RESUMO
Protein S-palmitoylation, a hydrophobic post-translational modification, is performed by protein acyltransferases that have a common DHHC Cys-rich domain (DHHC proteins), and provides a regulatory switch for protein membrane association. In this work, we analyzed the presence of DHHC proteins in the protozoa parasite Giardia lamblia and the function of the reversible S-palmitoylation of proteins during parasite differentiation into cyst. Two specific events were observed: encysting cells displayed a larger amount of palmitoylated proteins, and parasites treated with palmitoylation inhibitors produced a reduced number of mature cysts. With bioinformatics tools, we found nine DHHC proteins, potential protein acyltransferases, in the Giardia proteome. These proteins displayed a conserved structure when compared to different organisms and are distributed in different monophyletic clades. Although all Giardia DHHC proteins were found to be present in trophozoites and encysting cells, these proteins showed a different intracellular localization in trophozoites and seemed to be differently involved in the encystation process when they were overexpressed. dhhc transgenic parasites showed a different pattern of cyst wall protein expression and yielded different amounts of mature cysts when they were induced to encyst. Our findings disclosed some important issues regarding the role of DHHC proteins and palmitoylation during Giardia encystation.
Assuntos
Aciltransferases/análise , Aciltransferases/química , Giardia lamblia , Proteínas de Protozoários/análise , Proteínas de Protozoários/química , Animais , Biologia Computacional , Giardia lamblia/química , Giardia lamblia/enzimologia , Giardia lamblia/fisiologia , Processamento de Proteína Pós-TraducionalRESUMO
SUMOylation, a posttranslational modification of proteins, has been recently described as vital in eukaryotic cells. In a previous work, we analyzed the role of SUMO protein and the genes encoding the putative enzymes of the SUMOylation pathway in the parasite Giardia lamblia. Although we observed several SUMOylated proteins, only the enzyme Arginine Deiminase (ADI) was confirmed as a SUMOylated substrate. ADI is involved in the survival of the parasite and, besides its role in ATP production, it also catalyzes the modification of arginine residues to citrulline in the cytoplasmic tail of surface proteins. During encystation, however, ADI translocates to the nuclei and downregulates the expression of the Cyst Wall Protein 2 (CWP2). In this work, we made site-specific mutation of the ADI SUMOylation site (Lys101) and observed that transgenic trophozoites did not translocate to the nuclei at the first steps of encystation but shuttled in the nuclei late during this process through classic nuclear localization signals. Inside the nuclei, ADI acts as a peptidyl arginine deiminase, being probably involved in the downregulation of CWPs expression and cyst wall formation. Our results strongly indicate that ADI plays a regulatory role during encystation in which posttranslational modifications of proteins are key players.
Assuntos
Epigênese Genética , Giardia lamblia/genética , Giardia lamblia/metabolismo , Iminas/metabolismo , Proteínas de Protozoários/metabolismo , Esporos de Protozoários/metabolismo , Sumoilação , Sequência de Aminoácidos , Animais , Núcleo Celular/enzimologia , Simulação por Computador , Regulação para Baixo , Giardia lamblia/enzimologia , Hidrolases/química , Hidrolases/metabolismo , Lisina/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Sinais de Localização Nuclear , Processamento de Proteína Pós-Traducional , Transporte Proteico , Desiminases de Arginina em ProteínasRESUMO
The retromer is a pentameric protein complex that mediates the retrograde transport of acid hydrolase receptors between endosomes and the trans-Golgi network and is conserved across all eukaryotes. Unlike other eukaryotes, the endomembrane system of Giardia trophozoite is simple and is composed only of the endoplasmic reticulum and peripheral vesicles (PVs), which may represent an ancient organellar system converging compartments such as early and late endosomes and lysosomes. Sorting and trafficking of membrane proteins and soluble hydrolases from the endoplasmic reticulum to the PVs have been described as specific and conserved but whether the giardial retromer participates in receptor recycling remains elusive. Homologs of the retromer Vacuolar Protein Sorting (Vps35p, Vps26p, and Vps29p) have been identified in this parasite. Cloning the GlVPS35 subunit and antisera production enabled the localization of this protein in the PVs as well as in the cytosol. Tagged expression of the subunits was used to demonstrate their association with membranes, and immunofluorescence confocal laser scanning revealed high degrees of colabeling between the retromer subunits and also with the endoplasmic reticulum and PV compartment markers. Protein-protein interaction data revealed interaction between the subunits of GlVPS35 and the cytosolic domain of the hydrolase receptor GlVps. Altogether our data provide original information on the molecular interactions that mediate assembly of the cargo-selective retromer subcomplex and its involvement in the recycling of the acid hydrolase receptor in this parasite.
Assuntos
Giardia/metabolismo , Complexos Multiproteicos/metabolismo , Subunidades Proteicas/metabolismo , Proteínas de Protozoários/metabolismo , Vacúolos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Sequência de Aminoácidos , Animais , Transporte Biológico , Membrana Celular/metabolismo , Centrifugação , Camundongos , Camundongos Endogâmicos BALB C , Modelos Biológicos , Dados de Sequência Molecular , Ligação Proteica , Proteínas de Protozoários/química , Frações Subcelulares/metabolismoRESUMO
Post-translational modifications are able to regulate protein function and cellular processes in a rapid and reversible way. SUMOylation, the post-translational modification of proteins by the addition of SUMO, is a highly conserved process that seems to be present in modern cells. However, the mechanism of protein SUMOylation in earlier divergent eukaryotes, such as Giardia lamblia, is only starting to become apparent. In this work, we report the presence of a single SUMO gene encoding to SUMO protein in Giardia. Monoclonal antibodies against recombinant Giardia SUMO protein revealed the cytoplasmic localization of native SUMO in wild-type trophozoites. Moreover, the over-expression of SUMO protein showed a mainly cytoplasmic localization, though also neighboring the plasma membrane, flagella, and around and even inside the nuclei. Western blot assays revealed a number of SUMOylated proteins in a range between 20 and 120 kDa. The genes corresponding to putative enzymes involved in the SUMOylation pathway were also explored. Our results as a whole suggest that SUMOylation is a process conserved in the eukaryotic lineage, and that its study is significant for understanding the biology of this interesting parasite and the role of post-translational modification in its evolution.
RESUMO
BACKGROUND: To date, eight assemblages of Giardia lamblia have been described, but only assemblages A and B are known to infect humans. Despite the fact that the genomic, biological, and clinical differences found between these two assemblages has raised the possibility that they may be considered different species, there is relatively limited information on their phenotypic differences. In the present study, we developed monoclonal antibodies against alpha-1 and beta giardin, two immunodominant proteins produced during G. lamblia infection, and studied their expression and localization in WB (assemblage A) and GS trophozoites (assemblage B). RESULTS: The polyclonal antibodies generated against WB trophozoites, particularly those recognizing intracellular proteins as well as the proteins present at the plasma membrane (variable-specific surface proteins), showed cross-reactivity with intracellular proteins in GS trophozoites. The use of monoclonal antibodies against beta giardin indicated ventral disc localization, particularly at the periphery in WB trophozoites. Interestingly, although beta giardin was also restricted to the ventral disc in GS trophozoites, the pattern of localization clearly differed in this assemblage. On the other hand, monoclonal antibodies against alpha-1 giardin showed plasma membrane localization in both assemblages with the bare area of GS trophozoites also being distinguished. Moreover, the same localization at the plasma membrane was observed in Portland-1 (Assemblage A) and in P15 (Assemblage E) trophozoites. CONCLUSIONS: We found differences in localization of the beta giardin protein between assemblages A and B, but the same pattern of localization of alpha-1 giardin in strains from Assemblages A, B and E. These findings reinforce the need for more studies based on phenotypic characteristics in order to disclose how far one assemblage is from the other.
Assuntos
Proteínas do Citoesqueleto/genética , Regulação da Expressão Gênica , Giardia lamblia/genética , Giardíase/parasitologia , Proteínas de Protozoários/genética , Sequência de Aminoácidos , Animais , Membrana Celular , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/imunologia , Feminino , Giardia lamblia/classificação , Giardia lamblia/crescimento & desenvolvimento , Giardia lamblia/imunologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Transporte Proteico , Proteínas de Protozoários/química , Proteínas de Protozoários/imunologia , Alinhamento de Sequência , Trofozoítos/química , Trofozoítos/crescimento & desenvolvimento , Trofozoítos/metabolismoRESUMO
Acute infection with Trypanosoma cruzi, the aetiological agent of Chagas' disease, results in parasitaemia and polyclonal lymphocyte activation. It has been reported that polyclonal B-cell activation is associated with hypergammaglobulinaemia and delayed parasite-specific antibody response. In the present study we analysed the development of a B-cell response within the different microenvironments of the spleen during acute T. cruzi infection. We observed massive germinal centre (GC) and extrafollicular (EF) responses at the peak of infection. However, the EF foci were evident since day 3 post-infection (p.i.), and, early in the infection, they mainly provided IgM. The EF foci response reached its peak at 11 days p.i. and extended from the red pulp into the periarteriolar lymphatic sheath. The GCs were detected from day 8 p.i. At the peak of parasitaemia, CD138(+) B220(+) plasma cells in EF foci, red pulp and T-cell zone expressed IgM and all the IgG isotypes. Instead of the substantial B-cell response, most of the antibodies produced by splenic cells did not target the parasite, and parasite-specific IgG isotypes could be detected in sera only after 18 days p.i. We also observed that the bone marrow of infected mice presented a strong reduction in CD138(+) B220(+) cells compared with that of normal mice. Hence, in acute infection with T. cruzi, the spleen appears to be the most important lymphoid organ that lodges plasma cells and the main producer of antibodies. The development of a B-cell response during T. cruzi infection shows features that are particular to T. cruzi and other protozoan infection but different to other infections or immunization with model antigens.
Assuntos
Anticorpos/imunologia , Linfócitos B/imunologia , Baço/citologia , Baço/imunologia , Trypanosoma cruzi/imunologia , Animais , Anticorpos Antiprotozoários/imunologia , Doença de Chagas/imunologia , Doença de Chagas/parasitologia , Contagem de Linfócitos , Camundongos , Camundongos Endogâmicos BALB C , Trypanosoma cruzi/isolamento & purificaçãoRESUMO
BACKGROUND: B cells and antibodies are involved not only in controlling the spread of blood circulating Trypanosoma cruzi, but also in the autoreactive manifestations observed in Chagas disease. Acute infection results in polyclonal B cell activation associated with hypergammaglobulinemia, delayed specific humoral immunity and high levels of non-parasite specific antibodies. Since TNF superfamily B lymphocyte Stimulator (BAFF) mediates polyclonal B cell response in vitro triggered by T. cruzi antigens, and BAFF-Tg mice show similar signs to T. cruzi infected mice, we hypothesized that BAFF can mediate polyclonal B cell response in experimental Chagas disease. METHODOLOGY/PRINCIPAL FINDINGS: BAFF is produced early and persists throughout the infection. To analyze BAFF role in experimental Chagas disease, Balb/c infected mice were injected with BR3:Fc, a soluble receptor of BAFF, to block BAFF activity. By BAFF blockade we observed that this cytokine mediates the mature B cell response and the production of non-parasite specific IgM and IgG. BAFF also influences the development of antinuclear IgG and parasite-specific IgM response, not affecting T. cruzi-specific IgG and parasitemia. Interestingly, BAFF inhibition favors the parasitism in heart. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate, for the first time, an active role for BAFF in shaping the mature B cell repertoire in a parasite infection.
Assuntos
Anticorpos Antiprotozoários/biossíntese , Fator Ativador de Células B/metabolismo , Linfócitos B/imunologia , Doença de Chagas/imunologia , Baço/imunologia , Trypanosoma cruzi/imunologia , Animais , Fator Ativador de Células B/antagonistas & inibidores , Modelos Animais de Doenças , Imunoglobulina G/biossíntese , Imunoglobulina M/biossíntese , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Humoral immunity during experimental Chagas disease has been considered a double-edge sword, critical to control Trypanosoma cruzi spreading but also associated to tissue damage. Peritoneal B-1 cells have been linked to the pathogenesis of Chagas disease; however, they may also help to control the infection by providing a fast wave of antibodies. In the present work, we determined that peritoneal B-cell response to T. cruzi is characterized by a marked reduction of CD19(+) B cells due to plasma cell differentiation rather than to cell death. Both peritoneal B-2 and B-1 cells decrease after parasite infection, but with different kinetics. Thus, the reduction in B-2 cell number can be detected from day 4 postinfection while the number of B-1 cells decreases only after 15 days of infection. Differentiation of peritoneal B-1 and B-2 cells into IgM-secreting cells was triggered by parasites but not by cytokines produced by peritoneal cells. Electron microscopy studies showed that peritoneum of infected mice lodges plasma cells with typical morphology as well as atypical plasma cells named 'Mott-like cells' containing high number of cytoplasmatic Ig(+) granules. The plasma cells induced during the infection showed a phenotype that may allow their persistence in peritoneum and they may contribute to the high levels of antibodies exhibited at the chronic phase of infection. We also showed that the peritoneal B-cell response is scarcely specific for the invading pathogen and rather constitute an important source of non-parasite-specific IgM and IgG in the infected host.
Assuntos
Anticorpos/imunologia , Doença de Chagas/imunologia , Peritônio/citologia , Peritônio/imunologia , Plasmócitos/citologia , Plasmócitos/imunologia , Trypanosoma cruzi/imunologia , Animais , Antígenos CD19/imunologia , Diferenciação Celular , Células Cultivadas , Modelos Animais de Doenças , Cinética , Camundongos , Camundongos Endogâmicos BALB C , Plasmócitos/metabolismo , Sindecana-1/análise , Fatores de Transcrição/genética , Fatores de Transcrição/imunologia , Trypanosoma cruzi/isolamento & purificaçãoRESUMO
The whole life of a B-cell from a stem cell to a mature plasma cell is governed, among other factors, by cytokines and growth factors in their microenvironment. Remarkable progress in the understanding of the mechanisms of cytokines action on the B-cell compartment was achieved by analysis of gene-targeted mice. The generation of mice deficient for individual cytokines or their receptors has shed light on the in vivo function of cytokines in B-cell responses. This review focuses on the role of cytokines in the development, maturation and differentiation of different B-cell subsets into antibody-secreting cells or memory B-cells.
Assuntos
Subpopulações de Linfócitos B/imunologia , Diferenciação Celular/imunologia , Quimiocinas/imunologia , Citocinas/imunologia , Células-Tronco Hematopoéticas/imunologia , Plasmócitos/imunologia , Animais , Humanos , Memória ImunológicaRESUMO
Microorganisms with pathogen-associated molecular patterns (PAMP) activate B cells directly by binding to TLR and also indirectly by inducing APC to release cytokines such as BAFF that promote B cell survival. We found that murine B cells activated concomitantly with LPS (TLR-4 ligand) and BAFF are protected from spontaneous apoptosis, but are more susceptible to Fas/CD95-mediated cell death. This increased susceptibility to Fas-induced apoptosis is associated with a dramatic coordinated up-regulation of Fas/CD95 and IRF-4 expression through a mechanism mediated, at least in part, by inhibition of the MEK/ERK pathway. Up-regulation of Fas/CD95 by BAFF is restricted to B cells activated through TLR-4, but not through TLR-9, BCR or CD40. TLR ligands differ in the BAFF family receptors (R) they induce on B cells: BAFF-R is increased by the TLR4 ligand, LPS, but not by the TLR9 ligand, CpG-containing oligodeoxynucleotides, which, in contrast, strongly up-regulates transmembrane activator and CAML interactor (TACI). This suggests the up-regulation of Fas by BAFF is mediated by BAFF-R and not by TACI. Consistently, APRIL, which binds to TACI and B cell maturation antigen but not BAFF-R, did not enhance Fas expression on LPS-activated B cells. Increased susceptibility to Fas-mediated killing of B cells activated with LPS and BAFF may be a fail-safe mechanism to avoid overexpansion of nonspecific or autoreactive B cells.
Assuntos
Apoptose/imunologia , Fator Ativador de Células B/fisiologia , Subpopulações de Linfócitos B/citologia , Subpopulações de Linfócitos B/imunologia , Lipopolissacarídeos/farmacologia , Receptor fas/fisiologia , Animais , Subpopulações de Linfócitos B/metabolismo , Morte Celular/imunologia , Células Cultivadas , Inibidores do Crescimento/biossíntese , Inibidores do Crescimento/fisiologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Regulação para Cima/imunologia , Receptor fas/biossínteseRESUMO
B1 lymphocytes are an anatomically, phenotypically, and functionally distinct subset of B cells producing the bulk of natural serum IgM in the absence of any apparent stimulation by specific antigens. These cells are a dominant population of B cells in peritoneal and pleural cavities and they have characteristics of activated cells and higher cell size and cytoplasmic complexity than conventional B cells. B1 cells spontaneously secrete antibodies and operate under a differentiation program that is unique and differs from the paradigm associated with Ig-secreting B-2 cells. The antibodies produced by B1 cells may participate in a variety of physiological activities since they are involve in immune regulation, clearance of senescent and apoptotic cells and resistance to infection. However, it has been suggested that they are also involved in autoimmunity. Many advances have been made to describe the origin, development and differentiation of B1 cells, which will be examined here.
Assuntos
Autoimunidade , Linfócitos B/imunologia , Animais , Anticorpos/imunologia , Antígenos CD/imunologia , Linfócitos B/citologia , Humanos , Sistema Imunitário/fisiologia , Imunidade Celular , Imunoglobulinas/fisiologia , Cavidade Peritoneal/citologia , Cavidade Peritoneal/fisiologiaRESUMO
Las células B1, responsables de la producción de IgM sérica en ausencia de aparente estimulaciónantigénica, son linfocitos B maduros con ubicación anatómica y características fenotípicas y funcionalesparticulares. Los linfocitos B1 se ubican mayoritariamente en cavidad peritoneal y pleural, presentancaracterísticas de células activadas y son de mayor tamaño y complejidad citoplasmática que las células B convencionales.Mientras que estos últimos deben diferenciarse a células plasmáticas para poder secretarinmunoglobulinas, los linfocitos B1 liberan espontáneamente anticuerpos al medio extracelular operando bajoun programa de diferenciación particular. Los anticuerpos producidos por los linfocitos B1 tendrían un rol protector,ya que están implicados en la remoción de células envejecidas y apoptóticas, en mecanismos deinmunomodulación y en resistencia a infecciones, sin embargo su participación en procesos autoinmunes tambiénha sido sugerida. Muchos estudios han aportado información sobre el origen, desarrollo y diferenciaciónde los linfocitos B1, los cuales son analizados en esta revisión. (AU)
B1 lymphocytes are an anatomically, phenotypically, and functionally distinct subset ofB cells producing the bulk of natural serum IgM in the absence of any apparent stimulation by specific antigens.These cells are a dominant population of B cells in peritoneal and pleural cavities and they have characteristicsof activated cells and higher cell size and cytoplasmic complexity than conventional B cells. B1 cells spontaneouslysecrete antibodies and operate under a differentiation program that is unique and differs from the paradigmassociated with Ig-secreting B-2 cells. The antibodies produced by B1 cells may participate in a variety ofphysiological activities since they are involve in immune regulation, clearance of senescent and apoptotic cellsand resistance to infection. However, it has been suggested that they are also involved in autoimmunity. Manyadvances have been made to describe the origin, development and differentiation of B1 cells, which will beexamined here. (AU)
Assuntos
Humanos , Animais , Autoimunidade , Linfócitos B/imunologia , Cavidade Peritoneal , Anticorpos/imunologia , Sistema Imunitário/fisiologia , Antígenos CD/imunologia , Cavidade Peritoneal/citologia , Cavidade Peritoneal/fisiologia , Imunoglobulinas/fisiologiaRESUMO
Las células B1, responsables de la producción de IgM sérica en ausencia de aparente estimulaciónantigénica, son linfocitos B maduros con ubicación anatómica y características fenotípicas y funcionalesparticulares. Los linfocitos B1 se ubican mayoritariamente en cavidad peritoneal y pleural, presentancaracterísticas de células activadas y son de mayor tamaño y complejidad citoplasmática que las células B convencionales.Mientras que estos últimos deben diferenciarse a células plasmáticas para poder secretarinmunoglobulinas, los linfocitos B1 liberan espontáneamente anticuerpos al medio extracelular operando bajoun programa de diferenciación particular. Los anticuerpos producidos por los linfocitos B1 tendrían un rol protector,ya que están implicados en la remoción de células envejecidas y apoptóticas, en mecanismos deinmunomodulación y en resistencia a infecciones, sin embargo su participación en procesos autoinmunes tambiénha sido sugerida. Muchos estudios han aportado información sobre el origen, desarrollo y diferenciaciónde los linfocitos B1, los cuales son analizados en esta revisión. (AU)
B1 lymphocytes are an anatomically, phenotypically, and functionally distinct subset ofB cells producing the bulk of natural serum IgM in the absence of any apparent stimulation by specific antigens.These cells are a dominant population of B cells in peritoneal and pleural cavities and they have characteristicsof activated cells and higher cell size and cytoplasmic complexity than conventional B cells. B1 cells spontaneouslysecrete antibodies and operate under a differentiation program that is unique and differs from the paradigmassociated with Ig-secreting B-2 cells. The antibodies produced by B1 cells may participate in a variety ofphysiological activities since they are involve in immune regulation, clearance of senescent and apoptotic cellsand resistance to infection. However, it has been suggested that they are also involved in autoimmunity. Manyadvances have been made to describe the origin, development and differentiation of B1 cells, which will beexamined here. (AU)
Assuntos
Humanos , Animais , Autoimunidade , Linfócitos B/imunologia , Cavidade Peritoneal , Anticorpos/imunologia , Sistema Imunitário/fisiologia , Antígenos CD/imunologia , Cavidade Peritoneal/citologia , Cavidade Peritoneal/fisiologia , Imunoglobulinas/fisiologiaRESUMO
Las células B1, responsables de la producción de IgM sérica en ausencia de aparente estimulaciónantigénica, son linfocitos B maduros con ubicación anatómica y características fenotípicas y funcionalesparticulares. Los linfocitos B1 se ubican mayoritariamente en cavidad peritoneal y pleural, presentancaracterísticas de células activadas y son de mayor tamaño y complejidad citoplasmática que las células B convencionales.Mientras que estos últimos deben diferenciarse a células plasmáticas para poder secretarinmunoglobulinas, los linfocitos B1 liberan espontáneamente anticuerpos al medio extracelular operando bajoun programa de diferenciación particular. Los anticuerpos producidos por los linfocitos B1 tendrían un rol protector,ya que están implicados en la remoción de células envejecidas y apoptóticas, en mecanismos deinmunomodulación y en resistencia a infecciones, sin embargo su participación en procesos autoinmunes tambiénha sido sugerida. Muchos estudios han aportado información sobre el origen, desarrollo y diferenciaciónde los linfocitos B1, los cuales son analizados en esta revisión.
B1 lymphocytes are an anatomically, phenotypically, and functionally distinct subset ofB cells producing the bulk of natural serum IgM in the absence of any apparent stimulation by specific antigens.These cells are a dominant population of B cells in peritoneal and pleural cavities and they have characteristicsof activated cells and higher cell size and cytoplasmic complexity than conventional B cells. B1 cells spontaneouslysecrete antibodies and operate under a differentiation program that is unique and differs from the paradigmassociated with Ig-secreting B-2 cells. The antibodies produced by B1 cells may participate in a variety ofphysiological activities since they are involve in immune regulation, clearance of senescent and apoptotic cellsand resistance to infection. However, it has been suggested that they are also involved in autoimmunity. Manyadvances have been made to describe the origin, development and differentiation of B1 cells, which will beexamined here.