Original article
Heme modulates Trypanosoma cruzi bioenergetics inducing mitochondrial ROS production

https://doi.org/10.1016/j.freeradbiomed.2017.03.027Get rights and content

Highlights

  • Heme decreases oxygen consumption inhibiting cytochrome c oxidase activity.

  • Heme increases mitochondrial ROS and mitochondrial membrane potential in T. cruzi epimastigotes.

  • Mitochondrial ROS induced by heme favours epimastigote proliferation.

Abstract

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.

Introduction

Chagas disease, or American trypanosomiasis, is caused by Trypanosoma cruzi parasites [1]. In South and Central America, it is a major parasitic disease and worldwide it is the third major parasitic disease after malaria and schistosomiasis Currently, it is estimated that ten million people are infected worldwide, especially in Latin America where it is endemic [2], and the main cause of non-ischemic heart diseases, costing 1.2 billion dollars annually [3]. Hence, Chagas disease stands out as a public health problem not only in Latin America but also in other continents, mainly North America and Europe. This spreading is mainly due to immigration of infected individuals and the insect vector expansion over border areas [4]. This disease is among the 18 neglected tropical diseases in the world [2].

Trypanosoma cruzi is a hemoflagellate parasite that presents a complex life cycle, alternating between vertebrate and invertebrate hosts that involves proliferative (amastigote, epimastigote) and non-proliferative (metacyclic or bloodstream trypomastigotes) stages [5]. T. cruzi and other trypanosomatids have developed survival strategies and morphogenetic adaptations to cope with the pH, osmolarity, and nutritional changes in various host environments [6], [7].

Among the features unique to the parasite, unlike most eukaryotic cells, T. cruzi presents a single mitochondrion that display several peculiar features, such as the presence of a specific arrangement of mitochondrial DNA, named kinetoplast [8]. Mitochondria are organelles directly involved in cellular redox status, and play a central role in energy metabolism, representing the site of oxidative phosphorylation that drives ATP synthesis, nutrient oxidation, calcium homeostasis and control of apoptosis [9], [10], [11]. Mitochondria also represent the main sources of reactive oxygen species (ROS), which might have toxic effects when their levels are not controlled, but also contribute to a number of redox-dependent signalling cascades. Hydrogen peroxide (H2O2), which is the most studied mitochondrial ROS, acts as a signalling molecule in the cytosol, affecting multiple networks that control cell cycle, stress response, energy metabolism, and redox balance [12], [13]. The addition of exogenous H2O2 can cause the activation of the NF-κB transcription factor signalling [14]. Hydrogen peroxide has also been implicated in the activation of the ERK pathway by stimulants of the respiratory burst in macrophages, and the increase in tyrosine phosphorylation of several proteins [15]. Mitochondrial ROS production is regulated by the redox state of the electron transport system (ETS), and/or the magnitude of the mitochondrial membrane potential (ΔΨm) [10].

Heme (ferriprotoporphyrin-IX) is an iron-containing porphyrin that participates in many biological reactions, including electron transport, detoxification and oxygen transport [16], which are processes that are essentially mediated by heme proteins such as mitochondrial cytochromes, catalase, and hemoglobin. Besides its pro-oxidant effects, heme also participates in cell signalling processes including changes in the expression of genes related to antioxidant activities, energy metabolism and the cell cycle in Aedes aegypti [17]. Moreover, it is involved in the activation of a Toll-like receptor 4 and a calcium calmodulin kinase II-like, inducing the secretion of cytokines by macrophages [18], and the proliferation of T. cruzi epimastigotes, respectively [19]. However, unlike most organisms, trypanosomatids lack a complete route for heme synthesis, requiring its incorporation from the environment [20], [21].

It is known that during its life cycle T. cruzi is exposed to different redox environments inside the invertebrate and vertebrate hosts [22], [23] and the ability of T. cruzi to adapt to the redox state contributes to the success of the infection [24]. Additionally, in terms of a physiological approach, ROS play a vital role in T. cruzi – vector interactions, since heme, a molecule from the insect blood digestion, triggers epimastigote proliferation through a redox-sensitive signalling mechanism [19]. In this work, we demonstrate that the mitochondrial function in T. cruzi is affected by heme increasing ROS production without compromising the total ATP levels as the main mechanism used by the parasite insect stage to increase and maintain an oxidative environment favouring proliferation.

Section snippets

Parasites

All experiments were performed with epimastigotes forms of T. cruzi (Y strain) unless otherwise stated. Epimastigotes were maintained axenically at 28 °C for 7 days in liver infusion tryptose (LIT, BD Bacto, USA) and supplemented with 30 µM heme (Frontier Scientific, Utah, USA) and 10% (w/v) foetal calf serum (FCS, Vitrocell, Campinas, Brazil). The medium was changed weekly, and epimastigotes were harvested during the exponential growth phase (4–5 day old cultures). Parasite growth was monitored

The mitochondrion is a site of heme-induced ROS

Most of mitochondrial ROS is formed during respiration, in which oxygen can be partially reduced causing superoxide radicals (O2•-) that can either be spontaneously reduced to hydrogen peroxide (H2O2) or by the action of superoxide dismutase (SOD) [29]. In order to confirm that mitochondrion is the site of heme-induced oxidants, we evaluated ROS production on epimastigotes with the ROS-sensitive probe DHE by flow cytometry. Table 1 shows that heme increased parasite oxidant levels in about 94%

Discussion

Reactive oxygen species have historically known to cause damage in molecules inducing oxidative stress and pathological scenarios [35]. However, in the last decades, many evidences that ROS could act as intracellular signalling molecules have emerged, showing ROS controlling various physiological and pathological cell processes [36]. Because of that, redox signalling together with clinical studies in the redox biology field has changed perceptions of the role played by ROS. Notably,

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Author contributions

Conceived and designed the experiments: NPN, MCP, MFO. Performed the experiments: NPN, FMSS, MPO, JDFI, APMM, RFMB, GATL, EJLT. Analysed the data: NPN, MCP, MFO, FMSS, EEAA, RFMB, EJLT. Contributed reagents/materials/analytical tools: MCP, MFO, EEAA, RFMB, EJLT. Wrote the paper: NPAN, MCP, FMSS, EEAA, EJLT, RFMB, MFO.

Acknowledgments

We thank Fernando Almeida, for the excellent technical assistance on the confocal microscopy analyses realized with support of CENABIO-UFRJ. Supported by FAPERJ, CNPq and INCT-EM.

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