RESUMO
We have studied the genomic organization and expression of the gene encoding a high molecular mass (300 kDa) repetitive antigen associated with the cytoskeleton of Trypanosoma cruzi. Protease digestion of the native protein, restriction analysis of genomic DNA and sequencing of genomic and cDNA clones indicated that most of the protein is built up by tandemly arranged, nearly identical repeats of 68 amino acids. The gene size was estimated to be approx. 9.4 kb based on the sizes of the transcript and the native protein. The nucleotide sequence conservation among the repeats indicates that selective sequence homogenization, presumably through gene conversion, maintained the amino-acid sequence conservation. Two duplicated allelic forms of this gene were mapped in fragments of about 20 kb. In some strains an additional allele was located in a fragment of 9.4 kb. Our results suggest that this repetitive antigen is a structural protein which could be involved in the attachment of the flagellum to the cell body.
Assuntos
Antígenos de Protozoários/genética , Citoesqueleto/química , Genes de Protozoários/genética , Proteínas de Protozoários/genética , Trypanosoma cruzi/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Sequência Conservada/genética , Proteínas do Citoesqueleto/genética , Regulação da Expressão Gênica , Dados de Sequência Molecular , Polimorfismo de Fragmento de Restrição , Proteínas de Protozoários/análise , RNA Mensageiro/análise , RNA de Protozoário/análise , Sequências Repetitivas de Ácido Nucleico/genética , Mapeamento por Restrição , Análise de Sequência de DNA , Transcrição Gênica , Trypanosoma cruzi/imunologiaRESUMO
Tubercidin (TUB) is an adenosine analog with potent antiparasite action, unfortunately associated with severe host toxicity. Prevention of TUB toxicity can be reached associating nitrobenzylthioinosine (NBMPR), an inhibitor of the purine nucleoside transport, specifically target to the mammal cells. It was demonstrated that this nucleoside transport inhibitor has no significant effect in the in vitro uptake of TUB by Schistosoma mansoni and Trypanosoma gambiense. Seeking to evaluate if the association of these compounds is also effective against leishmania, we analyzed the TUB-NBMPR combined treatment in in vitro cultures of promastigote forms of Leishmania (L.) amazonensis, Leishmania (L.) chagasi, Leishmania (L.) major, and Leishmania (V.) braziliensis as well as in cultures of amastigote forms of L. (L.) amazonensis, mice macrophages infected with L. (L.) amazonensis, and in vivo tests in BALB/c mice infected with L. (L.) amazonensis. We demonstrated that TUB-NBMPR combined treatment can be effective against leishmania cells protecting mammalian cells from TUB toxicity.
Assuntos
Antiparasitários/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Leishmania/efeitos dos fármacos , Leishmaniose/tratamento farmacológico , Tioinosina/análogos & derivados , Tionucleotídeos/uso terapêutico , Tubercidina/uso terapêutico , Animais , Antiparasitários/farmacologia , Antiparasitários/toxicidade , Células Cultivadas , Quimioterapia Combinada , Inibidores Enzimáticos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Schistosoma mansoni/efeitos dos fármacos , Tioinosina/farmacologia , Tioinosina/uso terapêutico , Tionucleotídeos/farmacologia , Trypanosoma brucei gambiense/efeitos dos fármacos , Tubercidina/farmacologia , Tubercidina/toxicidadeRESUMO
We tested a general method for the identification of drug resistance loci in the trypanosomatid protozoan parasite Leishmania major. Genomic libraries in a multicopy episomal cosmid vector were transfected into susceptible parasites, and drug selections of these transfectant libraries yielded parasites bearing cosmids mediating resistance. Tests with two antifolates led to the recovery of cosmids encoding DHFR-TS or PTR1, two known resistance genes. Overexpression/selection using the toxic nucleoside tubercidin similarly yielded the TOR (toxic nucleoside resistance) locus, as well as a new locus (TUB2) conferring collateral hypersensitivity to allopurinol. Leishmania synthesize ergosterol rather than cholesterol, making this pathway attractive as a chemotherapeutic target. Overexpression/selection using the sterol synthesis inhibitors terbinafine (TBF, targeting squalene epoxidase) and itraconazole (ITZ, targeting lanosterol C(14)-demethylase) yielded nine new resistance loci. Several conferred resistance to both drugs; several were drug-specific, and two TBF-resistant cosmids induced hypersensitivity to ITZ. One TBF-resistant cosmid encoded squalene synthase (SQS1), which is located upstream of the sites of TBF and ITZ action in the ergosterol biosynthetic pathway. This suggests that resistance to "downstream" inhibitors can be mediated by increased expression of ergosterol biosynthetic intermediates. Our studies establish the feasibility of overexpression/selection in parasites and suggest that many Leishmania drug resistance loci are amenable to identification in this manner.
Assuntos
Resistência a Medicamentos/genética , Ergosterol/antagonistas & inibidores , Leishmania major/efeitos dos fármacos , Seleção Genética , Tubercidina/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Sequência de Bases , Primers do DNA , Ergosterol/metabolismo , Farnesil-Difosfato Farnesiltransferase/química , Farnesil-Difosfato Farnesiltransferase/genética , Humanos , Leishmania major/enzimologia , Leishmania major/genética , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Tubercidina/metabolismoRESUMO
We have isolated a clone of Trypanosoma cruzi genomic DNA, lambda 3b2-5, which contains sequences that are reiterated in the genome. Northern blot analysis showed that clone 3b2-5 hybridizes to 1,200-5,000 bases different mRNA species. The number of mRNAs species hybridized to clone 3b2-5 exceeds its coding capacity showing that this clone carries sequences that are common to several mRNAs species and conserved in the poly A(+) RNA. These sequences are not homologous to the T. cruzi spliced leader sequence, since clone 3b2-5 does not hybridize to a synthetic 20 nucleotide complementary to the spliced leader sequence. Clone 3b2-5 does not hybridize to DNA and RNA from several genera of Trypanosomatidae and other Trypanosoma species indicating that it carries T. cruzi species-specific sequences.
Assuntos
DNA/genética , RNA Mensageiro/genética , Sequências Repetitivas de Ácido Nucleico , Trypanosoma cruzi/genética , Animais , Northern Blotting , Southern Blotting , Biblioteca Genômica , Família Multigênica , Homologia de Sequência do Ácido Nucleico , Especificidade da EspécieRESUMO
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.
Assuntos
Antígenos de Protozoários/isolamento & purificação , Trypanosoma cruzi/metabolismo , Animais , Eletroforese em Gel de Poliacrilamida , Peso Molecular , Biossíntese de Proteínas , RNA Mensageiro/isolamento & purificação , Coelhos , Reticulócitos/parasitologiaRESUMO
A monospecific antiserum raised to a Trypanosoma cruzi recombinant antigen, with tandem repeat of 68 amino acids, was used to screen urine samples of chagasic and nonchagasic patients. The antiserum detected a specific 150-160 kDa antigen in urine of 60% of chronic chagasic patients, but not in urine samples from nonchagasic patients and healthy control individuals. The reactivity to 150-160 kDa urinary antigen could be abolished by adsorption with the recombinant repetitive antigen. These results suggest that 150-160 kDa urinary antigen is a T. cruzi-derived antigen and specific for Chagas' disease.
Assuntos
Antígenos de Protozoários/urina , Doença de Chagas/urina , Trypanosoma cruzi/imunologia , Adulto , Animais , Doença de Chagas/diagnóstico , Doença de Chagas/imunologia , Doença de Chagas/parasitologia , Epitopos , Feminino , Humanos , Soros Imunes , Masculino , Pessoa de Meia-Idade , Peso Molecular , Proteínas Recombinantes de Fusão/imunologia , Trypanosoma cruzi/isolamento & purificaçãoRESUMO
A genomic clone expressing a Trypanosoma cruzi antigen in Escherichia coli was identified using human chagasic sera. Chagasic antibodies affinity purified on extracts of this clone recognized a high-molecular-weight protein expressed in all developmental stages of the parasite life cycle, as well as in various T. cruzi strains. The antigen is associated with the cytoskeleton of the parasite and localizes along the attachment region between the flagellum and the cell body. Antibodies to the recombinant antigen were detected in the sera of 115 chagasic patients from different endemic regions, but not in sera of patients with leishmaniasis, T. rangeli infection, or other parasitic diseases. Our data suggest that the presence of antibodies to this antigen may be specifically associated with Chagas' disease.
Assuntos
Antígenos de Protozoários/genética , Doença de Chagas/imunologia , Trypanosoma cruzi/imunologia , Animais , Northern Blotting , Southern Blotting , Western Blotting , Clonagem Molecular , Citoesqueleto/imunologia , DNA/genética , Epitopos/imunologia , Escherichia coli/genética , Imunofluorescência , Expressão Gênica , Humanos , Soros Imunes/imunologia , Trypanosoma cruzi/genética , Trypanosoma cruzi/ultraestruturaRESUMO
A Trypanosoma cruzi DNA fragment encoding an immunodominant repetitive antigen (H49) was subcloned into a protein purification and expressed system. Purified H49 peptide reacted specifically in an enzyme-linked immunosorbent assay (ELISA) with sera from T. cruzi-infected patients, but not with sera from patients with other parasitic diseases such as leishmaniasis and T. rangeli-infection. The H49 recombinant ELISA was able to detect specific antibodies in 84% of chronic chagasic serum samples tested. One of the major advantage of the recombinant ELISA for serodiagnosis of chronic Chagas' disease resides in its high specificity (100%). Our data suggest that recombinant peptides could provide a practical basis for specific diagnosis tests for Chagas' disease.
Assuntos
Anticorpos Antiprotozoários/sangue , Doença de Chagas/imunologia , Epitopos Imunodominantes/imunologia , Proteínas Recombinantes/imunologia , Trypanosoma cruzi/imunologia , Animais , Antígenos de Protozoários/imunologia , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Humanos , Epitopos Imunodominantes/genética , Infecções por Protozoários/imunologia , Testes Sorológicos , Trypanosoma cruzi/genéticaRESUMO
Here we describe the characterization of a Trypanosoma cruzi DNA sequence (clone A13) that codes for a polypeptide recognized by IgM and IgG antibodies from sera of acute and congenital chagasic patients. Antibodies to A13 antigen are also detected in the sera of chronic patients with different clinical forms of Chagas' disease, but not in sera of patients with leishmaniasis or other parasitic diseases. The antigenic determinants encoded by clone A13 are found in amastigotes and trypomastigotes of several T. cruzi strains, but not in the noninfective epimastigotes. The DNA sequence of the recombinant clone reveals one open reading frame encoding 251 amino acids without tandemly repeated sequences. Our data suggest that the A13 antigen may be useful for the development of serodiagnostic procedures.