RESUMO
Trypanosoma cruzi I, a discrete typing unit (DTU) found in human infections in Venezuela and other countries of the northern region of South America and in Central America, has been recently classified into five intra-DTU genotypes (Ia, Ib, Ic, Id, Ie) based on sequence polymorphisms found in the spliced leader intergenic region. In this paper we report the genotype identification of T. cruzi human isolates from one outbreak of acute orally acquired Chagas disease that occurred in a non-endemic region of Venezuela and from T. cruzi triatomine and rat isolates captured at a guava juice preparation site which was identified as the presumptive source of infection. The genotyping of all these isolates as TcId supports the view of a common source of infection in this oral Chagas disease outbreak through the ingestion of guava juice. Implications for clinical manifestations and dynamics of transmission cycles are discussed.
Assuntos
Doença de Chagas/epidemiologia , Doença de Chagas/parasitologia , Surtos de Doenças , Genes de Protozoários , Trypanosoma cruzi/genética , Animais , Sequência de Bases , Bebidas/parasitologia , Doença de Chagas/transmissão , Genótipo , Técnicas de Genotipagem , Humanos , Dados de Sequência Molecular , Psidium , Ratos/parasitologia , Instituições Acadêmicas , Alinhamento de Sequência , Trypanosoma cruzi/classificação , Trypanosoma cruzi/isolamento & purificação , Venezuela/epidemiologiaRESUMO
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
In Venezuela six episodes of oral transmission of Chagas disease (OChD) have been described, being the one reported in 2007 with a total of 103 people infected the largest worldwide. This work shows the use of three molecular markers (mini-exon gene and domains 24Sα and 18S of the ribosomal RNA) to characterize the infecting Trypanosoma cruzi strain of patients, reservoirs and vectors involved in five of the six OChD outbreaks. For this, 28 T. cruzi isolates were characterized by PCR, and the products of these reactions cloned and sequenced to reveal the existence of different TcI SL-IR genotypes. We also describe a new PCR assay able to discriminate between TcIb and TcId parasite populations. In summary, we have identified mostly parasites with the TcId haplotype and multiclonal populations with predominance of haplotype TcId (65.2%). Additionally, populations of haplotypes TcIb, TcIa and mixtures (TcId+TcIb, TcId+TcIa, TcIb+TcIa) are recurrent in samples obtained from children. The analysis of the SL-IR motif showed two clones depicting a different motif that could be an evidence for a possible hybrid haplotype between TcIa and TcIb (haplotype TcIa/Ib). Interestingly, in a single patient haplotype differences between T.cruzi isolates obtained pre and post-treatment were found. In conclusion, our findings show that in order to understand the pathogenic mechanisms involved in the orally acquired Chagas disease there is a need to join efforts to study T. cruzi haplotypes, their tissue tropisms and their susceptibility to chemoteraphy.
Assuntos
Doença de Chagas/epidemiologia , Doença de Chagas/transmissão , Trypanosoma cruzi/genética , Animais , Sequência de Bases , Surtos de Doenças , Reservatórios de Doenças/parasitologia , Vetores de Doenças , Éxons , Genótipo , Haplótipos , Humanos , Repetições de Microssatélites , Tipagem Molecular , RNA Líder para Processamento , Trypanosoma cruzi/classificação , Trypanosoma cruzi/isolamento & purificação , Venezuela/epidemiologiaRESUMO
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
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
By screening a Leishmania braziliensis complementary DNA library with a pool of sera from leishmaniasis patients, the gene coding for L6 ribosomal protein was isolated. The sequence, genomic organization, and transcription of this gene are described in this article. The sequence analysis of the L. braziliensis L6 gene shows a single open reading frame, which codes for a protein of 192 amino acids (aa) with a hypothetical molecular mass of 20.9 kDa. The protein exhibits significant sequence similarity to L6 ribosomal proteins from higher eukaryotes and yeast. Thus, the L. braziliensis L6 protein contains 4 functional motifs, which are located at equivalent positions in other L6 ribosomal proteins described previously. Interestingly, the L6 ribosomal protein from L. braziliensis contains a specific region of 14 aa and a tyrosine kinase motif, which is absent in human and C. elegans L6 protein. The locus coding the L. braziliensis L6 ribosomal protein is formed by 2 gene copies arranged in tandem and located in a chromosome of approximately 0.9. Mb. The genes are actively transcribed as 2 polyadenylated transcripts of approximately 1.15 and 0.85 kb, which differ in their steady-state level and stability.
Assuntos
DNA Complementar/isolamento & purificação , Leishmania braziliensis/genética , Leishmaniose Cutânea/parasitologia , Proteínas Ribossômicas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Southern Blotting , DNA Complementar/genética , DNA de Protozoário/genética , DNA de Protozoário/isolamento & purificação , Eletroforese em Gel de Campo Pulsado , Biblioteca Gênica , Humanos , Soros Imunes/genética , Soros Imunes/imunologia , Leishmania braziliensis/química , Leishmania braziliensis/imunologia , Leishmaniose Cutânea/imunologia , Dados de Sequência Molecular , Proteínas Ribossômicas/química , Alinhamento de SequênciaRESUMO
The isolation and molecular characterization of the gene coding for L14 ribosomal protein from L. braziliensis is described. There are 2 copies of the gene per haploid genome, repeated in a head-to-tail tandem orientation and located in a single chromosome of approximately 950 kb. Northern blot analyses indicate the presence of a single transcript of 0.95 kb which is up-regulated when parasites reach the stationary growth phase. L. braziliensis L14 gene codes for a 175 amino acid long polypeptide showing 75-83% sequence identity with L14 proteins from trypanosomatids and approximately 25% with its counterparts from higher eukaryotic organisms. L14 ribosomal proteins from trypanosomatids and higher eukaryotes share along their molecules a similar distribution pattern of theoretically functional domains. L. braziliensis L14 recombinant protein is not recognized by sera from cutaneous leishmaniasis patients. Immunization of mice with one dose of L14 recombinant protein and a second dose of L14 protein covalently linked to the HSP70 from Trypanosoma cruzi induces a high antibody level against this L14 protein, which is mostly of the IgG2a subtype, as well as a strong increase in splenocyte proliferation index.
Assuntos
Leishmania braziliensis/genética , Leishmania braziliensis/imunologia , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antiprotozoários/sangue , Sequência de Bases , Divisão Celular/imunologia , DNA de Protozoário/química , DNA de Protozoário/genética , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Leishmaniose Cutânea/sangue , Leishmaniose Cutânea/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Alinhamento de Sequência , Análise de Sequência de DNA , Baço/citologia , Baço/imunologiaRESUMO
The technique of Random Amplification Polymorphic DNA allows fragments of the genome to be amplified by means of polymerase chain reaction (PCR) without previous knowledge of their sequences. The protozoa of the genus Leishmania present great genetic variability, making it difficult to characterize the different species. A method is developed with a single 10-mers long primer, which allows the species L. braziliensis, L. mexicana, L. infantum, L. tropica, L. chagasi, L. amazonensis and L. major to be differentiated. These products amplified by RAPD have also facilitated the design of some primers that amplify L. braziliensis DNA exclusively.
Assuntos
Leishmania/classificação , Leishmaniose/diagnóstico , Técnica de Amplificação ao Acaso de DNA Polimórfico/métodos , Animais , Clonagem Molecular , Primers do DNA , DNA de Protozoário/química , DNA de Protozoário/genética , Variação Genética , Humanos , Leishmania/genética , Hibridização de Ácido NucleicoRESUMO
The isolation and molecular characterization of the histone H1-encoding gene from Leishmania braziliensis was carried out. The gene is present in the genome as a single copy and transcribed as a polyadenylated transcript of 830 nucleotides. The deduced amino acid sequence has in its central region the DNA binding K-[K/R]-A-A-[A/P] motif, which is repeated in tandem 9 times.
Assuntos
Clonagem Molecular/métodos , Histonas/genética , Leishmania braziliensis/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Complementar/genética , DNA de Protozoário/análise , DNA de Protozoário/genética , Histonas/química , Histonas/metabolismo , Dados de Sequência Molecular , Análise de Sequência de DNARESUMO
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.
Assuntos
Genes de Protozoários , Genoma de Protozoário , Histonas/genética , Trypanosoma cruzi/genética , Animais , Northern Blotting , Southern Blotting , Brasil , Clonagem Molecular , Colômbia , DNA de Protozoário/análise , Eletroforese em Gel de Campo Pulsado , Escherichia coli/metabolismo , Dosagem de Genes , Vetores Genéticos , Histonas/biossíntese , Humanos , Polimorfismo Genético , RNA de Protozoário/análise , Proteínas Recombinantes/biossíntese , Elementos Nucleotídeos Curtos e DispersosRESUMO
Strategies to construct the physical map of the Trypanosoma cruzi nuclear genome have to capitalize on three main advantage of the parasite genome, namely (a) its small size, (b) the fact that all chromosomes can be defined, and many of them can be isolated by pulse field gel electrophoresis, and (c) the fact that simple Southern blots of electrophoretic karyotypes can be used to map sequence tagged sites and expressed sequence tags to chromosomal bands. A major drawback to cope with is the complexity of T. cruzi genetics, that hinders the construction of a comprehensive genetic map. As a first step towards physical mapping, we report the construction and partial characterization of a T. cruzi CL-Brener genomic library in yeast artificial chromosomes (YACs) that consists of 2.770 individual YACs with a mean insert size of 365 kb encompassing around 10 genomic equivalents. Two libraries in bacterial artificial chromosomes (BACs) have been constructed, BACI and BACII. Both libraries represent about three genome equivalents. A third BAC library (BAC III) is being constructed. YACs and BACs are invaluable tools for physical mapping. More generally, they have to be considered as a common resource for research in Chagas disease.
Assuntos
Animais , Mapeamento Cromossômico , Genoma de Protozoário , Trypanosoma cruzi/genética , Cromossomos Artificiais de Levedura , Células Clonais , Sitios de Sequências RotuladasRESUMO
Strategies to construct the physical map of the Trypanosoma cruzi nuclear genome have to capitalize on three main advantages of the parasite genome, namely (a) its small size, (b) the fact that all chromosomes can be defined, and many of them can be isolated by pulse field gel electrophoresis, and (c) the fact that simple Southern blots of electrophoretic karyotypes can be used to map sequence tagged sites and expressed sequence tags to chromosomal bands. A major drawback to cope with is the complexity of T. cruzi genetics, that hinders the construction of a comprehensive genetic map. As a first step towards physical mapping, we report the construction and partial characterization of a T. cruzi CL-Brener genomic library in yeast artificial chromosomes (YACs) that consists of 2,770 individual YACs with a mean insert size of 365 kb encompassing around 10 genomic equivalents. Two libraries in bacterial artificial chromosomes (BACs) have been constructed, BACI and BACII. Both libraries represent about three genome equivalents. A third BAC library (BAC III) is being constructed. YACs and BACs are invaluable tools for physical mapping. More generally, they have to be considered as a common resource for research in Chagas disease.
Assuntos
Mapeamento Cromossômico , Cromossomos Artificiais de Levedura , Cromossomos Bacterianos/genética , Clonagem de Organismos , Genoma de Protozoário , Trypanosoma cruzi/genética , Animais , Biblioteca GenômicaRESUMO
A novel multiprimer PCR method with the potential to identify mycobacteria in clinical samples is presented. The assay relies on the simultaneous amplification of three bacterial DNA genomic fragments by using different sets of oligonucleotide primers. The first set of primers amplifies a 506-bp fragment from the gene for the 32-kDa antigen of Mycobacterium tuberculosis, which is present in most of the species belonging to the genus Mycobacterium. The second set of primers amplifies a 984-bp fragment from the IS6110 insertion sequence of the bacteria belonging to the M. tuberculosis complex. The third set of primers, derived from an M. tuberculosis species-specific sequence named MTP40, amplifies a 396-bp genomic fragment. Thus, while the multiprimer system would render three amplification fragments from the M. tuberculosis genome and two fragments from the Mycobacterium bovis genome, a unique amplification fragment would be obtained from nontuberculous mycobacteria. The results obtained, using reference mycobacterial strains and typed clinical isolates, show that the multiprimer PCR method may be a rapid, sensitive, and specific tool for the differential identification of various mycobacterial strains in a single-step assay.