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Nogueira N.P.,Federal University of Rio de Janeiro | Nogueira N.P.,Instituto Nacional Of Ciencia E Tecnologia Entomologia Molecular Inct Em | Saraiva F.M.S.,Federal University of Rio de Janeiro | Oliveira M.P.,University Federal Do Rio Of Janeirorj | And 9 more authors.
Free Radical Biology and Medicine | Year: 2017

Trypanosoma cruzi is the causative agent of Chagas disease and has a single mitochondrion, an organelle responsible for ATP production and the main site for the formation of reactive oxygen species (ROS). T. cruzi is an obligate intracellular parasite with a complex life cycle that alternates between vertebrate and invertebrate hosts, therefore the development of survival strategies and morphogenetic adaptations to deal with the various environments is mandatory. Over the years our group has been studying the vector-parasite interactions using heme as a physiological oxidant molecule that triggered epimastigote proliferation however, the source of ROS induced by heme remained unknown. In the present study we demonstrate the involvement of heme in the parasite mitochondrial metabolism, decreasing oxygen consumption leading to increased mitochondrial ROS and membrane potential. First, we incubated epimastigotes with carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), an uncoupler of oxidative phosphorylation, which led to decreased ROS formation and parasite proliferation, even in the presence of heme, correlating mitochondrial ROS and T. cruzi survival. This hypothesis was confirmed after the mitochondria-targeted antioxidant ((2-(2,2,6,6 Tetramethylpiperidin-1-oxyl-4-ylamino)−2-oxoethyl) triphenylphosphonium chloride (MitoTEMPO) decreased both heme-induced ROS and epimastigote proliferation. Furthermore, heme increased the percentage of tetramethylrhodamine methyl ester (TMRM) positive parasites tremendously-indicating the hyperpolarization and increase of potential of the mitochondrial membrane (ΔΨm). Assessing the mitochondrial functional metabolism, we observed that in comparison to untreated parasites, heme-treated epimastigotes decreased their oxygen consumption, and increased the complex II-III activity. These changes allowed the electron flow into the electron transport system, even though the complex IV (cytochrome c oxidase) activity decreased significantly, showing that heme-induced mitochondrial ROS appears to be a consequence of the enhanced mitochondrial physiological modulation. Finally, the parasites that were submitted to high concentrations of heme presented no alterations in the ultrastructure. Consequently, our results suggest that heme released by the insect vector after the blood meal, modify epimastigote mitochondrial physiology to increase ROS as a metabolic mechanism to maintain epimastigote survival and proliferation. © 2017

Cupello M.P.,State University of Rio de Janeiro | Souza C.F.D.,State University of Rio de Janeiro | Buchensky C.,National University of Rosario | Soares J.B.R.C.,Federal University of Rio de Janeiro | And 5 more authors.
Acta Tropica | Year: 2011

Heme (iron protoporphyrin IX) is an important molecule involved in many biological reactions, including oxygen transport, respiration, photosynthesis and drug detoxification. Trypanosoma cruzi parasites, the etiological agent of Chagas' disease, take up heme from the environment to supply their nutritional needs because they do not synthesize this cofactor. However, the mechanisms involved in heme transport across biological membranes are poorly understood. Indeed, in T. cruzi, no heme transporter has yet been characterized. In the present work, we evaluate the heme uptake processes by T. cruzi epimastigotes using fluorescent heme-analogues. Heme uptake decreased significantly when cells were pretreated with different concentrations of SnPPIX, PdMPIX or ZnMPIX, this observed competition suggests that they are taken up by the same transport system. We studied the growth behavior of epimastigotes using the same heme-analogues and the treatments with SnPPIX or PdMPIX impaired cell growth but when heme was added to the culture medium the observed inhibition was partially reversed. In addition, we tested how the heme uptake processes are affected by the presence of different transporter inhibitors. When the cells were treated with inhibitors and then incubated with heme, heme uptake decreased significantly for all treatments. These results constitute a strong indication for the existence of a protein associated with porphyrin transport in T. cruzi, possibly ATP-binding cassette transporters (ABC-transporter). © 2011 Elsevier B.V.

Cupello M.P.,State University of Rio de Janeiro | Souza C.F.,State University of Rio de Janeiro | Menna-Barreto R.F.,Laboratorio Of Biologia Celular | Nogueira N.P.A.,State University of Rio de Janeiro | And 6 more authors.
Biochemical and Biophysical Research Communications | Year: 2014

Trypanosoma cruzi, the causal agent of Chagas disease, has a complex life cycle and depends on hosts for its nutritional needs. Our group has investigated heme (Fe-protoporphyrin IX) internalization and the effects on parasite growth, following the fate of this porphyrin in the parasite. Here, we show that epimastigotes cultivated with heme yielded the compounds α-meso- hydroxyheme, verdoheme and biliverdin (as determined by HPLC), suggesting an active heme degradation pathway in this parasite. Furthermore, through immunoprecipitation and immunoblotting assays of epimastigote extracts, we observed recognition by an antibody against mammalian HO-1. We also detected the localization of the HO-1-like protein in the parasite using immunocytochemistry, with antibody staining primarily in the cytoplasm. Although HO has not been described in the parasite's genome, our results offer new insights into heme metabolism in T. cruzi, revealing potential future therapeutic targets. © 2014 Elsevier Inc. All rights reserved.

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