RESUMO
The genes encoding a protein antigenic during the acute phase (SAPA) and another one antigenic during the chronic phase (antigen 30) of human Chagas' disease were analyzed in 15 Trypanosoma cruzi isolates and clones collected in distant geographical regions. These two genes had tandem repeats which were present in all parasites tested. However, large differences in the length of restriction fragments were observed among isolates. This was readily explained by variations in the number of repeat units present in homologous genes. This result was confirmed after analysis of 3 members of the SAPA gene family. In the case of antigen 30, we propose that differences in the number of repeat units result in size differences in the corresponding RNAs and proteins, which explains the large size heterogeneity in otherwise homologous T. cruzi antigens.
Assuntos
Antígenos de Protozoários/genética , Trypanosoma cruzi/genética , Animais , Southern Blotting , Western Blotting , Genes , Peso Molecular , RNA Mensageiro/genética , Sequências Repetitivas de Ácido Nucleico , Mapeamento por Restrição , Trypanosoma cruzi/imunologiaRESUMO
A Trypanosoma cruzi antigen which is shed into the culture medium by the trypomastigote stage of the parasite and detected in blood of acutely infected mice was cloned and characterized. We designate this antigen shed acute phase antigen (SAPA). Five protein bands with apparent molecular masses ranging from 160 to 200 kDa were detected by immunoblotting of plasma from infected mice and in supernatants of cultured trypomastigotes upon reaction with antibodies against SAPA. A serum obtained from a patient acutely infected with Chagas' disease revealed a similar set of polypeptides in supernatants of cultured trypomastigotes when tested by immunoblotting. SAPA seems thus to be a major shed protein during the acute period of the disease. Twenty-six of 28 sera from human acute cases of Chagas' disease tested reacted with SAPA. Conversely, only 8-10% of sera from chronic cases of the disease contained detectable levels of antibody against SAPA. Sera from rabbits infected with six different parasite strains all contained antibodies against SAPA. Antibodies against SAPA are detectable 15 days after the manifestation of acute Chagas' disease symptoms in humans and 15 days post-infection in sera from mice and rabbits. The nucleotide sequence of a genomic clone encoding the 3' end of the SAPA gene revealed the presence of 14 tandemly arranged 12-amino acid-long repeats. A 39-amino acid-long region that is very hydrophobic precedes the stop codon. Due to its early appearance it might be possible to design diagnostic assays which are based on SAPA for identification of recently infected cases of Chagas' disease.
Assuntos
Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/análise , Doença de Chagas/imunologia , Trypanosoma cruzi/imunologia , Doença Aguda , Adulto , Sequência de Aminoácidos , Animais , Anticorpos Antiprotozoários/biossíntese , Antígenos de Protozoários/genética , Sequência de Bases , Criança , DNA/genética , Feminino , Humanos , Immunoblotting , Masculino , Camundongos , Dados de Sequência Molecular , Coelhos , Sequências Repetitivas de Ácido Nucleico , Trypanosoma cruzi/genética , Células VeroRESUMO
Chromosomal DNA from Trypanosoma cruzi, the agent of the American trypanosomiasis (Chagas' disease), was used for construction of a DNA library, employing the expression vector lambda gt11. Nine clones encoding different parasite antigens were isolated from this library by screening with an antiserum from a Chagasic patient. Nucleotide sequence analysis showed that seven out of the nine isolated clones code for antigens which contain tandemly repeated amino acid sequence motifs. Each of the seven antigens contains a unique repeat, ranging in length between 5 and 68 amino acids. The length of the repeats is highly conserved within each clone. Fusion proteins, expressed from two of the clones, reacted with a large proportion of sera collected from Chagasic patients in Argentina, Brazil and Chile. These clones appear thus to encode antigens which are shared between different strains of T. cruzi. Immunofluorescence experiments with live parasites showed that three of the antigens were detectable on the surface of trypanosomes.
Assuntos
Antígenos de Protozoários/genética , Sequências Repetitivas de Ácido Nucleico , Trypanosoma cruzi/imunologia , Sequência de Aminoácidos , Animais , Doença de Chagas/imunologia , DNA , Imunofluorescência , Hibridização de Ácido Nucleico , Proteínas Recombinantes de Fusão/imunologia , Trypanosoma cruzi/genéticaRESUMO
Fifty-two isolates and several clones from Trypanosoma cruzi, the agent of Chagas' disease, were analyzed using cloned minicircles or total kinetoplast DNA as probes. Isolates were obtained from triatomines, guinea pigs and infected humans in the Central and Northern regions of Argentina and the North of Chile. 35% of all the randomly selected isolates could be identified with one cloned minicircle probe. This widely distributed T. cruzi group was detected on both sides of the Andes mountain range (Argentina and Chile) in Triatoma infestans as well as in human infections. Most of the other isolates could be grouped with four kinetoplast DNAs as probes, but their geographical distribution seems to be restricted as compared with the one mentioned above. These results confirm the heterogeneity of T. cruzi subspecies in nature and the usefulness of DNA probes to group them.