RESUMO
Trypanosoma cruzi can compromise the human central nervous system (CNS) during acute infection or reactivation in immune-suppressed hosts. Astrocytes have been identified as targets of T. cruzi's CNS infection in humans. Despite a high degree of parasitism and cellular lysis by T. cruzi in vitro the number of astrocytoma cells did not change when compared to uninfected cultures. This work evaluated cellular proliferation, changes in Major Histocompatibility Complex (MHC) expression as a reflection of antigen processing, and cytokine (IL-6 & IL-8) secretion in a human astrocytoma cell line exposed to a trypomastigote-derived antigen. Light microscopy was used to evaluate the number of cells; MHC molecule expression, cell cycle and cytokine secretion were assessed by flow cytometry. The number of astrocytoma cells increased proportional to the amount of antigen used and the percentage of cells in G2/M phase was higher when compared to control cultures. Antigen exposure increased expression of MHC class II, but not MHC class I in comparison to cultures incubated without antigen. Astrocytoma cell secretion of IL-6 and IL-8 was unaffected by antigen exposure. These results suggest the participation of a trypomastigote-derived mediator that induces astrocytoma cell proliferation without an inflammatory response; which may contribute to the pathogenesis of neurologic Chagas disease.
Assuntos
Antígenos de Protozoários/farmacologia , Trypanosoma cruzi/metabolismo , Astrocitoma/metabolismo , Astrocitoma/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Microscopia , Regulação para Cima/efeitos dos fármacosRESUMO
HSP70 protein is involved in Leishmania differentiation, apoptosis, antimony-resistance and host-immune response. Therefore, this protein and the regulatory mechanisms of HSP70 gene expression are promising targets for therapeutic intervention against leishmaniasis. The regulation of mRNA expression in trypanosomatids operates mostly through the interaction of trans-acting proteins, and elements located in the untranslated regions of mRNAs. The aim of this work was to identify protein factors interacting specifically with the Leishmania braziliensis HSP70 mRNAs. Thus, the 5' UTR and the two types of 3' UTRs (UTR-I and UTR-II) from L. braziliensis HSP70 genes were used as baits in pull down assays using total protein extracts from parasites cultured at 26 or 35°C. The captured proteins were resolved by two-dimensional gel electrophoresis (2-DE) and identified by mass spectrometry (MS) analysis. As a result, 52 different proteins were identified based on their binding to the L. braziliensis HSP70-mRNAs. As expected, several of the identified proteins were related to RNA metabolism (27%) and translation process (7%). In addition, five hypothetical conserved proteins having motifs related with RNA interaction were also identified (9.6%). Nevertheless, unexpected proteins, apparently unrelated to the mRNA expression, were also identified. The biological significance of these and others L. braziliensis detected proteins, including the HSP70 itself, is discussed. BIOLOGICAL SIGNIFICANCE: For the first time, a riboproteomic analysis of the proteins interacting with the untranslated regions of the heat shock protein 70 (HSP70) mRNA from Leishmania braziliensis was carried out. This work provides new insights related to protein factors putatively involved in the regulation of HSP70 gene expression in L. braziliensis, and thereby, contributes to a better understanding of the parasite biology, and ultimately to the development of novel therapeutic interventions for controlling the important diseases caused by this parasite.
Assuntos
Regiões 3' não Traduzidas/fisiologia , Regiões 5' não Traduzidas/fisiologia , Regulação da Expressão Gênica/fisiologia , Proteínas de Choque Térmico HSP70/biossíntese , Leishmania braziliensis/metabolismo , Proteínas de Protozoários/biossíntese , RNA de Protozoário/metabolismo , Proteínas de Choque Térmico HSP70/genética , Leishmania braziliensis/genética , Proteínas de Protozoários/genética , RNA de Protozoário/genéticaRESUMO
Calcineurin B, the regulatory subunit of calcineurin, a serine/threonine protein phosphatase, is highly conserved throughout the evolutionary scale including trypanosomatids such as Trypanosoma cruzi, and Leishmania major. Thus, in these flagellates the protein is required for mammalian host cell invasion and virulence and stress responses. With the aim of determining the presence of calcineurin B in Trypanosoma rangeli, a non-virulent trypanosome for mammals, the respective gene was amplified by PCR, cloned and sequenced. Two sequences of 531 bp in length showing a nucleotide polymorphism (314A>C) were obtained in spite of a single-copy gene was revealed by Southern blot. These sequences, probably the alleles from the gene, showed a 79% of identity with those from T. cruzi and clustered as the sister group of this trypanosome species in a Maximum Parsimony analysis. Deduced amino acid sequence comparison with trypanosomatids and other organisms through the phylogenetic scale as well as the obtained protein structural homology model suggested the presence of the four potential EF-hand regions and the corresponding calcium binding sites of the last three of these domains. Having assessed the expression of this protein in T. rangeli epimastigotes, and taking into account the following facts: (i) calcineurin inhibitors have inhibitory effect on the in vitro replication of T. cruzi, (ii) L. major promastigote growth is inhibited by chelating agents, and (iii) T. rangeli does not seem to productively infect mammalian cells, it is hypothesized herein that the function of this protein in T. rangeli is required for epimastigote growth.
Assuntos
Calcineurina/genética , Sequência Conservada/genética , Estágios do Ciclo de Vida/fisiologia , Modelos Moleculares , Trypanosoma rangeli/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Southern Blotting , Calcineurina/química , Clonagem Molecular , Estágios do Ciclo de Vida/genética , Modelos Genéticos , Dados de Sequência Molecular , Oligonucleotídeos/genética , Filogenia , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência , Trypanosoma rangeli/crescimento & desenvolvimentoRESUMO
The K1 peptide is a CD8(+)T cell HLA-A*0201-restricted epitope derived from the Trypanosoma cruzi KMP-11 protein. We have previously shown that this peptide induces IFN-gamma secretion by CD8(+)T cells. The aim of this study was to characterize the frequency of K1-specific CD8(+)T cells in chagasic patients. Nineteen HLA-A2(+)individuals were selected from 50 T. cruzi infected patients using flow cytometry and SSP-PCR assays. Twelve HLA-A*0201(+)noninfected donors were included as controls. Peripheral blood mononuclear cells were stained with HLA-A2-K1 tetramer, showing that 15 of 19 infected patients have K1-specific CD8(+)T cells (0.09-0.34% frequency) without differences in disease stages or severity. Of note, five of these responders were A*0205, A*0222, A*0226, A*0259 and A*0287 after molecular typing. Thus, a phenotypic and functional comparison of K1-specific CD8(+)T cells from non-HLA-A*0201 and HLA-A*0201(+)infected patients was performed. The results showed that both non-HLA-A*0201 and HLA-A*0201(+)individuals have a predominant effector memory CD8(+)T cell phenotype (CCR7-, CD62L-). Moreover, CD8(+)T cells from non-HLA-A*0201 and HLA-A*0201(+)individuals expressed IL-2, IFN-gamma and perforin without any differences. These findings support that K1 peptide is a promiscuous epitope presented by HLA-A2 supertype molecules and is highly recognized by chagasic patients.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Doença de Chagas/imunologia , Epitopos de Linfócito T/imunologia , Proteínas de Protozoários/imunologia , Trypanosoma cruzi/imunologia , Adulto , Idoso , Alelos , Feminino , Genótipo , Antígeno HLA-A2/genética , Antígeno HLA-A2/imunologia , Humanos , Interferon gama/biossíntese , Interleucina-2/biossíntese , Masculino , Pessoa de Meia-Idade , Perforina/biossínteseRESUMO
The K1 peptide is an HLA-A*0201-restricted cytotoxic epitope derived from the Trypanosoma cruzi KMP-11 protein, this being the etiological agent of Chagas' disease. This work describes the K1 peptide's secondary structure and its recognition by sera from chagasic patients. Circular dichroism and NMR spectroscopy analysis revealed that the K1 peptide adopts an alpha-helical conformation. Fifty-six percent of individuals had anti-K1 and 86% anti-KMP-11 antibodies by ELISA in the chronic Chagas' group and 28 and 68% in the indeterminate Chagas' group, respectively. By contrast, no reactivity was observed in sera from healthy individuals and tuberculosis patients. Antibody response subclass specificity to the K1 peptide was IgG1 and IgG3. Taken together these results support the idea that the K1 peptide acts as a B-cell-inducer epitope during Chagas' disease.
Assuntos
Antígenos de Protozoários/química , Proteínas de Protozoários/química , Proteínas de Protozoários/imunologia , Trypanosoma cruzi/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/genética , Doença de Chagas/imunologia , Epitopos/química , Epitopos/genética , Humanos , Imunoglobulina G/sangue , Isotipos de Imunoglobulinas/sangue , Modelos Moleculares , Estrutura Secundária de Proteína , Proteínas de Protozoários/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Trypanosoma cruzi/genéticaRESUMO
The cellular response mediated by MHC class I restricted CD8+ T cells has been shown to be crucial in the control of Chagas disease. The K1 peptide derived from T. cruzi KMP-11 protein has a high binding affinity to the HLA-A*0201 molecule. Nevertheless, it is not known whether this peptide is processed and displayed as an MHC class I epitope during natural infection by T. cruzi. The aim of this study was to evaluate, by ELISPOT assay, the ability of K1 peptide to activate CD8+ T lymphocytes to produce IFN-gamma. Therefore, CD8+ T lymphocytes from 22 HLA-A*0201+ individuals, 12 chronic chagasic patients and 10 uninfected controls, were analysed. The results revealed that two of the chagasic patients had IFN-gamma-secreting CD8+ T cells that were able to respond to K1 peptide with a relative frequency of 110 and 230 per million CD8+ T cells. In contrast, none of HLA-A*0201+ uninfected controls responded to K1 peptide. Responses to HLA-A*0201 restricted peptide from the influenza matrix protein were found in six chagasic patients and four uninfected controls with an average frequency of 175 and 111 cells per million CD8+ T cells, respectively. Moreover, a flow cytometric assay for degranulation showed that chagasic responders had K1-specific cytotoxic CD8+ T cells. It is shown here for the first time that the K1 peptide is efficiently processed, presented and recognized by CD8+ T lymphocytes during the natural course of Chagas disease.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Doença de Chagas/imunologia , Interferon gama/biossíntese , Glicoproteínas de Membrana/farmacologia , Fragmentos de Peptídeos/farmacologia , Proteínas de Protozoários/farmacologia , Trypanosoma cruzi/imunologia , Adulto , Idoso , Animais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Feminino , Antígenos HLA-A/imunologia , Antígeno HLA-A2 , Humanos , Imunofenotipagem , Interferon gama/imunologia , Masculino , Glicoproteínas de Membrana/imunologia , Pessoa de Meia-Idade , Fragmentos de Peptídeos/imunologia , Proteínas de Protozoários/imunologia , Estatísticas não ParamétricasRESUMO
Trypanosomatids are early divergent parasites which include several species of medical interest. Trypanosoma rangeli is not pathogenic for humans but shows a high immunological cross-reactivity with Trypanosoma cruzi, the causative agent of Chagas' disease that affects more than 17 million people throughout the world. Recent studies have suggested that T. cruzi KMP-11 antigen could be a good candidate for the induction of immunoprotective cytotoxic responses against T. cruzi natural infection. In the present paper the genes coding for the T. rangeli kinetoplastid membrane protein-11 have been characterized. The results show that the locus encoding this protein is formed by 4 gene units measuring 550 nucleotides in length, organized in tandem, and located in different chromosomes in KP1(+) and KP1(-) strains. The gene units are transcribed as a single mRNA of 530 nucleotides in length. Alignment of the T. rangeli KMP-11 deduced amino acid sequence with the homologous KMP-11 protein from T. cruzi revealed an identity of 97%. Interestingly, the T and B cell epitopes of the T. cruzi KMP-11 protein are conserved in the T. rangeli KMP-11 amino acid sequence.
Assuntos
Glicoproteínas de Membrana/química , Proteínas de Protozoários/química , Trypanosoma/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento Cromossômico , Expressão Gênica , Glicoproteínas de Membrana/genética , Dados de Sequência Molecular , Proteínas de Protozoários/genética , Alinhamento de Sequência , Trypanosoma/genéticaRESUMO
The sequence, genomic organization, and transcription of the gene encoding the H2A histone protein of the protozoan parasite Trypanosoma rangeli is described in this paper. The locus encoding the T. rangeli H2A protein is formed by at least 11 gene units measuring 790 nucleotides in length, organized in tandem, and located in a single chromosome of approximately 1.9 Mb. The gene units actively transcribe only one size class of mRNA measuring 0.7 kb in length. The T. rangeli H2A protein contains in the amino-terminal the AGLXFPV motif, which is conserved in a broad range of H2A proteins, and the RSAK motif, which is implicated in repression of the histone's basal transcription in yeast. The carboxyl-terminal of the protein contains a two-lysine residue described as the ubiquitin binding site and the histidine residue implicated in DNA binding.
Assuntos
Genes de Protozoários , Histonas/genética , Histonas/metabolismo , Trypanosoma/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA de Protozoário/análise , DNA de Protozoário/genética , Histonas/química , Dados de Sequência Molecular , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Trypanosoma/crescimento & desenvolvimento , Trypanosoma/metabolismoRESUMO
The analysis of three recombinant clones containing the histone H2A locus isolated from a genomic library of Trypanosoma cruzi DNA shows that the H2A gene loci are formed by 1.2 and 0.76 kb long intercalated units organized in a head-to-tail tandem array. The difference in length between the two gene units is due to the presence of a short interspersed nucleotide element (SINE)-like DNA sequence inserted at the 3' end of some of these units. Southern, northern and chromosomal blot analysis of a Brazilian Y strain and six Colombian strains demonstrated the existence of polymorphisms regarding the relative copy number of the H2A gene units, the relative abundance of the H2A transcripts and their chromosomal location. These results show the existence of a dynamic organization in the H2A loci among T. cruzi strains in which a SINE-like sequence may be involved and support the fact that T. cruzi has a high degree of plasticity in its genome.