Análise da região 5 não traduzida (5 utr) em sequências anotadas como trans-sialidase no genoma de Trypanosoma cruzi

Conselho Nacional de Desenvolvimento Científico e TecnológicoA transsialidase é uma glicoproteína de membrana pertencente a uma família de genes de cópia múltipla, envolvida no processo de invasão celular do Trypanosoma cruzi no hospedeiro vertebrado. Esta dissertação foi concebida com um amplo componente analítico que dependia de dados publicamente disponíveis, ou seja, as sequências oriundas do projeto genoma de T. cruzi e cDNAs de trans-sialidase depositadas no Genbank-dbEST. Este componente analítico necessitou ser complementado e ampliado com a obtenção experimental de novas sequências, a partir da metodologia baseada na transcrição reversa acoplada a PCR. Os fragmentos obtidos de cepas de T. cruzi Dm28c (T. cruzi I), Y (T. cruzi II), CL-Brener (T. cruzi II, cepa híbrida), INPA4167 (zimodema III), 3663 (zimodema III) e Colombiana (zimodema III) foram clonados, sequenciados e analisados composicionalmente. Essas sequências foram editadas e alinhadas usando-se o software CLUSTAL X. Em uma seção específica do Genbank, denominada dbEST, buscamos os cDNAs homólogos a trans-sialidase. Esta busca por similaridade foi realizada individualmente com os números de acesso referentes às seqüências supracitadas contra o dbEST utilizando o BLAST a fim de obtermos informações funcionais e evolutivas. Em seguida, desenvolvemos metodologias experimentais que nos permitiu avaliar segmentos da 5 UTR, tais como os sítios de trans-splicing adicionais ou múltiplos em TS e seus respectivos sinais (região rica em polipirimidina), variação composicional e tamanho da região das sequências entre diferentes linhagens de T. cruzi. O resultado dessa averiguação também nos mostrou a quantidade de cDNAs relacionados com a transsialidase no dbEST bem como a relação desses cDNAs com o mini-exon. As cepas do zimodema III apresentaram tamanho médio dos fragmentos de 312 bases, enquanto T. cruzi I e T. cruzi II apresentaram, respectivamente 209 e 218. Trans splicing adicional ou duplicações gênicas com mutações no sítio primário de trans splicing não parece ser um fenômeno exclusivo de algum grupo populacional, embora seja mais evidente em T. cruzi zimodema III

Os sítios adicionais de trans-splicing na 5' UTR dos genes trans-sialidase de Trypanosoma cruzi e avaliação de seus efeitos sobre a tradução

Made available in DSpace on 2015-11-04T13:39:10Z (GMT). No. of bitstreams: 2 tainah_paula_ioc_dout_2013.pdf: 4720016 bytes, checksum: abb2c1119571eec6ea090739b5bbc8a7 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2015-10-29Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil.A regulação gênica em tripanossomatídeos é policistrônica e ocorre de maneira pós-transcricional. Nas extremidades 5' e 3' dos RNA mensageiros existem segmentos que podem conter elementos regulatórios chamados UTR (Untranslated Regions), os quais são regiões transcritas, porém não-traduzidas. Para verificar se UTR de tamanhos diferentes possuem impacto na transcrição e, por conseguinte, na tradução de proteínas, selecionou-se genes da família de trans-sialidases, devido à importância destas no processo de invasão celular. A metodologia envolveu a extração de RNA total de T. cruzi CL-Brener, seguido da síntese de cDNA, PCR, clonagem dos produtos amplificados e seqüenciamento por Sanger e pelo uso do 454 Junior (Next Generation Sequencing \2013 NSG). Como as trans-sialidases correspondem a uma família gênica de cópias múltiplas, usou-se a estratégia de sequenciamento de alto-desempenho na tentativa de cobrir o maior número possível de genes desta família. Para isso foram obtidos cDNAs de trans-sialidases de CL-Brener nas formas epimastigota e tripomastigota com o iniciador 5\2019UTRTCNA, os quais apresentaram UTR com tamanhos variados entre 65 \2013 187 pb, além da obtenção de cDNAs para esta família de proteínas, em epimastigotas CL-Brener, com o iniciador 5\2019TcTS, apresentando UTR variando de 171 a 221 pb, com similaridade de sequências entre elas. Com o intuito de avaliar a correspondência entre a transcrição dos RNAs de trans-sialidases e a sua tradução, houve a necessidade de identificação das proteínas. Desenvolvemos uma metodologia de lise celular e produção de extrato proteico (denominado TcS12) a partir de células de T. cruzi no estágio epimastigota. As cepas selecionadas para esse estudo foram CL-Brener (TcVI), Dm28c (TcI), Y (TcII) e 4167 (TcIV) O processo de lise celular foi otimizado para 107 parasitos/mL ressuspensos em 200 \03BCL de tampão de lise hipotônica (por 30 minutos a 40C), associado ao sonicador de banho (por 30 minutos a 40C). A eficiência da metodologia foi validada pela citometria de fluxo mostrando que aproximadamente 72% das células foram marcadas com iodeto de propídeo (PI). A qualidade dos extratos proteicos foi analisada por LCMS/MS usando-se a estratégia MSE label free para quantificação relativa de proteínas. Foram identificadas 1153 proteínas totais, cuja expressão proteica das cepas 4167, Dm28c e Y, quando comparada à CL-Brener (cepa referência), apresentou 32, 51 e 73 proteínas up-expressed, enquanto que 80, 92 e 60 proteínas mostraram-se down\2013expressed, respectivamente. Entre as trans-sialidases identificadas, a única cópia encontrada no extrato TcS12 (número de acesso no UniProt - Q4DGV8) apresentou seu RNAm correspondente no banco de dados de cDNA de CL-Brener, com UTR de 214 pb, estando, portanto, na faixa de tamanhos das outras UTR de trans-sialidases obtidas neste estudo. Esse resultado sugere que outros fatores, não necessariamente o tamanho per se das UTR, podem influenciar no fenômeno de tradução nesses tripanossomatídeos. Em relação aos extratos proteicos de epimastigotas gerados a partir das outras cepas, a quantidade de trans-sialidases identificadas foram de 1 (cepa 4167), 2 (cepa Dm28c) e 117 (cepa Y) cópias, indicando assim a tradução satisfatória de pelo menos uma das muitas cópias desta família gênicaGene regulation in trypanosomatids is polycistronic and occurs in a post - transcriptional way. There are also regulatory elements named UTRs (Untranslated Regions) that are transcribed regions, but not translated. To verify the impact of UTRs presenting different sizes in the transcription machinery and protein translation, genes f rom trans - sialidas e family were selected due to its importance in the cell invasion process. The methodological strategies involved the extraction of total RNA from T. cruzi CL - Brener strain , followed by cDNA synthesis, PCR, cloning and sequencing of amplified products by Sanger and by using the 454 Junior (Next Generation Sequencing – N G S ). Considering that trans - sialidase is a multi - copy gene family, this high - throughput sequencing strategy was employed in an attempt to cover the largest number of trans - sialidase genes. Trans - sialidase cDNAs from CL - Brener epimastigote and tripomastigote were obtained with 5`UTRTCNA primer show ing UT R sizes between 65 - 187 bp. The cDNA from this protein fam ily were also obtained with the 5’TcTS primer from CL - Brener epimastigote s , generating UTRs with 171 - 221 bp . Both 5’UTR presented sequence similarit ies between them. In order to evaluate the correspondence between trans - sialidase gene transcription and t ranslation, it was necessary to accomplish the identification of proteins. Therefore, we developed a methodology for cell disruption, which resulted in a protein extract ( referred as TcS12 ) from epimastigote T. cruzi cells. The strains selected for this st udy were CL - Brener (TcVI), Dm28c (TcI), Y (TcII) and 4167 (TcIV). The process for lysing the cells was optimized to 10 7 parasites/mL resuspended in 200 μL hypotonic lysis buffer (30 minutes at 4 o C) , followed by water bath sonication (30 minut e s a t 4 o C) . The process effic acy was confirmed by FACS , showing that near 72% of the cells were successfully stained with propidium iodide solution (PI). The quality of the protein extracts was analyzed by LCMS/MS using the strategy MS E label free for relative quant ification of proteins. A totality of 1153 proteins were identified and the comparison of the expression profiles between the strains 4167, Dm28c and Y , using the CL - Brener as reference, showed 32, 51 and 73 proteins up - expressed, and 80, 92 and 60 proteins were shown to be down - expressed, respectively. We observed that 117 trans - sialidase s were identified in Y strain, whilst in 4167, Dm28c and CL - Brener were found 1, 2 and 1 trans - sialidases, respectively. Moreover only one copy of trans - sialidase found in the TcS12 extract (UniProt accession number - Q4DGV8), also met its corresponding mRNA in the CL - Brener cDNA database, presenting an UTR of 214 bp, in the size range of the others trans - sialidase UTRs obtained herein. This result suggests th at other facto r s, but not exclusively the UTR sizes per se , could be related to the translation phenomenon in these trypanosome

Trypanosoma cruzi I genotype among isolates from patients with chronic Chagas disease followed at the Evandro Chagas National Institute of Infectious Diseases (FIOCRUZ, Brazil)

ABSTRACT INTRODUCTION: Trypanosoma cruzi is the etiologic agent of Chagas disease in humans, mainly in Latin America. Trypanosome stocks were isolated by hemoculture from patients followed at Evandro Chagas National Institute of Infectious Diseases (FIOCRUZ) and studied using different approaches. METHODS: For species and genotype identification, the stocks were analyzed by parasitological techniques, polymerase chain reaction assays targeted to specific DNA sequences, isoenzyme patterns, besides sequencing of a polymorphic locus of TcSC5D gene (one stock). RESULTS: The isolates presented typical T. cruzi morphology and usually grew well in routine culture media. Metacyclic trypomastigotes were found in cultures or experimentally infected Triatoma infestans. All isolates were pure T. cruzi cultures, presenting typical 330-bp products from kinetoplast DNA minicircles, and 250 or 200-bp amplicons from the mini-exon non-transcribed spacer. Their genetic type assignment was resolved by their isoenzyme profiles. The finding of TcI in one asymptomatic patient from Paraíba was confirmed by the sequencing assay. TcVI was found in two asymptomatic individuals from Bahia and Rio Grande do Sul. TcII was identified in six patients from Pernambuco, Bahia and Minas Gerais, who presented different clinical forms: cardiac (2), digestive with megaesophagus (1), and indeterminate (3). CONCLUSIONS: The main T. cruzi genotypes found in Brazilian chronic patients were identified in this work, including TcI, which is less frequent and usually causes asymptomatic disease, unlike that in other American countries. This study emphasizes the importance of T. cruzi genotyping for possible correlations between the parasite and patient’ responses to therapeutic treatment or disease clinical manifestations

Trypanosoma cruzi I genotype among isolates from patients with chronic Chagas disease followed at the Evandro Chagas National Institute of Infectious Diseases (FIOCRUZ, Brazil)

<div><p>ABSTRACT INTRODUCTION: Trypanosoma cruzi is the etiologic agent of Chagas disease in humans, mainly in Latin America. Trypanosome stocks were isolated by hemoculture from patients followed at Evandro Chagas National Institute of Infectious Diseases (FIOCRUZ) and studied using different approaches. METHODS: For species and genotype identification, the stocks were analyzed by parasitological techniques, polymerase chain reaction assays targeted to specific DNA sequences, isoenzyme patterns, besides sequencing of a polymorphic locus of TcSC5D gene (one stock). RESULTS: The isolates presented typical T. cruzi morphology and usually grew well in routine culture media. Metacyclic trypomastigotes were found in cultures or experimentally infected Triatoma infestans. All isolates were pure T. cruzi cultures, presenting typical 330-bp products from kinetoplast DNA minicircles, and 250 or 200-bp amplicons from the mini-exon non-transcribed spacer. Their genetic type assignment was resolved by their isoenzyme profiles. The finding of TcI in one asymptomatic patient from Paraíba was confirmed by the sequencing assay. TcVI was found in two asymptomatic individuals from Bahia and Rio Grande do Sul. TcII was identified in six patients from Pernambuco, Bahia and Minas Gerais, who presented different clinical forms: cardiac (2), digestive with megaesophagus (1), and indeterminate (3). CONCLUSIONS: The main T. cruzi genotypes found in Brazilian chronic patients were identified in this work, including TcI, which is less frequent and usually causes asymptomatic disease, unlike that in other American countries. This study emphasizes the importance of T. cruzi genotyping for possible correlations between the parasite and patient’ responses to therapeutic treatment or disease clinical manifestations.</p></div

Molecular diagnosis of cutaneous leishmaniasis in an endemic area of Acre State in the Amazonian Region of Brazil

Abstract INTRODUCTION This study proposes to identify the Leishmania species found in the skin lesions of cutaneous leishmaniasis (CL) patients from Brasiléia municipality (Acre). METHODS Skin biopsy imprints or biopsy fragments were assayed via kDNA-PCR/RFLP and FRET-real-time PCR. RESULTS Of individuals with suspected CL, 18 were positive for Leishmania kDNA. Leishmania (Viannia) braziliensis (61.1%) and Leishmania (Viannia) guyanensis (5.5%) were identified in the positive samples. CONCLUSIONS These results are congruent with the previous reports in Acre and Bolivia, revealing L. braziliensis as the most prevalent species. L. guyanensis identification also corroborates with the epidemiology of the disease in the Amazon Basin