RESUMO
The intracellular protozoan parasite Trypanosoma cruzi causes Chagas' disease, which affects millions of people in Latin America. T. cruzi enters a large number of cell types by an unusual mechanism that involves Ca(2+)-triggered fusion of lysosomes with the plasma membrane. Here we show that synaptotagmin VII (Syt VII), a ubiquitously expressed synaptotagmin isoform that regulates exocytosis of lysosomes, is localized on the membranes of intracellular vacuoles containing T. cruzi. Antibodies against the C(2)A domain of Syt VII or recombinant peptides including this domain inhibit cell entry by T. cruzi, but not by Toxoplasma gondii or Salmonella typhimurium. The C(2)A domains of other ubiquitously expressed synaptotagmin isoforms have no effect on T. cruzi invasion, and mutation of critical residues on Syt VII C(2)A abolish its inhibitory activity. These findings indicate that T. cruzi exploits the Syt VII-dependent, Ca(2+)-regulated lysosomal exocytic pathway for invading host cells.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Exocitose/imunologia , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Trypanosoma cruzi/patogenicidade , Células 3T3 , Animais , Células CHO , Proteínas de Ligação ao Cálcio/genética , Cricetinae , Células HeLa , Humanos , Glicoproteínas de Membrana/genética , Camundongos , Proteínas do Tecido Nervoso/genética , SinaptotagminasRESUMO
An early event in the Trypanosoma cruzi cell invasion process, the recruitment of host lysosomes, led us to investigate the involvement of signal transduction. Infective trypomastigotes were found to contain a soluble Ca2+-signaling activity for mammalian cells that is sensitive to protease inhibitors. Inhibitor and substrate utilization profiles were used to purify a candidate peptidase for involvement in this process, from which we isolated a full-length cDNA clone. The sequence revealed a novel enzyme, denominated T. cruzi oligopeptidase B, which is homologous to members of the prolyl oligopeptidase family of serine hydrolases, known to participate in the maturation of biologically active peptides. The T. cruzi oligopeptidase B was expressed as a fully active product in Escherichia coli, and antibodies to the recombinant enzyme inhibited both peptidase activity and Ca2+ signaling induced in normal rat kidney cells by trypomastigote extracts. Our data suggest that the T. cruzi oligopeptidase B participates in processing events in the cytoplasm of the parasites, generating a factor with Ca2+-signaling activity for mammalian cells.
Assuntos
Cálcio/metabolismo , Proteínas de Protozoários/metabolismo , Serina Endopeptidases/metabolismo , Transdução de Sinais/fisiologia , Trypanosoma cruzi/enzimologia , Sequência de Aminoácidos , Animais , Anticorpos/farmacologia , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Citoplasma/metabolismo , Citosol/enzimologia , DNA Complementar , Dosagem de Genes , Expressão Gênica , Cobaias , Hidrolases/química , Hidrólise/efeitos dos fármacos , Dados de Sequência Molecular , Prolil Oligopeptidases , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Serina Endopeptidases/química , Serina Endopeptidases/genéticaRESUMO
Plasma membrane wounds are repaired by a mechanism involving Ca(2+)-regulated exocytosis. Elevation in intracellular [Ca(2+)] triggers fusion of lysosomes with the plasma membrane, a process regulated by the lysosomal synaptotagmin isoform Syt VII. Here, we show that Ca(2+)-regulated exocytosis of lysosomes is required for the repair of plasma membrane disruptions. Lysosomal exocytosis and membrane resealing are inhibited by the recombinant Syt VII C(2)A domain or anti-Syt VII C(2)A antibodies, or by antibodies against the cytosolic domain of Lamp-1, which specifically aggregate lysosomes. We further demonstrate that lysosomal exocytosis mediates the resealing of primary skin fibroblasts wounded during the contraction of collagen matrices. These findings reveal a fundamental, novel role for lysosomes: as Ca(2+)-regulated exocytic compartments responsible for plasma membrane repair.
Assuntos
Cálcio/metabolismo , Membrana Celular/metabolismo , Membrana Celular/patologia , Exocitose , Lisossomos/metabolismo , Regeneração/fisiologia , Animais , Antígenos CD/química , Antígenos CD/metabolismo , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/metabolismo , Divisão Celular , Linhagem Celular , Membrana Celular/química , Células Cultivadas , Colágeno/metabolismo , Cricetinae , Fibroblastos/metabolismo , Fibroblastos/patologia , Citometria de Fluxo , Humanos , Proteínas de Membrana Lisossomal , Lisossomos/química , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Camundongos , Microinjeções , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Estrutura Terciária de Proteína , Ratos , Pele/metabolismo , Pele/patologia , Sinaptotagminas , Ferimentos e Lesões/metabolismo , Ferimentos e Lesões/patologiaRESUMO
Cell invasion by the protozoan parasite Trypanosoma cruzi involves activation of host signaling pathways and the recruitment and fusion of lysosomes at the parasite entry site. A major signaling pathway regulating invasion of fibroblasts, epithelial cells, and myoblasts involves mobilization of Ca(2+) from intracellular stores and requires the activity of a T. cruzi serine peptidase, oligopeptidase B (OPB). Deletion of the OPB gene results in a marked defect in trypomastigote virulence, consistent with a greatly reduced cell invasion capacity. Here we show that uptake by macrophages, on the other hand, is largely independent of OPB expression and sensitive to inhibition of by cytochalasin D. The residual invasion capacity of OPBnull trypomastigotes in fibroblasts still involves lysosome recruitment, although in a significantly delayed fashion. Transient elevations in intracellular Ca(2+) concentrations were observed in host cells exposed to both wild-type and OPBnull trypomastigotes, but the signals triggered by the mutant parasites were less vigorous and delayed. The capacity of triggering elevation in host cell cyclic AMP (cAMP), however, was unaltered in OPBnull trypomastigotes. Modulation in cAMP levels preferentially affected the residual cell invasion capacity of OPBnull parasites, suggesting that this signaling pathway can play a dominant role in promoting cell invasion in the absence of the major OPB-dependent pathway.
Assuntos
Cálcio/metabolismo , AMP Cíclico/biossíntese , Serina Endopeptidases/fisiologia , Trypanosoma cruzi/patogenicidade , Actinas/metabolismo , Animais , Linhagem Celular , Lisossomos/metabolismo , Camundongos , Ratos , Fatores de Virulência de Bordetella/farmacologiaRESUMO
Mammalian cell invasion by the intracellular protozoan parasite Trypanosoma cruzi is mediated by recruitment and fusion of host cell lysosomes, an unusual process that has been proposed to be dependent on the ability of parasites to trigger intracellular free calcium concentration ([Ca2+]i) transients in host cells. Previous work implicated the T.cruzi serine hydrolase oligopeptidase B in the generation of Ca2+-signaling activity in parasite extracts. Here we show that deletion of the gene encoding oligopeptidase B results in a marked defect in host cell invasion and in the establishment of infections in mice. The invasion defect is associated with the inability of oligopeptidase B null mutant trypomastigotes to mobilize Ca2+ from thapsigargin-sensitive stores in mammalian cells. Exogenous recombinant oligopeptidase B reconstitutes the oligopeptidase B-dependent Ca2+ signaling activity in null mutant parasite extracts, demonstrating that this enzyme is responsible for the generation of a signaling agonist for mammalian cells.
Assuntos
Serina Endopeptidases/metabolismo , Trypanosoma cruzi/patogenicidade , Animais , Sinalização do Cálcio , Feminino , Deleção de Genes , Haplorrinos , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos C3H , Modelos Biológicos , Músculos/citologia , Músculos/parasitologia , Mutagênese , Proteínas de Protozoários/metabolismo , Ratos , Serina Endopeptidases/genética , Trypanosoma cruzi/genéticaRESUMO
Trypanosoma brucei contains a soluble serine oligopeptidase (OP-Tb) that is released into the host bloodstream during infection, where it has been postulated to participate in the pathogenesis of African trypanosomiasis. Here, we report the identification of a single copy gene encoding the T. brucei oligopeptidase and a homologue from the related trypanosomatid pathogen Leishmania major. The enzymes encoded by these genes belong to an emerging subgroup of the prolyl oligopeptidase family of serine hydrolases, referred to as oligopeptidase B. The trypanosomatid oligopeptidases share 70% amino acid sequence identity with oligopeptidase B from the intracellular pathogen Trypanosoma cruzi, which has a demonstrated role in mammalian host cell signaling and invasion. OP-Tb exhibited no activity toward the prolyl oligopeptidase substrate H-Gly-Pro-7-amido-4-methylcoumarin. Instead, it had activity toward substrates of trypsin-like enzymes, particularly those that have basic amino acids in both P(1) and P(2) (e.g. benzyloxycarbonyl-Arg-Arg-7-amido-4-methylcoumarin k(cat)/K(m) = 529 s(-1) microM(-1)). The activity of OP-Tb was enhanced by reducing agents and by polyamines, suggesting that these agents may act as in vivo regulators of OP-Tb activity. This study provides the basis of the characterization of a novel subgroup of serine oligopeptidases from kinetoplastid protozoa with potential roles in pathogenesis.