Modificações pós-traducionais durante o processo de amastigogênese do Trypanosoma cruzi / Post-translational modifications during the amastigogenesis of Trypanosoma cruzi

A doença de Chagas é uma doença negligenciada causada pelo protozoário Trypanosoma cruzi constituindo-se em um problema de saúde pública em vários países da América Latina. No seu complexo ciclo de vida, o protozoário passa por quatro estágios diferentes: tripomastigota metacíclica, amastigota, tripomastigota sanguíneo e epimastigota, que permitem sua sobrevivência nos diferentes ambientes com os quais o parasita entra em contato. A diferenciação dos tripomastigotas de T. cruzi em amastigotas (amastigogênese) ocorre com grandes mudanças morfológicas, estruturais e metabólicas no parasita e pode ser reproduzido in vitro por exemplo, pela acidificação do meio extracelular. Apesar dos vários trabalhos descritos na literatura, o processo ainda não é totalmente compreendido. A participação de NO na transdução de sinal durante a amastigogênese, sugerida por dados não publicados de nosso grupo, assim como a via de sinalização dependente de AMPc, foram o foco do presente estudo. A indução da amastigogênese foi obtida por incubação de tripomastigotas em meio de cultura acidificado (pH 6,0) e os parâmetros estudados comparados com parasitas controle (meio de cultura, pH 7,4). Estudamos a variação no perfil de nucleotídios cíclicos (AMPc, GMPc), de quinases (PKA, MAPK- ERK1/2), de uma fosfatase (PP2A), assim como o perfil de proteínas fosforiladas, S-nitrosiladas e nitradas até 6 h do início da amastigogênese. O processo foi dividido nas etapas: inicial (até 60 minutos) e tardio (em torno de 3-4 h), caracterizados por um aumento de formas amastigotas na etapa tardia. Houve um aumento de aproximadamente 17 vezes no nível de AMPc nos primeiros 15 minutos da amastigogênese (meio pH 6,0), seguido por aumento discreto no nível de PKA fosforilada, utilizado como indicador de atividade enzimática, este mais evidente na etapa tardia (360 minutos). Quanto à subunidade catalítica fosforilada da MAPK (ativa), há uma aparente diminuição no nível de fosforilação na fase inicial (30 minutos) e aumento na etapa tardia (120 minutos) do processo de amastigogênese. Quanto ao perfil geral de fosforilação de proteínas, há uma diminuição de fosforilação em torno de 30 minutos, seguida de aumento de fosforilação em proteínas de aproximadamente 5 e 100 kDa, mas de maneira geral, não se observaram grandes mudanças nesse perfil com a metodologia utilizada. Quanto às modificações por NO e seus derivados, foram observadas modificações por S-nitrosilação e nitração das proteínas, além do aumento de GMPc em torno de 60 minutos. Embora essas modificações modulem a atividade biológica de uma grande diversidade de proteínas, seu papel biológico não foi explorado.8 Em resumo, nossos resultados apontam para uma variação no perfil de fosforilação, S-nitrosilação e nitração de proteínas, além do aumento de AMPc e GMPc ao longo do processo de amastigogênese in vitro, com a via de sinalização dependente de quinases/ fosfatases e de óxido nítrico ocorrendo ao longo do processo de amastigogênese

Chagas disease is a neglected disease caused by the parasite Trypanosoma cruzi and is a public health problem in several Latin American countries. In its complex life cycle, the protozoan goes through four different stages: metacyclic trypomastigote, amastigote, blood trypomastigote and epimastigote, which allow its survival in the different environments which the parasite comes into contact. The differentiation of T. cruzi trypomastigotes into amastigotes (amastigogenesis) occurs with large morphological, structural and metabolic changes in the parasite and can be reproduced in vitro by, for example, acidification of the extracellular medium. Despite the many data described in the literature, the process is not yet fully understood. The participation of NO in signal transduction during amastigogenesis, suggested by unpublished data from our group, as well as the cAMP-dependent signaling pathway, were the focus of the present study. The induction of amastigogenesis was obtained by incubating trypomastigotes in acidified culture medium (pH 6.0) and the studied parameters compared with control parasites (culture medium, pH 7.4). We studied the variation in the profile of cyclic nucleotides (cAMP, cGMP), kinases (PKA, MAPK-ERK1 / 2), phosphatase (PP2A), as well as the profile of phosphorylated, S-nitrosylated and nitrated proteins up to 6 h. onset of amastigogenesis. The process was divided into early (up to 60 minutes) and late (around 3-4 hours), characterized by an increase in amastigote forms in the late stage. There was an approximately 17-fold increase in cAMP level in the first 15 minutes of amastigogenesis (pH 6.0 medium), followed by a slight increase in phosphorylated PKA level, most evident in the late stage (360 minutes). As for the phosphorylated catalytic subunit of MAPK (active), there is an apparent decrease in the phosphorylation level in the early phase (30 minutes) and increase in the late stage (120 minutes) of the amastigogenesis process. As for the general protein phosphorylation profile, there is a decrease in phosphorylation around 30 minutes, followed by an increase in phosphorylation of proteins (approximately 5 and 100 kDa), but overall, no major changes were observed in this profile with the methodology used. As for modifications by NO and its derivatives, modifications were observed by S-nitrosylation and protein nitration, besides the increase of cGMP around 60 minutes. Although these modifications modulate the biological activity of a wide range of proteins, their biological role has not been explored. In summary, our results point to a variation in phosphorylation, S-nitrosylation and nitration profile of proteins, as well as an increase in cAMP and cGMP along the amastigogenesis process, implicating kinases / phosphatases and nitric oxide dependent signaling pathways in this differentiation

Trypanosoma cruzi: analysis of two different strains after piplartine treatment

ABSTRACT The hemoflagellate protozoan, Trypanosoma cruzi, mainly transmitted by triatomine insects through blood transfusion or from mother-to-child, causes Chagas' disease. This is a serious parasitic disease that occurs in Latin America, with considerable social and economic impact. Nifurtimox and benznidazole, drugs indicated for treating infected persons, are effective in the acute phase, but poorly effective during the chronic phase. Therefore, it is extremely urgent to find innovative chemotherapeutic agents and/or effective vaccines. Since piplartine has several biological activities, including trypanocidal activity, the present study aimed to evaluate it on two T. cruzi strains proteome. Considerable changes in the expression of some important enzymes involved in parasite protection against oxidative stress, such as tryparedoxin peroxidase (TXNPx) and methionine sulfoxide reductase (MSR) was observed in both strains. These findings suggest that blocking the expression of the two enzymes could be potential targets for therapeutic studies.

Costos de la prueba de tamización para la enfermedad de Chagas en donantes de dos bancos de sangre de Colombia, 2015 / Costs of Chagas' disease screening test in blood donors in two Colombian blood banks, 2015

Resumen Introducción . La transfusión es un mecanismo de transmisión de la enfermedad de Chagas. No se han hecho estudios de costos de la prueba de tamización en bancos de sangre de Colombia. Objetivo. Estimar los costos de la prueba de tamización para la enfermedad de Chagas en donantes de bancos de sangre de Colombia, 2015. Materiales y métodos. Se hizo un estudio de costos desde la perspectiva del prestador de servicios en los bancos de sangre de la Cruz Roja, seccional Bolívar, y del Hospital de Yopal, Casanare, que incluyó: 1) gastos administrativos, es decir, costos de servicios públicos y seguros asignados según los metros cuadrados de las instalaciones del banco de sangre; 2) costos de capital, es decir, edificación y equipos, anualizados con una tasa de descuento de 3 % y considerando una vida útil de 20 y cinco años, respectivamente; 3) costos de insumos y materiales ajustados al nivel de producción, y 4) costos del recurso humano encargado del procesamiento de las pruebas. Se reportó, asimismo, el costo de las bolsas y de las pruebas de inmunohematología. Resultados. En el banco de sangre de la Cruz Roja, seccional Bolívar, el costo de la prueba fue de COP$ 37.804 (USD$ 12), mientras que la bolsa y la prueba de inmunohematología costaron COP$ 25.942 (USD$ 8,2) y COP$ 6.800 (USD$ 2,2), respectivamente. En el banco de sangre del Hospital de Yopal, los costos ascendieron a COP$ 77.384 (USD$ 24,6), COP$ 30.141 (USD$ 9,6) y COP$ 12.627 (USD$ 4), respectivamente. La mayor participación en el costo de la prueba correspondió al recurso humano (47,5 % en Cartagena y 55,7 % en Yopal). Conclusiones. Estos resultados son importantes para la planificación de los servicios y los análisis de costo-efectividad de la prueba de tamización para la enfermedad de Chagas en los bancos de sangre.

Abstract Introduction: Transfusion is a mechanism of transmission of Chagas' disease. There are no studies on the costs of the screening test in Colombian blood banks. Objective: To estimate the costs of the screening test for Chagas' disease among blood donors in two Colombian blood banks, 2015. Materials and methods: We conducted a micro-costing study from the perspective of the health care provider to estimate the cost of Chagas' disease testing in two blood banks, Banco de Sangre de la Cruz Roja, Seccional Bolívar, and Banco de Sangre del Hospital de Yopal, Casanare, taking into account four cost categories: 1) Administrative costs: public services and insurance costs were calculated based on the blood bank area in square meters; 2) capital costs: building and equipment costs that were annualized using a 3% discount rate and a lifespan of 20 years for building and five for equipment; 3) costs of Chagas' disease test materials and reagents adjusted by blood bank production level, and 4) costs of staff in charge of Chagas' disease test processing. The costs of transfusion bags and immunohematology tests are also reported. Results: The cost of Chagas' disease test in the blood bank of Seccional Bolívar was COP$ 37,804 (USD$ 12), and the blood bag and immunohematology test costs were COP$ 25,941 (USD$ 8.2) and COP$ 6,800 (USD$ 2.2), respectively. In the blood bank of Yopal, Casanare, the costs were COP$ 77,384 (USD$ 24.6), COP$ 30,141 (USD$ 9.6) and COP$ 12,627 (USD$ 4), respectively. Personnel cost accounted for the highest percentage of the total cost for both blood banks (47.5% in Seccional Bolívar, and 55.7% in Yopal, Casanare). Conclusion: Our results are an important input for the planning of services and cost-effectiveness studies for screening tests for Chagas' disease in Colombian blood banks.

Subcellular localisation of FLAG tagged enzymes of the dynamic protein S-palmitoylation cycle of Trypanosoma cruzi epimastigotes

Dynamic S-palmitoylation of proteins is the addition of palmitic acid by zDHHC palmitoyl transferases (PATs) and depalmitoylation by palmitoyl protein thioesterases (PPTs). A putative PAT (TcPAT1) has been previously identified in Trypanosoma cruzi, the etiological agent of Chagas disease. Here we analyse other 14 putative TcPATs and 2 PPTs in the parasite genome. T. cruzi cell lines expressing TcPATs and TcPPTs plus a FLAG tag at the C terminus were produced for most enzymes, with positive detection by indirect immunofluorescence. Overexpressed TcPATs were mostly found as single spots at the parasite anterior end, while the TcPPTs were dispersed throughout the parasite body.

Reconhecimento molecular na doença de chagas do ponto de vista do parasita e do hospedeiro / Molecular recognition in Chagas disease from the point of view of the parasite and the host

A doença de Chagas, causada pelo parasita protozoário Trypanosoma cruzi, afeta milhões de pessoas, a maioria delas vivendo na América latina. Apesar dos avanços da medicina e da biotecnologia, ainda existem poucas opções de tratamento para indivíduos com a doença. Assim, é importante compreendermos os detalhes moleculares da infecção parasitária, para que novas alternativas terapêuticas e de diagnóstico possam ser desenvolvidas para esses pacientes. Neste trabalho estudamos esta doença em duas frentes, uma do ponto de vista do parasita, e a outra, da resposta do hospedeiro. Utilizando bioinformática, identifcamos um peptídeo conservado (denominado TS9) presente nas proteínas de superfície gp85/transsialidases do parasita. Este peptídeo é capaz de promover adesão celular e, na sua forma sintética, inibe a entrada do T. cruzi na célula hospedeira. Análise da estrutura proteica revelou que o peptídeo TS9 encontra-se num domínio do tipo laminina-G, lado-a-lado com o peptídeo FLY, outro peptídeo conservado desta grande família, previamente descrito pelo nosso grupo. Juntos, eles formam um sítio de adesão a citoqueratinas e proteínas de flamento intermediário. Na segunda parte, investigamos os antígenos e epítopos reconhecidos pelas imunoglobulinas de pacientes portadores da doença nas suas diferentes formas clínicas: assintomática e cardiomiopatias, leve ou grave. Criamos uma biblioteca de phage display contendo, virtualmente, todos os fragmentos proteicos existentes no T. cruzi, que foi varrida contra imunoglobulinas para a construção de um mapa da resposta humoral dos pacientes com a doença de Chagas. Nossos resultados mostram que a resposta dos pacientes é complexa, e mais de dois mil epítopos foram mapeados. Muitos deles, como os antígenos B13, SAPA e FRA já foram previamente descritos, validando nosso método. Porém, um grande número de novos epítopos, inclusive contra proteína descritas como hipotéticas ou sem função conhecida, também foram encontrados. Seus papéis na infecção e resposta imune da doença merecem, portanto, atenção. Em resumo, as abordagens e técnicas utilizadas nesta tese são inovadoras, e permitiram a identificação de peptídeos e moléculas que poderão ser úteis para o desenvolvimento de novos métodos diagnósticos e terapêuticos para a doença de Chagas

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, afects millions of people, most of them living in Latin America. Despite advances in medicine and biotechnology, there are still few treatment options for individuals with the disease. Thus, it is important to understand the molecular details of the parasitic infection, so that new therapeutic and diagnostic alternatives can be developed for these patients. In this work, we study this disease in two fronts, one from the point of view of the parasite, and the other, of the response of the host. Using bioinformatics, we identifed a conserved peptide (called TS9) present in the surface proteins gp85 / trans-sialidases of the parasite. This peptide is capable of promoting cell adhesion and, in its synthetic form, inhibits the entry of T. cruzi into the host cell. Analysis of the protein structure revealed that the TS9 peptide is in a laminin-G-like domain, side-by-side with the peptide FLY, another conserved peptide of this large family, previously described by our group. Together, they form an adhesion site to cytokeratins and intermediate flament proteins. In the second part, we investigated the antigens and epitopes recognized by the immunoglobulins of patients with the disease in their diferent clinical forms: asymptomatic and cardiomyopathies, mild or severe. We created a phage display library containing virtually all existing protein fragments in T. cruzi. This library was screened against immunoglobulins for the construction of a humoral response map of patients with Chagas disease. Our results show that the response of the patients is complex, and more than 2,000 epitopes have been mapped. Many of them, such as the B13, SAPA and FRA antigens have been previously described, validating our method. However, a large number of new epitopes, including many against proteins described as hypothetical or with no known function, were also found. Their roles in infection and immune response of the disease deserve, therefore, attention. In summary, the approaches and techniques used in this thesis are innovative and have allowed the identifcation of new peptides and molecules that may be useful for the development of new diagnostic and therapeutic methods for Chagas disease

Mir-190b negatively contributes to the Trypanosoma cruzi- infected cell survival by repressing PTEN protein expression

Chagas disease, which is caused by the intracellular protozoanTrypanosoma cruzi, is a serious health problem in Latin America. The heart is one of the major organs affected by this parasitic infection. The pathogenesis of tissue remodelling, particularly regarding cardiomyocyte behaviour after parasite infection, and the molecular mechanisms that occur immediately following parasite entry into host cells are not yet completely understood. Previous studies have reported that the establishment of parasitism is connected to the activation of the phosphatidylinositol-3 kinase (PI3K), which controls important steps in cellular metabolism by regulating the production of the second messenger phosphatidylinositol-3,4,5-trisphosphate. Particularly, the tumour suppressor PTEN is a negative regulator of PI3K signalling. However, mechanistic details of the modulatory activity of PTEN on Chagas disease have not been elucidated. To address this question, H9c2 cells were infected with T. cruzi Berenice 62 strain and the expression of a specific set of microRNAs (miRNAs) were investigated. Our cellular model demonstrated that miRNA-190b is correlated to the decrease of cellular viability rates by negatively modulating PTEN protein expression in T. cruzi-infected cells.

The TcI and TcII Trypanosoma cruzi experimental infections induce distinct immune responses and cardiac fibrosis in dogs

Trypanosoma cruzi infection may be caused by different strains with distinct discrete typing units (DTUs) that can result in variable clinical forms of chronic Chagas disease. The present study evaluates the immune response and cardiac lesions in dogs experimentally infected with different T. cruzi strains with distinct DTUs, namely, the Colombian (Col) and Y strains of TcI and TcII DTU, respectively. During infection with the Col strain, increased levels of alanine aminotransferase, erythrocytes, haematocrit and haemoglobin were observed. In addition, CD8+ T-lymphocytes isolated from the peripheral blood produced higher levels of interleukin (IL)-4. The latter suggests that during the acute phase, infection with the Col strain may remain unnoticed by circulating mononuclear cells. In the chronic phase, a significant increase in the number of inflammatory cells was detected in the right atrium. Conversely, infection with the Y strain led to leucopoenia, thrombopoenia, inversion of the ratio of CD4+/CD8+ T-lymphocytes and alterations in monocyte number. The Y strain stimulated the production of interferon-γ by CD4+ and CD8+ T-lymphocytes and IL-4 by CD8+ T-cells. In the chronic phase, significant heart inflammation and fibrosis were observed, demonstrating that strains of different DTUs interact differently with the host.

Avaliação do efeito do composto derivado de B-lapachona R72 em formas tripomastigotas de trypanozoma cruzi e na infecção de macrófagos pelo Trypanosoma cruzi e na infecção de macrófagos pelo T. cruzi in vitro / Effect of the compound derived from B-lapachone R72 in the trypanozoma cruzi trypomastigotes and macrophage infection by Trypanosoma cruzi and infection of macrophages in vitro with T. cruzi

A tripanossomíase americana também conhecida como doença de Chagas é um importante problema de saúde pública, uma doença de origem parasitária que afeta 8 milhões de pessoas na América Latina (OMS, 2013). Atualmente existem apenas dois fármacos utilizados na prática clínica: o benzonidazol e o nifurtimox. Esse último não é usado no Brasil. Esses fármacos apresentam efeitos colaterais e são confrontados com cepas resistentes (FILARDI e BRENER, 1987; MEJIA et al., 2012). Desta forma, torna-se necessária a busca de compostos que apresentem maior eficácia e menor toxicidade. No presente trabalho foi avaliado o efeito do composto derivado de β–lapachona, R72, em formas tripomastigotas de T. cruzi e em macrófagos infectados in vitro. Observamos que a R72 reduziu, significativamente (p<0,05)...

The American trypanosomiasis also known as Chagas disease is a major public health problem, a parasitic disease caused that affects 8 million people in Latin America (OMS, 2013). Currently there are only two drugs used in clinical practice: the benznidazole and the nifurtimox. The latter is not used in Brazil. These drugs are faced with considerable side effects and resistant strains (FILARDI e BRENER, 1987; MEJIA et al., 2012). Thus the search for drugs effective in the chronic phase of the disease and lower toxicity is required. In this study the effect of the β-lapachone derivative R72 in trypomastigote forms of T. cruzi and upon the infection of macrophage by T. cruzi in vitro. We observed that the R72 significantly inhibited (p<0,05)...

Induction of chagasic-like arrhythmias in the isolated beating hearts of healthy rats perfused with Trypanosoma cruzi-conditioned medium

Chagas' myocardiopathy, caused by the intracellular protozoan Trypanosoma cruzi, is characterized by microvascular alterations, heart failure and arrhythmias. Ischemia and arrythmogenesis have been attributed to proteins shed by the parasite, although this has not been fully demonstrated. The aim of the present investigation was to study the effect of substances shed by T. cruzi on ischemia/reperfusion-induced arrhythmias. We performed a triple ischemia-reperfusion (I/R) protocol whereby the isolated beating rat hearts were perfused with either Vero-control or Vero T. cruzi-infected conditioned medium during the different stages of ischemia and subsequently reperfused with Tyrode's solution. ECG and heart rate were recorded during the entire experiment. We observed that triple I/R-induced bradycardia was associated with the generation of auricular-ventricular blockade during ischemia and non-sustained nodal and ventricular tachycardia during reperfusion. Interestingly, perfusion with Vero-infected medium produced a delay in the reperfusion-induced recovery of heart rate, increased the frequency of tachycardic events and induced ventricular fibrillation. These results suggest that the presence of parasite-shed substances in conditioned media enhances the arrhythmogenic effects that occur during the I/R protocol.

Parameters for evaluation of clinical trial in mice infected with Trypanosoma cruzi / Parâmetros para avaliação clínica de camundongos infectados pelo Trypanosoma cruzi em experimentação

The effectiveness of clinical parameters in the evaluation of Trypanosoma cruzi infection was analyzed in male Swiss mice at 8 weeks old Animals were divided into HG (healthy) and IG (1400 trypomastigotes, intraperitoneally, Y strain - Trypanosoma cruzi). Quantitative and qualitative parameters were evaluated in non-consecutive days in the period, from 7th-11th and 15th-18th days of infection. There were significant differences (P<0.05) between both groups in both periods regarding water consumption, abdominal circumference and weight. The second group presented differences in amount of excreta, body temperature, move-up and mortality. There was no difference (P>0.05) between the groups in food consumption, exploration of self-cleaning and skin staining. The fecal feature differed between the groups in the second period. The occurrence of isolation was not practical. Differences were observed in the hair between groups, although the parameter had been interfered by fights between animals. The consumption of water, feed, excreta production, characteristic of the faeces, body temperature, abdominal circumference, move up, weight and mortality parameters are easy to be measured and effective in clinical differentiation of healthy mice infected with T. cruzi, elected in protocols for clinical study with mice, which is the first work to gather information of qualitative and quantitative clinical parameters evaluated in these animals.

Analisou-se a eficiência de parâmetros clínicos na avaliação da infecção pelo Trypanosoma cruzi em camundongos suíços, machos de 8 semanas. Os grupos foram divididos em GS (sadios) e GI (1400 tripomastigotas, intraperitoneal, cepa Y - Trypanosoma cruzi). Avaliaram-se parâmetros quantitativos e qualitativos em dias não consecutivos nos períodos, 7º-11º e 15º-18º dias de infecção. Observaram-se diferenças (P<0.05) significativas entre os grupos, nos dois períodos: consumo de água, circunferência abdominal e peso; apenas no segundo período: quantidade de excretas, temperatura corporal, movimento-levantar e mortalidade. Não houve diferença (P>0.05) entre os grupos: consumo de ração, exploração de auto-limpeza e coloração da pele. As fezes diferiram entre os grupos no segundo período. A ocorrência de isolamento não se mostrou prática. Diferenças no pêlo foram observadas entre os grupos, embora o parâmetro sofra interferência de brigas entre os animais. O consumo de água, ração, produção de excretas, característica das fezes, temperatura corporal, circunferência abdominal, movimento-levantar, peso e mortalidade são parâmetros fáceis de serem medidos e eficientes na diferenciação da clínica de camundongos sadios e infectados pelo T. cruzi, eleitos para protocolos de estudos clínicos com camundongos, sendo este o primeiro trabalho a reunir informações de parâmetros clínicos qualitativos e quantitativos avaliados nesses animais.

The overexpression of the trypanosomatid-exclusive TcRBP19 RNA-binding protein affects cellular infection by Trypanosoma cruzi

To characterise the trypanosomatid-exclusive RNA-binding protein TcRBP19, we analysed the phenotypic changes caused by its overexpression. Although no evident changes were observed when TcRBP19 was ectopically expressed in epimastigotes, the metacyclogenesis process was affected. Notably, TcRBP19 overexpression also led to a decrease in the number of infected mammalian cells. These findings suggest that TcRBP19 may be involved in the life cycle progression of the Trypanosoma cruzi parasite.

The zinc finger protein TcZFP2 binds target mRNAs enriched during Trypanosoma cruzi metacyclogenesis

Trypanosomes are parasitic protozoa in which gene expression is primarily controlled through the regulation of mRNA stability and translation. This post-transcriptional control is mediated by various families of RNA-binding proteins, including those with zinc finger CCCH motifs. CCCH zinc finger proteins have been shown to be essential to differentiation events in trypanosomatid parasites. Here, we functionally characterise TcZFP2 as a predicted post-transcriptional regulator of differentiation in Trypanosoma cruzi. This protein was detected in cell culture-derived amastigotes and trypomastigotes, but it was present in smaller amounts in metacyclic trypomastigote forms of T. cruzi. We use an optimised recombinant RNA immunopreciptation followed by microarray analysis assay to identify TcZFP2 target mRNAs. We further demonstrate that TcZFP2 binds an A-rich sequence in which the adenosine residue repeats are essential for high-affinity recognition. An analysis of the expression profiles of the genes encoding the TcZFP2-associated mRNAs throughout the parasite life cycle by microarray hybridisation showed that most of the associated mRNAs were upregulated in the metacyclic trypomastigote forms, also suggesting a role for TcZFP2 in metacyclic trypomastigote differentiation. Knockdown of the orthologous Trypanosoma brucei protein levels showed ZFP2 to be a positive regulator of specific target mRNA abundance.

Expresión diferencial entre estadios de Trypanosoma cruzi I en el aislamiento de un paciente con cardiomiopatía chagásica crónica de zona endémica de Santander, Colombia / Differential protein expression in developmental stages of Trypanosoma cruzi I isolated from a patient with chronic chagasic cardiomyopathy

Introducción. Trypanosoma cruzi es el agente causal de la enfermedad de Chagas. Durante la infección en los huéspedes mamíferos, se observan dos formas del parásito: tripomastigotes y amastigotes. En el curso de la diferenciación del parásito cada estadio expresa un patrón de proteínas específicas de fase, las cuales son responsables de sus características morfológicas, bioquímicas y biológicas, que podrían estar determinando un papel importante en la capacidad infecciosa, virulencia y supervivencia del parásito. Objetivo. Analizar la expresión diferencial entre los estadios tripomastigote y amastigote de un aislamiento de T. cruzi I, utilizando la electroforesis en dos dimensiones y la identificación de las proteínas diferencialmente expresadas mediante espectrometría de masas. Materiales y métodos. Se utilizó un clon del aislamiento MHOM/07/338 de T. cruzi I y, mediante electroforesis en dos dimensiones, se compararon los perfiles proteicos de los estadios tripomastigote y amastigote del parásito. Las imágenes se analizaron con el software PDQuest y las proteínas diferencialmente expresadas se identificaron por MALDI TOF o LC MS/MS. Resultados. Los geles bidimensionales mostraron un promedio de 325 manchas proteicas en cada estadio. En los análisis comparativos se detectaron 21 manchas "sobre expresadas" en el estadiotripomastigote y 30, en el estadio amastigote. Se seleccionaron 16 proteínas para identificación por espectrometría de masas y se clasificaron en diferentes categorías funcionales. Conclusiones. Las proteínas exclusivas de T. cruzi relacionadas, principalmente, con metabolismo glucolítico y ensamble del citoesqueleto, fueron las que presentaron una mayor expresión diferencial entre los estadios tripomastigote y amastigote del parásito. Estas proteínas podrían ser utilizadas para el diseño de fármacos.

Introduction. Trypanosoma cruzi is the causative agent of Chagas disease. During infection inmammalian hosts, two main forms of the parasite are observed: trypomastigotes and amastigotes. During differentiation, each stage of the parasite expresses a pattern of proteins specific to each phase-proteins which are responsible for the cell’s morphological, biochemical and biological properties. These properties ultimately govern the infectivity, virulence and survival of the parasite. Objective. A differential expression analysis was conducted to compare trypomastigote and amastigote stages of T. cruzi I isolate, and to identify proteins differentially expressed by means of mass spectrometry. Materials and methods. A T. cruzi clone of the strain MHOM/07/338 was used to analyze the differential expression between trypomastigote stages of a T. cruzi isolate, using two-dimensional electrophoresis and identification of diferentially expressed proteins by mass spectrometry. The protein profiles of the stages of the parasite were obtained by two-dimensional gel electrophoresis and visualized in gels dyed with Coomassie blue. The images were analyzed with PDQuest software and the differential expression of the proteins was identified by MALDI TOF or LC MS/MS. Results. The two-dimensional gels revealed an average of 325 protein spots in each stage. The comparative analyses detected 21 spots that were over expressed in the trypomastigote stage and 30 in the amastigote stage. Sixteen of the over expressed proteins were selected for identification by mass spectrometry and classified in several functional categories. Mass spectrophotometry determined that the proteins were associated mainly with glucolytic metabolism and assembly of the cytoskeleton constituents. Conclusions. The differential expression between trypomastigote and amastigote stages consisted of proteins specific to T. cruzi and are potential targets for the design of treatment drugs.

A new approach for potential drug target discovery through in silico metabolic pathway analysis using Trypanosoma cruzi genome information

The current drug options for the treatment of chronic Chagas disease have not been sufficient and high hopes have been placed on the use of genomic data from the human parasite Trypanosoma cruzi to identify new drug targets and develop appropriate treatments for both acute and chronic Chagas disease. However, the lack of a complete assembly of the genomic sequence and the presence of many predicted proteins with unknown or unsure functions has hampered our complete view of the parasite's metabolic pathways. Moreover, pinpointing new drug targets has proven to be more complex than anticipated and has revealed large holes in our understanding of metabolic pathways and their integrated regulation, not only for this parasite, but for many other similar pathogens. Using an in silicocomparative study on pathway annotation and searching for analogous and specific enzymes, we have been able to predict a considerable number of additional enzymatic functions in T. cruzi. Here we focus on the energetic pathways, such as glycolysis, the pentose phosphate shunt, the Krebs cycle and lipid metabolism. We point out many enzymes that are analogous to those of the human host, which could be potential new therapeutic targets.

Proline racemases: insights into Trypanosoma cruzi peptides containing D-proline

Trypanosoma cruzi proline racemases (TcPRAC) are homodimeric enzymes that interconvert the L and D-enantiomers of proline. At least two paralogous copies of proline racemase (PR) genes are present per parasite haploid genome and they are differentially expressed during T. cruzi development. Non-infective epimastigote forms that overexpress PR genes differentiate more readily into metacyclic infective forms that are more invasive to host cells, indicating that PR participates in mechanisms of virulence acquisition. Using a combination of biochemical and enzymatic methods, we show here that, in addition to free D-amino acids, non-infective epimastigote and infective metacyclic parasite extracts possess peptides composed notably of D-proline. The relative contribution of TcPRAC to D-proline availability and its further assembly into peptides was estimated through the use of wild-type parasites and parasites over-expressing TcPRAC genes. Our data suggest that D-proline-bearing peptides, similarly to the mucopeptide layer of bacterial cell walls, may be of benefit to T. cruzi by providing resistance against host proteolytic mechanisms.

Contribuição para o diagnóstico da doença de chagas pelas reações de imunidade: II: Isolamento de polissacarídeos, seu comportamento nas reações de fixação do complemento e de hipersensibilidade: os testes de floculação (sublimado e formol-gel) / Contribution to the diagnosis of Chagas disease by the reactions of immunity: II: isolation of polysaccharides, their behavior in the reactions of complement fixation and hypersensitivity: the flocculation tests (and sublimated formol gel)

Revisões históricas aos avanços científicos para o controle da doença, o Simpósio Internacional Comemorativo do Centenário da Descoberta da Doença de Chagas (1909-2009).

Antichagásicos potenciais: busca racional de compostos com ação seletiva pela cruzaína / Potential antichagasic agents: rational design of compounds with selective action in cruzain

A doença de Chagas, parasitose endêmica causada pelo Trypanosoma cruzi, se apresenta como grande causa de morbimortalidade, afetando cerca de 18 milhões de pessoas no continente americano e causando 21.000 mortes, a cada ano. Atualmente, estima-se que 100 milhões de pessoas estejam sob risco de contaminação nos 18 países da área endêmica da doença. A quimioterapia contra a tripanossomíase americana é constituída por apenas dois fármacos, nifurtimox e benznidazol, que não apresentam ação adequada na fase crônica da doença. Por estas razões, é premente a necessidade de novas e mais eficazes alternativas terapêuticas. A cruzaína, mais abundante cisteíno-protease do parasita, é enzima essencial em todos os estágios de modificação celular do parasita e está, também, presente no processo de invasão e modificação do sistema imune do hospedeiro. Além disso, apresenta diferenças em relação a enzimas dessa categoria no hospedeiro. Trata-se, pois, de excelente alvo bioquímico para a pesquisa de novos agentes contra o T. cruzi. Face ao exposto e tendo-se em vista a especificidade da cruzaína, considera-se de grande interesse a compreensão do mecanismo de interação de compostos com essa enzima com o intuito de planejar derivados com atividade antichagásica potencial. O presente trabalho teve o objetivo de elucidar a afinidade de diferentes classes de compostos pela enzima, na busca racional de novos tripanomicidas. Assim, pró-fármacos peptidicos recíprocos, derivados de primaquina(PQ) e de nitrofural(NF), anteriormente sintetizados, e pró-fármacos peptidicos duplos do hidroximetilnitrofural (NFOH) planejados, e cuja síntese foi estudada, foram submetidos a estudos de modelagem molecular e de docking...

Trypanosoma cruzi: Transporte de metabolitos esenciales obtenidos del hospedador / Trypanosoma cruzi: Transport of essential metabolites acquired from the host

El Trypanosoma cruzi es el agente causal de la enfermedad de Chagas, endémica en Argentina y en toda América Latina. Presenta numerosas características metabólicas diferenciales respecto a sus hospedadores insectos y mamíferos. Algunas de estas diferencias fueron consecuencia de millones de años de adaptación al parasitismo en los cuales estos organismos protozoarios reemplazaron, a lo largo de su evolución, muchas rutas metabólicas de biosíntesis por sistemas de transporte de metabolitos desde el hospedador. En esta revisión se describen los avances en el conocimiento de los sistemas de transporte tanto bioquímicos como también de las moléculas involucradas en dichos procesos. Se aborda con especial énfasis los transportadores de aminoácidos y poliaminas de T. cruzi de la familia AAAP (Amino Acid/Auxin Permeases) ya que parece ser exclusiva de los tripanosomátidos. Teniendo en cuenta que estas moléculas se encuentran completamente ausentes en mamíferos podrían ser consideradas como potenciales blancos contra el Trypanosoma cruzi.

Trypanosoma cruzi is the etiological agent of Chagas disease, a disease endemic not only in Argentina but also in all of Latinamerica. T. cruzi presents several metabolic characteristics which are completely absent in its insect vectors and in mammalian hosts. Some of these differences were acquired after millions of years of adaptation to parasitism, during which this protozoan replaced many biosynthetic routes for transport systems. In the present review, we describe the advances in the knowledge of T. cruzi transport processes and the molecules involved. In particular, we focus on aminoacid and polyamine transporters from the AAAP family (Amino Acid/Auxin Permeases), because they seem to be exclusive transporters from trypanosomatids. Taking into account that these permeases are completely absent in mammals, they could be considered as a potential target against Trypanosoma cruzi.

Estudio cariométrico de placentas de ratones con infección aguda por diferentes cepas de Trypanosoma cruzi / Karyometric study of placentas from mice with acute infection by different strains of Trypanosoma cruzi

El objetivo de este trabajo fue evaluar cariometricamente las alteraciones causadas por diferentes cepas de T. cruzi en la placenta del ratón. Ratones hembras de 60 días, grávidas, fueron inoculadas, intraperitonealmente, con 2 x 10(5) tripomastigotes sanguíneos de las cepas colombiana, Y, Solivia o RC del T. cruzi. Fueron observadas claras diferencias en las alteraciones cariométricas de las células trofoblásticas gigantes y de las células trofoblásticas de la zona esponjosa. Los resultados demostraron que las cepas colombiana y RC causan alteraciones tanto en las células trofoblásticas gigantes como en las células del trofoblasto esponjoso, mientras que las cepas Y y Bolivia provocan alteraciones solamente en las células trofoblásticas gigantes. Es posible concluir que cada cepa posee características propias y que, a pesar del tipo similar de transmisión, presenta matices diferenciales en el proceso de la patogénesis placentaria.

The objective of this work was to evaluate karyometrically the alterations caused by different strains of Trypanosoma cruzi in the mouse placenta. Pregnant mice, 60-day old, were intraperitoneally inoculated with 2 x 10(5) bloodstream trypomastigotes of the Colombian, Y, Bolivia or RC strain of T cruzi. There were observed clear differences in the karyometric alterations of the trophoblast giant cells and in the spongiotrophoblast cells. The results demonstrate that the Colombian and RC strains cause alterations both in the trophoblast giant cells and in the spongiotrophoblast cells, whereas the Y and Bolivia strains provoke alterations only in the trophoblast giant cells. It is possible concluding that each strain has its own characteristics and that, in spite of the similar type of transmission, it show differential nuances in the placental pathogenic process.

Transferrin uptake may occur through detergent-resistant membrane domains at the cytopharynx of Trypanosoma cruzi epimastigote forms

Uptake of transferrin by epimastigote forms of the protozoan Trypanosoma cruzi occurs mainly through a cytostome/ cytopharynx, via uncoated endocytic vesicles that bud off from the bottom of the cytopharynx. We have here examined whether detergent-resistant membrane (DRM) domains might be involved in this process. Purified whole cell membrane fractions were assayed for cholesterol levels and used in dot blot analyses. Detergent-resistant membrane markers (cholera B toxin and anti-flotillin-1 antibody) presented positive reaction by dot blots in cholesterol-rich/ protein-poor membrane sub-fractions. The positive dot blot fraction was submitted to lipid composition analysis, showing composition similar to that of raft fractions described for other eukaryotic cells. Immunofluorescence assays allowed the localization of punctual positive signal for flotillin-1, matching the precise cytostome/ cytopharynx location. These data were confirmed by immunofluorescence assays with the co-localization of flotillin-1 and the transferrin uptake site. Our data suggest that DRM domains occur and are integrated at the cytostome/ cytopharynx of T. cruzi epimastigotes, being the main route for transferrin uptake.