Redox Paradox: The Contribution of Reactive Oxygen Species (ROS) in the Proliferation and Differentiation of Trypanosoma cruzi (original) (raw)
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Redox Metabolism in Mitochondria of Trypanosomatids
Antioxidants & Redox Signaling, 2013
Significance: In the single mitochondrion of protozoan trypanosomatid parasites there are several sites for the generation and elimination of reactive oxygen species (ROS), a class of molecules that exhibit a dual role in cells, either as regulatory mediators or as cytotoxic effectors. Recent Advances: Formation of ROS in trypanosomatid mitochondria can be induced by various drug compounds. Importantly, it can also be triggered by specific physiologic stimuli, indicating that this phenomenon may occur in living parasites as well. Elimination of ROS in these organelles is attributed to the activity of two iron-dependent superoxide dismutases (FeSODs) and up to three different peroxidases (a cytochrome c peroxidase and two thiol peroxidases). Critical Issues: Data regarding the formation of ROS in trypanosomatid mitochondria are limited and nonsystematic. Another critical issue refers to the exact contribution of mitochondrial FeSODs and peroxidases for ROS removal, given that their antioxidant activity is not essential when abrogated individually. This suggests some level of functional overlapping or that ROS produced in mitochondria under normal conditions can be removed noncatalytically. Also still unsolved is the mechanism by which mitochondrial thiol peroxidases are regenerated to their reduced (active) form. Future Directions: The production of intramitochondrial ROS under physiologic conditions and their implication in parasite biology should be further clarified. The relative importance of enzymatic versus nonenzymatic mechanisms for ROS elimination in trypanosomatid mitochondria also requires investigation. Simultaneous depletion of several redundant antioxidant enzymes and determination of noncatalytic antioxidants are possible ways to achieve this. Antioxid. Redox Signal. 19, 696-707.
Mitochondrial Redox Metabolism in Trypanosomatids Is Independent of Tryparedoxin Activity
PLoS ONE, 2010
Tryparedoxins (TXNs) are oxidoreductases unique to trypanosomatids (including Leishmania and Trypanosoma parasites) that transfer reducing equivalents from trypanothione, the major thiol in these organisms, to sulfur-dependent peroxidases and other dithiol proteins. The existence of a TXN within the mitochondrion of trypanosomatids, capable of driving crucial redox pathways, is considered a requisite for normal parasite metabolism. Here this concept is shown not to apply to Leishmania. First, removal of the Leishmania infantum mitochondrial TXN (LiTXN2) by gene-targeting, had no significant effect on parasite survival, even in the context of an animal infection. Second, evidence is presented that no other TXN is capable of replacing LiTXN2. In fact, although a candidate substitute for LiTXN2 (LiTXN3) was found in the genome of L. infantum, this was shown in biochemical assays to be poorly reduced by trypanothione and to be unable to reduce sulfurcontaining peroxidases. Definitive conclusion that LiTXN3 cannot directly reduce proteins located within inner mitochondrial compartments was provided by analysis of its subcellular localization and membrane topology, which revealed that LiTXN3 is a tail-anchored (TA) mitochondrial outer membrane protein presenting, as characteristic of TA proteins, its N-terminal end (containing the redox-active domain) exposed to the cytosol. This manuscript further proposes the separation of trypanosomatid TXN sequences into two classes and this is supported by phylogenetic analysis: i) class I, encoding active TXNs, and ii) class II, coding for TA proteins unlikely to function as TXNs. Trypanosoma possess only two TXNs, one belonging to class I (which is cytosolic) and the other to class II. Thus, as demonstrated for Leishmania, the mitochondrial redox metabolism in Trypanosoma may also be independent of TXN activity. The major implication of these findings is that mitochondrial functions previously thought to depend on the provision of electrons by a TXN enzyme must proceed differently.
Heme modulates Trypanosoma cruzi bioenergetics inducing mitochondrial ROS production
Free Radical Biology and Medicine, 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.
Journal of Bioenergetics and Biomembranes, 2011
Trypanosoma cruzi is a hemoflagellate protozoan that causes Chagas' disease. The life cycle of T. cruzi is complex and involves different evolutive forms that have to encounter different environmental conditions provided by the host. Herein, we performed a functional assessment of mitochondrial metabolism in the following two distinct evolutive forms of T. cruzi: the insect stage epimastigote and the freshly isolated bloodstream trypomastigote. We observed that in comparison to epimastigotes, bloodstream trypomastigotes facilitate the entry of electrons into the electron transport chain by increasing complex II-III activity. Interestingly, cytochrome c oxidase (CCO) activity and the expression of CCO subunit IV were reduced in bloodstream forms, creating an "electron bottleneck" that favored an increase in electron leakage and H 2 O 2 formation. We propose that the oxidative preconditioning provided by this mechanism confers protection to bloodstream trypomastigotes against the host immune system. In this scenario, mitochondrial remodeling during the T. cruzi life cycle may represent a key metabolic adaptation for parasite survival in different hosts.
Biochemical Journal, 2007
Trypanosoma cruzi undergo PCD (programmed cell death) under appropriate stimuli, the mechanisms of which remain to be established. In the present study, we show that stimulation of PCD in T. cruzi epimastigotes by FHS (fresh human serum) results in rapid (< 1 h) externalization of phosphatidylserine and depletion of the low molecular mass thiols dihydrotrypanothione and glutathione. Concomitantly, enhanced generation of oxidants was established by EPR and immuno-spin trapping of radicals using DMPO (5,5-dimethylpyrroline-N-oxide) and augmentation of the glucose flux through the pentose phosphate pathway. In the early period (< 20 min), changes in mitochondrial membrane potential and inhibition of respiration, probably due to the impairment of ADP/ATP exchange with the cytosol, were observed, conditions that favour the generation of O 2 ᭹− . Accelerated rates of mitochondrial O 2 ᭹− production were detected by the inactivation of the re-dox-sensitive mitochondrial aconitase and by oxidation of a mitochondrial-targeted probe (MitoSOX). Importantly, parasites overexpressing mitochondrial FeSOD (iron superoxide dismutase) were more resistant to the PCD stimulus, unambiguously indicating the participation of mitochondrial O 2 ᭹− in the signalling process. In summary, FHS-induced PCD in T. cruzi involves mitochondrial dysfunction that causes enhanced O 2 ᭹− formation, which leads to cellular oxidative stress conditions that trigger the initiation of PCD cascades; moreover, overexpression of mitochondrial FeSOD, which is also observed during metacyclogenesis, resulted in cytoprotective effects.
Microbes and Infection, 2011
Because of its severe side effects and variable efficacy, the current treatment for Chagas disease is unsatisfactory. Natural compounds are good alternative chemotherapeutic agents for the treatment of this infection. Recently, our group reported the antiproliferative activity and morphological alterations in epimastigotes and intracellular amastigotes of Trypanosoma cruzi treated with eupomatenoid-5, a neolignan isolated from leaves of Piper regnellii var. pallescens. Here, we demonstrate that eupomatenoid-5 exhibited activity against trypomastigotes, the infective form of T. cruzi (EC 50 40.5 mM), leading to ultrastructural alteration and lipoperoxidation in the cell membrane. Additionally, eupomatenoid-5 induced depolarization of the mitochondrial membrane, lipoperoxidation and increased G6PD activity in epimastigotes of T. cruzi. These findings support the possibility that different mechanisms may be targeted, according to the form of the parasite, and that the plasma membrane and mitochondria are the structures that are most affected in trypomastigotes and epimastigotes, respectively. Thus, the trypanocidal action of eupomatenoid-5 may be associated with mitochondrial dysfunction and oxidative damage, which can trigger destructive effects on biological molecules of T. cruzi, leading to parasite death.
Journal of Bioenergetics and Biomembranes, 2009
In trypanosomatids the involvement of mitochondrial complex I in NADH oxidation has long been debated. Here, we took advantage of natural Trypanosoma cruzi mutants which present conspicuous deletions in ND4, ND5 and ND7 genes coding for complex I subunits to further investigate its functionality. Mitochondrial bioenergetics of wild type and complex I mutants showed no significant differences in oxygen consumption or respiratory control ratios in the presence of NADH-linked substrates or FADH 2 -generating succinate. No correlation could be established between mitochondrial membrane potentials and ND deletions. Since release of reactive oxygen species occurs at complex I, we measured mitochondrial H 2 O 2 formation induced by different substrates. Significant differences not associated to ND deletions were observed among the parasite isolates, demonstrating that these mutations are not important for the control of oxidant production. Our data support the notion that complex I has a limited function in T. cruzi.
BioMed research international, 2014
The pathogenic trypanosomatids Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp. are the causative agents of African trypanosomiasis, Chagas disease, and leishmaniasis, respectively. These diseases are considered to be neglected tropical illnesses that persist under conditions of poverty and are concentrated in impoverished populations in the developing world. Novel efficient and nontoxic drugs are urgently needed as substitutes for the currently limited chemotherapy. Trypanosomatids display a single mitochondrion with several peculiar features, such as the presence of different energetic and antioxidant enzymes and a specific arrangement of mitochondrial DNA (kinetoplast DNA). Due to mitochondrial differences between mammals and trypanosomatids, this organelle is an excellent candidate for drug intervention. Additionally, during trypanosomatids' life cycle, the shape and functional plasticity of their single mitochondrion undergo profound alterations, reflecting adapta...
PLOS ONE, 2015
Trypanosoma cruzi proliferate and differentiate inside different compartments of triatomines gut that is the first environment encountered by T. cruzi. Due to its complex life cycle, the parasite is constantly exposed to reactive oxygen species (ROS). We tested the influence of the pro-oxidant molecules H 2 O 2 and the superoxide generator, Paraquat, as well as, metabolism products of the vector, with distinct redox status, in the proliferation and metacyclogenesis. These molecules are heme, hemozoin and urate. We also tested the antioxidants NAC and GSH. Heme induced the proliferation of epimastigotes and impaired the metacyclogenesis. β-hematin, did not affect epimastigote proliferation but decreased parasite differentiation. Conversely, we show that urate, GSH and NAC dramatically impaired epimastigote proliferation and during metacyclogenesis, NAC and urate induced a significant increment of trypomastigotes and decreased the percentage of epimastigotes. We also quantified the parasite loads in the anterior and posterior midguts and in the rectum of the vector by qPCR. The treatment with the antioxidants increased the parasite loads in all midgut sections analyzed. In vivo, the group of vectors fed with reduced molecules showed an increment of trypomastigotes and decreased epimastigotes when analyzed by differential counting. Heme stimulated proliferation by increasing the cell number in the S and G2/M phases, whereas NAC arrested epimastigotes in G1 phase. NAC greatly increased the percentage of trypomastigotes. Taken together, these data show a shift in the triatomine gut microenvironment caused by the redox status may also influence T. cruzi biology inside the vector. In this scenario, oxidants act to turn on epimastigote proliferation while antioxidants PLOS ONE |
Free Radical Biology and Medicine, 2009
Trypanosoma cruzi is a hemoflagellate protozoan that causes Chagas' disease. The life cycle of T. cruzi is complex and involves different evolutive forms that have to encounter different environmental conditions provided by the host. Herein, we performed a functional assessment of mitochondrial metabolism in the following two distinct evolutive forms of T. cruzi: the insect stage epimastigote and the freshly isolated bloodstream trypomastigote. We observed that in comparison to epimastigotes, bloodstream trypomastigotes facilitate the entry of electrons into the electron transport chain by increasing complex II-III activity. Interestingly, cytochrome c oxidase (CCO) activity and the expression of CCO subunit IV were reduced in bloodstream forms, creating an "electron bottleneck" that favored an increase in electron leakage and H 2 O 2 formation. We propose that the oxidative preconditioning provided by this mechanism confers protection to bloodstream trypomastigotes against the host immune system. In this scenario, mitochondrial remodeling during the T. cruzi life cycle may represent a key metabolic adaptation for parasite survival in different hosts.