Assuntos
Acinetobacter baumannii/metabolismo , Acinetobacter baumannii/patogenicidade , Proteínas de Bactérias/metabolismo , Proteínas do Sistema Complemento/metabolismo , Plasminogênio/metabolismo , Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Membrana Celular/química , Proteínas do Sistema Complemento/química , Humanos , Plasminogênio/química , Ligação Proteica
5.
Microsc Microanal ; 21(3): 680-8, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25739645

RESUMO

Borrelia burgdorferi sensu lato, the causative agent of Lyme disease, is transmitted to humans through the bite of infected Ixodes spp. ticks. Successful infection of vertebrate hosts necessitates sophisticated means of the pathogen to escape the vertebrates' immune system. One strategy employed by Lyme disease spirochetes to evade adaptive immunity involves a highly coordinated regulation of the expression of outer surface proteins that is vital for infection, dissemination, and persistence. Here we characterized the expression pattern of bacterial surface antigens using different microscopy techniques, from fluorescent wide field to super-resolution and immunogold-scanning electron microscopy. A fluorescent strain of B. burgdorferi spirochetes was labeled with monoclonal antibodies directed against various bacterial surface antigens. Our results indicate that OspA is more evenly distributed over the surface than OspB and OspC that were present as punctate areas.


Assuntos
Antígenos de Bactérias/análise , Borrelia burgdorferi/química , Proteínas de Membrana/análise , Técnicas Microbiológicas/métodos , Anticorpos Monoclonais/metabolismo , Imunofluorescência , Microscopia
6.
J Biol Chem ; 288(35): 25229-25243, 2013 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-23861404

RESUMO

The Lyme disease spirochete Borrelia burgdorferi lacks endogenous, surface-exposed proteases. In order to efficiently disseminate throughout the host and penetrate tissue barriers, borreliae rely on recruitment of host proteases, such as plasmin(ogen). Here we report the identification of a novel plasminogen-binding protein, BBA70. Binding of plasminogen is dose-dependent and is affected by ionic strength. The BBA70-plasminogen interaction is mediated by lysine residues, primarily located in a putative C-terminal α-helix of BBA70. These lysine residues appear to interact with the lysine-binding sites in plasminogen kringle domain 4 because a deletion mutant of plasminogen lacking that domain was unable to bind to BBA70. Bound to BBA70, plasminogen activated by urokinase-type plasminogen activator was able to degrade both a synthetic chromogenic substrate and the natural substrate fibrinogen. Furthermore, BBA70-bound plasmin was able to degrade the central complement proteins C3b and C5 and inhibited the bacteriolytic effects of complement. Consistent with these functional activities, BBA70 is located on the borrelial outer surface. Additionally, serological evidence demonstrated that BBA70 is produced during mammalian infection. Taken together, recruitment and activation of plasminogen could play a beneficial role in dissemination of B. burgdorferi in the human host and may possibly aid the spirochete in escaping the defense mechanisms of innate immunity.


Assuntos
Proteínas de Bactérias/metabolismo , Borrelia burgdorferi/metabolismo , Plasminogênio/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Borrelia burgdorferi/química , Borrelia burgdorferi/genética , Borrelia burgdorferi/imunologia , Complemento C3b/química , Complemento C3b/genética , Complemento C3b/imunologia , Complemento C3b/metabolismo , Complemento C5/química , Complemento C5/genética , Complemento C5/imunologia , Complemento C5/metabolismo , Fibrinolisina/química , Fibrinolisina/genética , Fibrinolisina/imunologia , Fibrinolisina/metabolismo , Humanos , Imunidade Inata , Doença de Lyme/genética , Doença de Lyme/imunologia , Doença de Lyme/metabolismo , Plasminogênio/química , Plasminogênio/genética , Plasminogênio/imunologia , Ligação Proteica , Estrutura Terciária de Proteína , Ativador de Plasminogênio Tipo Uroquinase/química , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/imunologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismo
7.
Infect Immun ; 82(1): 380-92, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24191298

RESUMO

CspA of the Lyme disease spirochete Borrelia burgdorferi represents a key molecule in immune evasion, protecting borrelial cells from complement-mediated killing. As previous studies focused almost exclusively on CspA of B. burgdorferi, here we investigate the different binding capacities of CspA orthologs of Borrelia burgdorferi, B. afzelii, and B. spielmanii for complement regulator factor H and plasminogen and their ability to inhibit complement activation by either binding these host-derived plasma proteins or independently by direct interaction with components involved in formation of the lethal, pore-like terminal complement complex. To further examine their function in serum resistance in vivo, a serum-sensitive B. garinii strain was used to generate spirochetes, ectopically producing functional CspA orthologs. Irrespective of their species origin, all three CspA orthologs impart resistance to complement-mediated killing when produced in a serum-sensitive B. garinii surrogate strain. To analyze the inhibitory effect on complement activation and to assess the potential to inactivate C3b by binding of factor H and plasminogen, recombinant CspA orthologs were also investigated. All three CspA orthologs simultaneously bound factor H and plasminogen but differed in regard to their capacity to inactivate C3b via bound plasmin(ogen) and inhibit formation of the terminal complement complex. CspA of B. afzelii binds plasmin(ogen) and inhibits the terminal complement complex more efficiently than CspA of B. burgdorferi and B. spielmanii. Taken together, CspA orthologs of serum-resistant Lyme disease spirochetes act as multifunctional evasion molecules that inhibit complement on two central activation levels, C3b generation and assembly of the terminal complement complex.


Assuntos
Proteínas de Bactérias/fisiologia , Borrelia burgdorferi/fisiologia , Proteínas do Sistema Complemento/metabolismo , Doença de Lyme/microbiologia , Análise de Variância , Bacteriólise/fisiologia , Atividade Bactericida do Sangue , Borrelia/fisiologia , Células Cultivadas , Complemento C3b/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Doença de Lyme/imunologia , Plasminogênio/metabolismo , Ligação Proteica/fisiologia
8.
J Virol ; 86(16): 8713-9, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22674984

RESUMO

The T cell granule exocytosis pathway is essential to control hepatotropic lymphocytic choriomeningitis virus strain WE (LCMV-WE) but also contributes to the observed pathology in mice. Although effective antiviral T cell immunity and development of viral hepatitis are strictly dependent on perforin and granzymes, the molecular basis underlying induction of functionally competent virus-immune T cells, including participation of the innate immune system, is far from being resolved. We demonstrate here that LCMV-immune T cells of interleukin-1 receptor (IL-1R)-deficient mice readily express transcripts for perforin and granzymes but only translate perforin, resulting in the lack of proapoptotic potential in vitro. LCMV is not cleared in IL-1R-deficient mice, and yet the infected mice develop neither splenomegaly nor hepatitis. These results demonstrate that IL-1R signaling is central to the induction of proapoptotic CD8 T cell immunity, including viral clearance and associated tissue injuries in LCMV infection.


Assuntos
Infecções por Arenaviridae/imunologia , Linfócitos T CD8-Positivos/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Receptores de Interleucina-1/imunologia , Animais , Infecções por Arenaviridae/patologia , Infecções por Arenaviridae/virologia , Modelos Animais de Doenças , Hepatite/imunologia , Hepatite/patologia , Hepatite/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Interleucina-1/deficiência , Esplenomegalia/imunologia , Esplenomegalia/patologia , Esplenomegalia/virologia
9.
Artigo em Inglês | MEDLINE | ID: mdl-23722839

RESUMO

Borrelia burgdorferi has evolved many mechanisms of evading the different immune systems across its range of reservoir hosts, including the capture and presentation of host complement regulators factor H and factor H-like protein-1 (FHL-1). Acquisition is mediated by a family of complement regulator-acquiring surface proteins (CRASPs), of which the atomic structure of CspA (BbCRASP-1) is known and shows the formation of a homodimeric species which is required for binding. Mutagenesis studies have mapped a putative factor H binding site to a cleft between the two subunits. Presented here is a new atomic structure of CspA which shows a degree of flexibility between the subunits which may be critical for factor H scavenging by increasing access to the binding interface and allows the possibility that the assembly can clamp around the bound complement regulators.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Borrelia burgdorferi , Fator H do Complemento/química , Fator H do Complemento/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Cristalografia por Raios X , Ligação Proteica/fisiologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
10.
Exp Dermatol ; 21(1): 72-5, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22151394

RESUMO

The promise of mesotherapy is maintenance and/or recovery of a youthful skin with a firm, bright and moisturized texture. Currently applied medications employ microinjections of hyaluronic acid, vitamins, minerals and amino acids into the superficial layer of the skin. However, the molecular and cellular processes underlying mesotherapy are still elusive. Here we analysed the effect of five distinct medication formulas on pivotal parameters involved in skin ageing, that is collagen expression, cell proliferation and morphological changes using normal human skin fibroblast cultures in vitro. Whereas in the presence of hyaluronic acid, NCTF135(®) and NCTF135HA(®) , cell proliferation was comparable to control cultures; however, with higher expression of collagen type-1, matrix metalloproteinase-1 and tissue inhibitor of matrix metalloproteinase-1, addition of Soluvit(®) N and Meso-BK led to apoptosis and/or necrosis of human fibroblasts. The data indicate that bioactive reagents currently applied for skin rejuvenation elicit strikingly divergent physiological processes in human skin fibroblast in vitro.


Assuntos
Fibroblastos/efeitos dos fármacos , Ácido Hialurônico/farmacologia , Mesoterapia , Envelhecimento da Pele/efeitos dos fármacos , Vitaminas/farmacologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Colágeno Tipo I/metabolismo , Humanos , Metaloproteinase 1 da Matriz/metabolismo , Projetos Piloto , Inibidor Tecidual de Metaloproteinase-1/metabolismo
11.
Cell Microbiol ; 13(5): 731-41, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21276171

RESUMO

Some Borrelia species are the causative agents of tick-borne Lyme disease responsible for different disabilities depending on species and hosts. Borrelia are highly motile bacterial cells, and light microscopy shows that these spirochetes can associate with each other during movement. Using cryo-electron tomography, we observed closely associated Borrelia cells. Some of these showed a single outer membrane surrounding two longitudinally arranged cytoplasmic cylinders. We also observed fusion of two cytoplasmic cylinders and differences in the surface layer density of fused spirochetes. These processes could play a role in the interaction of Borrelia species with the host's immune system.


Assuntos
Borrelia/ultraestrutura , Membrana Celular/ultraestrutura , Variação Antigênica/genética , Variação Antigênica/imunologia , Borrelia/genética , Borrelia/metabolismo , Fusão Celular , Membrana Celular/metabolismo , Tomografia com Microscopia Eletrônica , Transferência Genética Horizontal
12.
Clin Dev Immunol ; 2012: 349657, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22400034

RESUMO

Borrelia burgdorferi evades complement-mediated killing by interacting with complement regulators through distinct complement regulator-acquiring surface proteins (CRASPs). Here, we extend our analyses to the contribution of CRASP-4 in mediating complement resistance of B. burgdorferi and its interaction with human complement regulators. CRASP-4 (also known as ErpC) was immobilized onto magnetic beads and used to capture proteins from human serum. Following Western blotting, factor H (CFH), CFH-related protein 1 (CFHR1), CFHR2, and CFHR5 were identified as ligands of CRASP-4. To analyze the impact of native CRASP-4 on mediating survival of serum-sensitive cells in human serum, a B. garinii strain was generated that ectopically expresses CRASP-4. CRASP-4-producing bacteria bound CFHR1, CFHR2, and CFHR5 but not CFH. In addition, transformed spirochetes deposited significant amounts of lethal complement components on their surface and were susceptible to human serum, thus indicating that CRASP-4 plays a subordinate role in complement resistance of B. burgdorferi.


Assuntos
Proteínas de Bactérias/imunologia , Borrelia burgdorferi/imunologia , Evasão da Resposta Imune , Doença de Lyme/imunologia , Proteínas de Membrana/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Western Blotting , Borrelia burgdorferi/genética , Proteínas Inativadoras do Complemento C3b/imunologia , Proteínas Inativadoras do Complemento C3b/metabolismo , Fator H do Complemento/imunologia , Fator H do Complemento/metabolismo , Proteínas do Sistema Complemento/imunologia , Proteínas do Sistema Complemento/metabolismo , Farmacorresistência Bacteriana , Humanos , Proteínas Imobilizadas/genética , Proteínas Imobilizadas/imunologia , Proteínas Imobilizadas/metabolismo , Doença de Lyme/metabolismo , Doença de Lyme/microbiologia , Magnetismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Plasmídeos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Transformação Genética
13.
J Biol Chem ; 285(24): 18918-27, 2010 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-20395300

RESUMO

Granule-associated perforin and granzymes (gzms) are key effector molecules of cytotoxic T lymphocytes (Tc cells) and natural killer cells and play a critical role in the control of intracellular pathogens and cancer. Based on the notion that many gzms, including A, B, C, K, H, and M exhibit cytotoxic activity in vitro, all gzms are believed to serve a similar function in vivo. However, more recent evidence supports the concept that gzms are not unidimensional but, rather, possess non-cytotoxic potential, including stimulation of pro-inflammatory cytokines and anti-viral activities. The present study shows that isolated mouse gzmB cleaves the actin-severing mouse protein, cytoplasmic gelsolin (c-gelsolin) in vitro. However, when delivered to intact target cells by ex vivo immune Tc cells, gzmB mediates c-gelsolin proteolysis via activation of caspases 3/7. The NH(2)-terminal c-gelsolin fragment generated by either gzmB or caspase 3 in vitro constitutively severs actin filaments without destroying the target cells. The observation that gzmB secreted by Tc cells initiates a caspase cascade that disintegrates the actin cytoskeleton in target cells suggests that this intracellular process may contribute to anti-viral host defense.


Assuntos
Caspase 3/metabolismo , Citoesqueleto/metabolismo , Gelsolina/química , Granzimas/metabolismo , Linfócitos T Citotóxicos/metabolismo , Animais , Apoptose , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/virologia , Fibroblastos/metabolismo , Gelsolina/metabolismo , Vírus da Coriomeningite Linfocítica/metabolismo , Camundongos , Microscopia de Fluorescência/métodos , Modelos Biológicos , RNA Mensageiro/metabolismo , Transcrição Gênica
14.
Blood ; 114(12): 2439-47, 2009 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-19528535

RESUMO

Homozygous deletion of a 84-kb genomic fragment in human chromosome 1 that encompasses the CFHR1 and CFHR3 genes represents a risk factor for hemolytic uremic syndrome (HUS) but has a protective effect in age-related macular degeneration (AMD). Here we identify CFHR1 as a novel inhibitor of the complement pathway that blocks C5 convertase activity and interferes with C5b surface deposition and MAC formation. This activity is distinct from complement factor H, and apparently factor H and CFHR1 control complement activation in a sequential manner. As both proteins bind to the same or similar sites at the cellular surfaces, the gain of CFHR1 activity presumably is at the expense of CFH-mediated function (inhibition of the C3 convertase). In HUS, the absence of CFHR1 may result in reduced inhibition of terminal complex formation and in reduced protection of endothelial cells upon complement attack. These findings provide new insights into complement regulation on the cell surface and biosurfaces and likely define the role of CFHR1 in human diseases.


Assuntos
Convertases de Complemento C3-C5/antagonistas & inibidores , Proteínas Inativadoras do Complemento C3b/fisiologia , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Animais , Células Cultivadas , Convertases de Complemento C3-C5/metabolismo , Complemento C3b/metabolismo , Complemento C3d/metabolismo , Fator H do Complemento/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Ensaio de Imunoadsorção Enzimática , Eritrócitos/metabolismo , Citometria de Fluxo , Imunofluorescência , Humanos , Técnicas Imunoenzimáticas , Imunoprecipitação , Rim/citologia , Rim/metabolismo , Microscopia Confocal , Retina/citologia , Retina/metabolismo , Ovinos , Veias Umbilicais/citologia , Veias Umbilicais/metabolismo
15.
J Cell Biol ; 174(4): 509-19, 2006 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-16893972

RESUMO

Aspergillus fumigatus infections cause high levels of morbidity and mortality in immunocompromised patients. Gliotoxin (GT), a secondary metabolite, is cytotoxic for mammalian cells, but the molecular basis and biological relevance of this toxicity remain speculative. We show that GT induces apoptotic cell death by activating the proapoptotic Bcl-2 family member Bak, but not Bax, to elicit the generation of reactive oxygen species, the mitochondrial release of apoptogenic factors, and caspase-3 activation. Activation of Bak by GT is direct, as GT triggers in vitro a dose-dependent release of cytochrome c from purified mitochondria isolated from wild-type and Bax- but not Bak-deficient cells. Resistance to A. fumigatus of mice lacking Bak compared to wild-type mice demonstrates the in vivo relevance of this GT-induced apoptotic pathway involving Bak and suggests a correlation between GT production and virulence. The elucidation of the molecular basis opens new strategies for the development of therapeutic regimens to combat A. fumigatus and related fungal infections.


Assuntos
Apoptose/fisiologia , Aspergilose/metabolismo , Aspergillus fumigatus/metabolismo , Gliotoxina/toxicidade , Imunidade Inata/genética , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Aspergilose/imunologia , Aspergilose/fisiopatologia , Aspergillus fumigatus/patogenicidade , Caspase 3 , Caspases/metabolismo , Linhagem Celular Transformada , Citocromos c/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Fibroblastos/química , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Virulência , Proteína Killer-Antagonista Homóloga a bcl-2/genética
16.
J Infect Dis ; 202(3): 490-8, 2010 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-20565259

RESUMO

Lyme disease-causing Borrelia burgdorferi spirochetes express up to 5 complement regulator-acquiring surface proteins (CRASPs). To better define how CRASP-1 contributes to infection, we aimed to identify novel CRASP-1-binding host proteins. Here, we identified a number of novel human CRASP-1-binding proteins, including bone morphogenic protein 2, collagen I, collagen III, collagen IV, fibronectin, laminin, and plasminogen. The plasminogen-binding regions were located in 2 separate regions of CRASP-1. Our results demonstrated that plasminogen-bound CRASP-1 can be converted to plasmin by the urokinase-type plasminogen activator and that proteolytically active plasmin cleaves the synthetic chromogenic substrate S-2251 and the natural substrate fibrinogen. In conclusion, CRASP-1 is a multifunctional protein of B. burgdorferi that binds to several human extracellular matrix proteins and plasminogen. These interactions may contribute to adhesion, bacterial colonization, and organ tropism and may allow dissemination of B. burgdorferi in the host.


Assuntos
Proteínas de Bactérias/metabolismo , Proteína Morfogenética Óssea 2/metabolismo , Borrelia burgdorferi/patogenicidade , Proteínas da Matriz Extracelular/metabolismo , Interações Hospedeiro-Patógeno , Proteínas de Membrana/metabolismo , Plasminogênio/metabolismo , Fatores de Virulência/metabolismo , Citometria de Fluxo , Técnica Direta de Fluorescência para Anticorpo , Humanos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Ressonância de Plasmônio de Superfície
17.
J Struct Biol ; 169(1): 54-61, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19699799

RESUMO

Bacteria can be propelled in liquids by flagellar filaments that are attached to and moved by flagellar motors. These motors are rotary nanomachines that use the electrochemical potential from ion gradients. The motor can spin in both directions with specific proteins regulating the direction in response to chemotactic stimuli. Here we investigated the structure of flagellar motors of Borrelia spirochetes, the causative agents of Lyme disease in humans. We revealed the structure of the motor complex at 4.6-nm resolution by sub-volume averaging of cryo-electron tomograms and subsequently imposing rotational symmetry. This allowed direct visualisation of individual motor components, the connection between the stator and the peptidoglycan as well as filamentous linkers between the stator and the rod. Two different motor assemblies seem to co-exist at a single bacterial pole. While most motors were completely assembled, a smaller fraction appeared to lack part of the C-ring, which plays a role in protein export and switching the directionality of rotation. Our data suggest a novel mechanism that bacteria may use to control the direction of movement.


Assuntos
Proteínas de Bactérias/ultraestrutura , Borrelia/ultraestrutura , Flagelos/ultraestrutura , Proteínas Motores Moleculares/ultraestrutura , Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Humanos , Modelos Biológicos , Peptidoglicano/ultraestrutura
18.
Infect Immun ; 78(5): 2199-208, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20231403

RESUMO

In North America, tick-borne relapsing fever is caused by the species Borrelia hermsii, B. parkeri, and B. turicatae, which are transmitted to humans through the bite of the respective infected tick vectors. Here we describe the identification and functional characterization of a surface lipoprotein of B. parkeri, designated BpcA, that binds the human complement regulators factor H and factor H-related protein 1 and, simultaneously, the host protease plasminogen. In contrast, the homologous B. turicatae protein failed to bind human factor H and factor H-related protein 1 but retained its plasminogen binding capacity. Factor H bound to BpcA maintains its regulatory capacity to control C3b deposition and C3 convertase activity. Ectopic expression of BpcA in a serum-sensitive B. burgdorferi strain protects transformed cells from complement-mediated killing. Furthermore, bound plasminogen/plasmin endows B. parkeri and B. turicatae with the potential to degrade extracellular matrix components. These findings expand our understanding of the putative recent evolutionary separation of Borrelia parkeri and Borrelia turicatae, provide evidence that B. parkeri differs from B. turicatae in its ability to resist complement attack, and may help in understanding the pathological processes underlying tick-borne relapsing fever.


Assuntos
Proteínas de Bactérias/imunologia , Proteínas Sanguíneas/imunologia , Borrelia/imunologia , Fator H do Complemento/imunologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Atividade Bactericida do Sangue , Proteínas Sanguíneas/metabolismo , Borrelia/genética , Fator H do Complemento/metabolismo , DNA Bacteriano/química , DNA Bacteriano/genética , Humanos , Dados de Sequência Molecular , América do Norte , Plasminogênio/metabolismo , Ligação Proteica , Alinhamento de Sequência , Análise de Sequência de DNA
19.
Infect Immun ; 78(11): 4467-76, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20823202

RESUMO

Spirochetes belonging to the Borrelia burgdorferi sensu lato complex differ in resistance to complement-mediated killing by human serum. Here, we characterize complement sensitivity of a panel of B. lusitaniae isolates derived from ticks collected in Germany and Portugal as well as one patient-derived isolate, PoHL. All isolates are highly susceptible to complement-mediated lysis in human serum and activate complement predominantly by the alternative pathway, leading to an increased deposition of complement components C3, C6, and the terminal complement complex. Interestingly, serum-sensitive B. lusitaniae isolates were able to bind immune regulator factor H (CFH), and some strains also bound CFH-related protein 1 (CFHR1) and CFHR2. Moreover, CFH bound to the surface of B. lusitaniae was inefficient in mediating C3b conversion. Furthermore, the identification and characterization of a potential CFH-binding protein, OspE, revealed that this molecule possesses a significantly reduced binding capacity for CFH compared to that of CFH-binding OspE paralogs expressed by various serum-resistant Borrelia species. This finding suggests that a reduced binding capability of CFH is associated with an increased serum sensitivity of B. lusitaniae to human complement.


Assuntos
Infecções por Borrelia/imunologia , Borrelia/metabolismo , Ativação do Complemento/imunologia , Fator H do Complemento/metabolismo , Via Alternativa do Complemento/imunologia , Animais , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Atividade Bactericida do Sangue , Proteínas Sanguíneas , Borrelia/classificação , Borrelia/genética , Borrelia/isolamento & purificação , Infecções por Borrelia/microbiologia , Proteínas Inativadoras do Complemento C3b , Fator H do Complemento/química , Alemanha , Humanos , Ixodes/microbiologia , Dados de Sequência Molecular , Portugal , Análise de Sequência de DNA
20.
Infect Immun ; 78(1): 39-48, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19858303

RESUMO

Acquisition of complement regulator factor H (CFH) and factor H-like protein 1 (CFHL1) from human serum enables Borrelia spielmanii, one of the etiological agents of Lyme disease, to evade complement-mediated killing by the human host. Up to three distinct complement regulator-acquiring surface proteins (CRASPs) may be expressed by serum-resistant B. spielmanii, each exhibiting an affinity for CFH and/or CFHL1. Here, we describe the functional characterization of the 15-kDa CRASPs of B. spielmanii, members of the polymorphic Erp (OspE/F-related) protein family, that bind two distinct host complement regulators, CFH and factor H-related protein 1 (CFHR1), but not CFHL1. CFH bound to the B. spielmanii CRASPs maintained cofactor activity for factor I-mediated C3b inactivation. Three naturally occurring alleles of this protein bound CFH and CFHR1 while a fourth natural allele could not. Comparative sequence analysis of these protein alleles identified a single amino acid, histidine-79, as playing a significant role in CFH/CFHR1 binding, with substitution by an arginine completely abrogating ligand binding. The mutation of His-79 to Arg did not inhibit binding of plasminogen, another known ligand of this group of borrelial outer-surface proteins.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Borrelia/metabolismo , Fator H do Complemento/metabolismo , Plasminogênio/metabolismo , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/genética , Ativação do Complemento , Fator H do Complemento/genética , Meios de Cultura , Regulação Bacteriana da Expressão Gênica , Humanos , Dados de Sequência Molecular , Ligação Proteica , Soro/química
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