RESUMO
We report here the isolation and characterisation of genomic and cDNA clones encoding a Serine-, Alanine-, and Proline-rich protein (SAP) of Trypanosoma cruzi metacyclic trypomastigotes. The deduced peptides translated from these clones were characterised by a high content of residues of alanine, proline, serine, glycine, valine, and threonine distributed in several repeats: P(2-4), S(2-3), A(2-3), AS, SA, PA, AP, SP, PS, and TP. The repeats are partially homologous to the serine-, alanine-, and proline-containing motifs of Leishmania major and Leishmania mexicana proteophosphoglycans. Genes coding for SAP are part of a polymorphic family whose members are linked to members of gp85/sialidase and mucin-like gene families. This is consistent with the hypothesis that this genetic organisation could be a means by which T. cruzi co-ordinates the expression of major surface proteins.
Assuntos
Genoma de Protozoário , Proteínas de Protozoários/genética , Trypanosoma cruzi/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar , Dados de Sequência Molecular , Família Multigênica , Proteínas de Protozoários/química , Proteínas de Protozoários/isolamento & purificação , Proteínas de Protozoários/metabolismo , Análise de Sequência de DNA , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/metabolismoRESUMO
To further investigate the immunological properties of the stage-specific 82-kDa glycoprotein (gp82) of Trypanosoma cruzi metacyclic trypomastigotes, previously shown to induce antigen-specific humoral and T-cell responses in mice, we performed a series of experiments with recombinant proteins containing sequences of gp82 fused to glutathione S-transferase. Of five fusion proteins tested, only J18b and J18b1, the carboxyproximal peptides containing amino acids 224 to 516 and 303 to 516, respectively, were recognized by monoclonal antibody 3F6 as well as by various anti-T. cruzi antisera and, when administered to mice, were capable of eliciting antibodies directed to the native gp82. The amino-terminal peptide and other carboxyterminal recombinant proteins lacking the central domain of gp82 (amino acids 224 to 356), which is exposed on the surface of live metacyclic forms, did not display any of these properties. Spleen cells derived from mice immunized with any of the five recombinant proteins proliferated in vitro in the presence of native gp82.J18b was the most stimulatory, whereas J18b3, the peptide containing amino acids 408 to 516, elicited the weakest response. When BALB/c mice immunized with J18b antigen plus A1(OH)3 as adjuvant were challenged 10 5 metacyclic trypomastigotes, 85% of them resisted acute infection, in comparison with control mice that received glutathione S-transferase plus adjuvant. Antibodies induced by J18b protein lacked agglutinating or complement-dependent lytic activity and failed to neutralize parasite infectivity. On the other hand, CD4+T cells from the spleens of J18b-immunized mice displayed an intense proliferative activity upon stimulation with 1.25 microgram of native gp82 per ml, which resulted in increased production of gamma interferon, a cytokine associated with resistance to T. cruzi infection.
Assuntos
Anticorpos Antiprotozoários/biossíntese , Antígenos de Protozoários/imunologia , Doença de Chagas/imunologia , Trypanosoma cruzi/imunologia , Doença Aguda , Animais , Feminino , Imunização , Interferon gama/biossíntese , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Peso Molecular , Proteínas Recombinantes/imunologiaRESUMO
A workshop organized by the Ibero-American Project of Biotechnology evaluated the diagnostic potential of several cloned Trypanosoma cruzi recombinant antigens for Chagas' disease serodiagnosis. A set of recombinants, Antigen 2, Antigen 13, SAPA, H49, A13, JL5, JL7, JL8, JL9, and RA1 provided by three different South American laboratories were probed with a panel of 236 South American serum samples. Antigens JL7, H49, Antigen 2, and A13 scored as the best diagnostic recombinant reagents. The results suggested that the main advantage of using cloned peptides for chronic Chagas' disease diagnosis resided in their highly specific immunoreactive properties.
Assuntos
Antígenos de Protozoários/imunologia , Doença de Chagas/diagnóstico , Trypanosoma cruzi/imunologia , Animais , Anticorpos Antiprotozoários/imunologia , Humanos , Proteínas Recombinantes/imunologia , Testes SorológicosRESUMO
Intact RNAs were isolated from epimastigote forms of different Trypanosoma cruzi strains. Translation of the mRNAs using rabbit reticulocyte lysate and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed protein profiles comparable to those observed by labeling cells in vivo. No major interstrain differences were observed in the patterns of the polypeptides synthesized in vitro and in vivo, indicating that metabolic proteins are similar among distinct strains. Several T. cruzi polypeptides produced in the rabbit reticulocyte lysate system were immunoprecipitated by specific antisera. The patterns of antigenic polypeptides recognized by antisera raised against epimastigotes from different strains were also very similar.