VARIABILIDADE INTRAESPECÍFICA DA PROTEÍNA FLAGELAR LIGADORA DE CÁLCIO (FCABP) DE TRYPANOSOMA RANGELI

 A doença de Chagas constitui um dos principais problemas de saúde pública nas Américas. Seu agente etiológico, o Trypanosoma cruzi Chagas, 1909, compartilha com Trypanosoma rangeli Tejera, 1920, regiões geográficas, além de hospedeiros, sendo comum ocorrerem infecções mistas dificultando o diagnóstico da doença. Dessa forma, a reatividade cruzada gera resultados falso-positivos com T. cruzi e T. rangeli dificultando o diagnóstico sorológico da doença de Chagas. No entanto, algumas diferenças moleculares destes parasitos podem ser localizadas e utilizadas como biomarcadores específicos. Diante disso, este trabalho buscou avaliar a variabilidade intraespecífica da Proteína Flagelar Ligadora de Cálcio (FCaBP) de T. rangeli em quatro diferentes cepas, pois essa proteína se apresenta como possível alvo molecular para o diagnóstico diferencial dos parasitos. Para isso, foram desenhados iniciadores para a amplificação mediante a Reação em Cadeia da Polimerase (PCR) e posterior sequenciamento do gene de FCaBP de T. rangeli (TR-FCaBP-F 5’ AGG CGA TAG TAG CAC AAT TCA G 3’) e (TR-FCaBP-R 5’ TCT GTC CAG CTC CTT GAA C 3’). A análise in silico do gene da FCaBP nas cepas Choachí, SC58, Macias e LDG de T. rangeli revelou alta similaridade entre as cepas deste parasito. Contudo, observou-se a ausência de seis resíduos de aminoácidos na região N-terminal quando comparada com a proteína homóloga de T. cruzi. Essa região confere a distinção de um epitopo exclusivo ao T. rangeli (SKSSAGNKDGKSATD), tornando-se um potencial marcador específico ao T. rangeli em diagnóstico sorológico diferencial ao T. cruzi.Palavras-chave: Doença de Chagas. Trypanosoma cruzi. Proteína de ligação ao cálcio. Diagnóstico diferencial. PCR

Trypanosoma cruzi I and IV Stocks from Brazilian Amazon Are Divergent in Terms of Biological and Medical Properties in Mice

Background: In the Brazilian Amazon, clinical and epidemiological frameworks of Chagas disease are very dissimilar in relation to the endemic classical areas of transmission, possibly due to genetic and biological characteristics of the circulating Trypanosoma cruzi stocks. Twenty six T. cruzi stocks from Western Amazon Region attributed to the TcI and TcIV DTUs were comparatively studied in Swiss mice to test the hypothesis that T. cruzi clonal structure has a major impact on its biological and medical properties. Methodology/Principal Findings: Seventeen parameters were assayed in mice infected with 14 T. cruzi strains belonging to DTU TcI and 11 strains typed as TcIV. In comparison with TcI, TcIV stocks promoted a significantly shorter pre-patent period (p<0.001), a longer patent period (p<0.001), higher values of mean daily parasitemia (p = 0.009) and maximum of parasitemia (p = 0.015), earlier days of maximum parasitemia (p<0.001) and mortality (p = 0.018), higher mortality rates in the acute phase (p = 0.047), higher infectivity rates (p = 0.002), higher positivity in the fresh blood examination (p<0.001), higher positivity in the ELISA at the early chronic phase (p = 0.022), and a higher positivity in the ELISA at the late chronic phase (p = 0.003). On the other hand TcI showed higher values of mortality rates in the early chronic phase (p = 0.014), higher frequency of mice with inflammatory process in any organ (p = 0.005), higher frequency of mice with tissue parasitism in any organ (p = 0.027) and a higher susceptibility to benznidazole (p = 0.002) than TcIV. Survival analysis showing the time elapsed from the day of inoculation to the beginning of the patent period was significantly shorter for TcIV strains and the death episodes triggered following the infection with TcI occurred significantly later in relation to TcIV. The notable exceptions come from positivity in the hemocultures and PCR, for which the results were similar. Conclusion/Significance: T. cruzi stocks belonging to TcI and TcIV DTUs from Brazilian Amazon are divergent in terms of biological and medical properties in mice.publishersversionpublishe

Caracterização molecular e funcional das amastinas e tuzina do trypanosoma rangeli

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Biotecnologia e Biociências, Florianópolis, 2017.O Trypanosoma rangeli, é um protozoário de ciclo heteroxênico, e para que esta alternância de hospedeiros ocorra, o parasito possui uma regulação gênica em nível pós- transcricional, que permite rápida mudança nos padrões de expressão gênica, possibilitando uma adaptação do parasito a diferentes ambientes. As amastinas constituem uma família multigênica, subdividida em quatro grupos: a, ß, ? e d. Genes representativos de amastinas estão presentes no genoma do T. rangeli, mas o seu padrão de expressão, localização celular, envolvimento na interação com células do hospedeiro neste parasito ainda precisam ser investigados. Sendo assim, este estudo, têm como objetivo avaliar o ciclo celular do Trypanosoma rangeli com a finalidade de atribuir o possível papel das amastinas e tuzina na interação parasito/hospedeiro. Neste trabalho foram identificados 21 (sendo quatro genes truncados) genes pertencentes à família das amastinas, divididos em sete grupos, e um gene referente à tuzina e ambos com localização membranar. Através de uma análise filogenética, classificar esses genes de amastinas como pertencentes ao grupo das a, ß e d-amastinas. Analisamos dois genes de duas diferentes subfamílias TrAma7_1 (ß ?amastina) e TrAma4_1 (a-amastina) quanto a produção dos níveis de transcritos e de expressão proteica, observamos que ambos os genes produzem níveis de transcritos nas formas evolutivas epimastigotas e tripomastigotas com diferença entre as mesmas, mas, apenas para TrAma4_1 foi observada a expressão proteica no Western blot. Para Tuzina, observamos que as mesmas encontram-se intercaladas no genoma com as d-amastinas, e apresar de os níveis de transcritos serem diferentes entre diferentes formas evolutivas analisadas, os níveis de proteína expressa são iguais entre todas as formas, tanto para T. rangeli e T. cruzi. Com isso, observamos então que o nível de proteína expressa não é estágio dependendo como a produção dos níveis de transcritos, para ambos os genes. Parasitos da cepa Choachí de T. rangeli foram transfectados com amastinas de T. cruzi de duas diferentes subfamílias e com a TrAma4_1 de T. rangeli fusionadas com GFP, e com base nos resultados encontrados no estudo in vivo da infecção experimental com essas cepas transfectadas em comparação com a cepa parental pudemos observar que não houveram diferenças significativas na evolução da infecção.Abstract : Trypanosoma rangeli is a protozoan of heteroxenic cycle and for this host alternation to occur the parasite has a post-transcriptional level of gene regulation that allows a rapid change in the gene expression patterns, allowing an adaptation of the parasite to different environments. The amastins constitute a multigenic family, subdivided into four groups: a, ß, ? and d. Representative amastin genes are present in the T. rangeli genome, but their pattern of expression, cell localization, involvement with host cells interaction in this parasite still needs to be investigated. Thus, this study aims to evaluate the cell cycle of Trypanosoma rangeli in order to attribute the possible role of amastines and tuzin in the parasite/host interaction. In this work 21 genes (those: four are truncated genes) were identified belonging to the amastine family, divided into seven groups, and one gene related to the tuzin and both of them with membrane localization. Through a phylogenetic analysis, these amastine genes were classify belonging to the a, ß and d-amastins group. We analyzed two different genes from two different amastin subfamilies: TrAma7_1 (ß-amastine) and TrAma4_1 (a -amastine) for the production of transcript levels and protein expression, and we observed that both genes produce levels of transcripts in epimastigote and trypomastigote evolutionary forms with difference between thenselves but only for TrAma4_1 was observed protein expression in the Western blot. For Tuzin, we observed that they are intercalated in the genome with the d-amastines, and the transcript levels are different between different evolutionary forms analyzed, expressed protein levels are equal between all forms, both for T. rangeli and T. cruzi. So, until now we observed that the protein expression levels are not stage specific, diferent os the transcriptions levels. Parasites of T. rangeli strain Choachi were transfected with four different T. cruzi amastins from two different subfamilies and with T. rangeli TrAma4_1 and fused with GFP, and based on the results found in the in vivo study of the experimental infection with these strains transfected into comparison with the parental strain showed that there were no significant differences in the evolution of the infection

VARIABILIDADE INTRAESPECÍFICA DA PROTEÍNA FLAGELAR LIGADORA DE CÁLCIO (FCaBP) DE Trypanosoma rangeli

A doença de Chagas constitui um dos principais problemas de saúde pública nas Américas. Seu agente etiológico, o Trypanosoma cruzi Chagas, 1909, compartilha com Trypanosoma rangeli Tejera, 1920, regiões geográficas, assim como hospedeiros, sendo comum ocorrerem infecções mistas, o que dificulta o diagnóstico da doença; a reatividade cruzada gera resultados falso-positivos com T. cruzi e T. rangeli, dificultando o diagnóstico sorológico da doença de Chagas. No entanto, algumas diferenças moleculares desses parasitos podem ser localizadas e utilizadas como biomarcadores específicos. Diante disso, este trabalho buscou avaliar a variabilidade intraespecífica da Proteína Flagelar Ligadora de Cálcio (FCaBP) de T. rangeli em quatro diferentes cepas, pois essa proteína se apresenta como possível alvo molecular para diagnóstico diferencial dos parasitos. Para isso, foram desenhados iniciadores para a amplificação por meio da Reação em Cadeia da Polimerase (PCR) e posterior sequenciamento do gene de FCaBP de T. rangeli (TR-FCaBP-F 5' AGG CGA TAG TAG CAC AAT TCA G 3') e (TR-FCaBP-R 5' TCT GTC CAG CTC CTT GAA C 3'). A análise in silico do gene da FCaBP nas cepas Choachí, SC58, Macias e LDG de T. rangeli, revelou alta similaridade entre as cepas desse parasito, contudo, observou-se a ausência de seis resíduos de aminoácidos na região N-terminal quando comparada à proteína homóloga de T. cruzi. Essa região confere a distinção de um epítopo exclusivo ao T. rangeli (SKSSAGNKDGKSATD), tornando-se um potencial marcador específico para o T. rangeli em diagnóstico sorológico diferencial ao T. cruzi.

Differential parasitological, molecular, and serological detection of Trypanosoma cruzi I, II, and IV in blood of experimentally infected mice.

Trypanosoma cruzi is the etiological agent of American trypanosomiasis (Chagas' disease), which affects 6 e7 million people worldwide, mainly in Latin America. It presents great genetic and biological variability that plays an important role in the clinical and epidemiological features of the disease. Our working hypothesis is that the genetic diversity of T. cruzi has an important impact on detection of the parasite using diagnostic techniques. The present study evaluated the diagnostic performance of parasitological, molecular, and serological techniques for detecting 27 strains of T. cruzi that belonged to discrete typing units (DTUs) TcI (11 strains), TcII (four strains), and TcIV (12 strains) that were obtained from different hosts in the states of Amazonas and Paran a, Brazil. Blood samples were taken from experimentally infected mice and analyzed by fresh blood examination, hemoculture in Liver Infusion Tryptose (LIT) medium, polymerase chain reaction (PCR), and enzyme-linked immunosorbent assay (ELISA). Polymerase chain reaction presented the best detection of TcI, with 80.4% positivity. For all of the detection methods, the animals that were inoculated with TcII presented the highest positivity rates (94.1e100%). ELISA that was performed 7 months after inoculation presented a higher detection ability (95.4%) for TcIV. Intra-DTU comparisons showed that the reproducibility of the majority of the results that were obtained with the different methods was weak for TcI and good for TcII and TcIV. Our data indicate that the detection capability of different techniques varies with the DTUs of the parasites in mammalian blood. The implications of these findings with regard to the diagnosis of human T. cruzi infection are discussed

Trypanosoma cruzi: Evaluation of PCR as a Laboratory Tool to Follow up the Evolution of Parasite Load

Background: The study evaluated qualitative PCR, primers 121-122 as a tool to follow up evolution parasite load of Trypanosoma cruzi. Methods: The study was conducted at the State University of Maringa, in 2015. Step 1, dilutions 1/10 were performed from T. cruzi-Y strain to obtain preparations of 50,000-0.05 parasites/mL from which DNA were extracted, quantified, and amplified. Step 2, the extracted DNA in the dilutions 5-0.05 parasites/mL was re-diluted 1/10, 1/100, 1/1000, quantified, and amplified. Polyacrylamide gels were photographed and thicknesses of the 330 bp kDNA fragments were measured. Results: Step 1, in the dilutions 50,000-50 parasites/mL kDNA fragments had same thickness and, dilutions 5-0.05 parasites/mL showed progressive decrease in thicknesses and staining intensity of the 330 bp fragments. Step 2, demonstrated that dilutions of five (re-dilutions 1/10 and 1/100) and 0.5 (1/10) parasites/mL produced similar thicknesses of the 330 bp fragments obtained in Step 1. However, very dilute DNA samples make difficult to reproduce the fragments thicknesses. Conclusion: PCR, despite its limitations, was able to detect progressive decrease in thicknesses/staining intensity of kDNA fragments in the dilutions 5-0.05 parasites/mL. Hence, has the potential to be used to follow-up evolution of parasite load, not by quantifying the number of parasites, but by dynamic evolution of the fragments thicknesses during etiological treatment

Virulence parameters obtained in Swiss mice from <i>Trypanosoma cruzi</i> I and IV strains.

a<p>Cumulative mortality from inoculation until the 90^th day after inoculation (d.a.i);</p>b<p>Cumulative mortality from the 90^th until the 180^th d.a.i.;</p>c<p>Percentage of animals presenting positive fresh blood examination within the first two months following inoculation and/or positive hemoculture and/or PCR on the 55^th d.a.i.;</p>d<p>Percentage of animals presenting positive ELISA in the 115^th d.a.i.;</p>e<p>Percentage of animals presenting positive ELISA in the 205^th d.a.i..</p>f<p>p<0.05: significant difference; N.S.: not significant.</p

Curves of mean parasitemia obtained from <i>Trypanosoma cruzi</i> I and IV strains.

<p>Curves of mean parasitemia obtained from <i>Trypanosoma cruzi</i> I and IV strains.</p

Number of mice infected with <i>Trypanosoma cruzi</i> I and IV strains presenting histopathological alterations.

<p>Number of mice infected with <i>Trypanosoma cruzi</i> I and IV strains presenting histopathological alterations.</p