Mitochondrial Ca²⁺ and Reactive Oxygen Species in Trypanosomatids

Abstract

Significance:

Millions of people are infected with trypanosomatids and new therapeutic approaches are needed. Trypanosomatids possess one mitochondrion per cell and its study has led to discoveries of general biological interest. These mitochondria, as in their animal counterparts, generate reactive oxygen species (ROS) and have evolved enzymatic and nonenzymatic defenses against them. Mitochondrial calcium ion (Ca²⁺) overload leads to generation of ROS and its study could lead to relevant information on the biology of trypanosomatids and to novel drug targets.

Recent Advances:

Mitochondrial Ca²⁺ is normally involved in maintaining the bioenergetics of trypanosomes, but when Ca²⁺ overload occurs, it is associated with cell death. Trypanosomes lack key players in the mechanism of cell death described in mammalian cells, although mitochondrial Ca²⁺ overload results in collapse of their membrane potential, production of ROS, and cytochrome c release. They are also very resistant to mitochondrial permeability transition, and cell death after mitochondrial Ca²⁺ overload depends on generation of ROS.

Critical Issues:

In this review, we consider the mechanisms of mitochondrial oxidant generation and removal and the involvement of Ca²⁺ in trypanosome cell death.

Future Directions:

More studies are required to determine the reactions involved in generation of ROS by the mitochondria of trypanosomatids, their enzymatic and nonenzymatic defenses against ROS, and the occurrence and composition of a mitochondrial permeability transition pore. Antioxid. Redox Signal. 36, 969–983.

Introduction

Trypanosomatids include the genera, Trypanosoma and Leishmania, with species that cause several human diseases: Chagas disease (Trypanosoma cruzi), African trypanosomiasis (Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense), and leishmaniasis (several Leishmania species). These pathogens have intricate life cycles that include an insect and a mammalian host (Fig. 1).

FIG. 1.

Life cycle stages of Trypanosoma cruzi, Trypanosoma brucei, and Leishmania spp. in the vector and mammalian hosts. Color images are available online.

T. cruzi exists in two vector forms: epimastigotes and metacyclic trypomastigotes, while intracellular amastigotes and cell-derived trypomastigotes are present in the mammalian host. Metacyclic trypomastigotes are nonreplicative and are released by the vector and invade nucleated mammalian cells, where they differentiate into amastigotes. Amastigotes live in the cytosol of host cells and differentiate back into trypomastigotes. These cell-derived trypomastigotes are also nonreplicative and are released into the blood circulation to invade other cells or to be ingested by the vector (Fig. 1, T. cruzi). The T. brucei group of pathogens includes several stages, the procyclic trypomastigotes that reproduce in the insect vector, converting first into epimastigotes and then into metacyclic trypomastigotes, and the slender bloodstream trypomastigotes that replicate in the blood of the mammalian host and differentiate into stumpy forms that the vector can take up (Fig. 1, T. brucei). Leishmania spp. have three life cycle stages: the promastigotes that replicate in the vector; the metacyclic promastigotes that infect the animal host macrophages, and the amastigotes that replicate within a parasitophorous vacuole of phagocytic cells and can be released and infect other macrophages or be taken up by the vector (Fig. 1, Leishmania spp.).

All trypanosomatids possess only one mitochondrion per cell (Fig. 2), with peculiarities not found in the mammalian hosts, for example, the presence of their DNA in a network known as kinetoplast DNA (kDNA), comprising minicircles and maxicircles (31); a complex process of mitochondrial RNA editing (99); the need to import nuclear-encoded transfer RNAs (tRNAs) (134); and unique features of the mitochondrial ribosomes (36), to mention just a few. The capacity of these mitochondria to generate endogenous levels of reactive oxygen species (ROS) has not been studied with the same level of detail as that of the mammalian mitochondria, although most trypanosomes possess potential sites in their respiratory chains to generate superoxide anion (O₂^•−) and hydrogen peroxide (H₂O₂). The mitochondrial defenses against ROS are, however, better identified. Mitochondrial calcium ion (Ca²⁺) overload is an important mechanism involved in generation of ROS that could end up in cell death. We will review recent work on the role of the mitochondria of trypanosomes in generation of ROS, their defense mechanisms, and the relevance of Ca²⁺ in these pathways.

FIG. 2.

Single mitochondrion of trypanosomes. A procyclic trypomastigote is observed by DIC microscopy (left panel) or with the single mitochondrion stained with MT (in red, right panel). DAPI staining of the nucleus and kinetoplast (blue). Bars = 10 μm. DIC, differential interference contrast; MT, MitoTracker. Modified from the study by Huang et al. (77) with permission. Color images are available online.

Mitochondrial Generation of O₂^•− and H₂O₂

Mammalian cells have been proposed to possess 11 sites linked to substrate oxidation and oxidative phosphorylation that could transfer electrons to partially reduced molecular oxygen to produce O₂^•− or H₂O₂: oxoacid dehydrogenase complexes, which provide electrons to NAD⁺, respiratory complexes I and III, and dehydrogenases, including complex II, which uses ubiquinone as an acceptor (20). Most of these sites involve flavoprotein intermediates, which undergo one-electron reduction reactions, and it is therefore not unexpected that leak of electrons to oxygen could generate O₂^•−.

Trypanosomes possess several of these sites and lack others. Several 2-oxoacid dehydrogenase complexes, such as alpha-ketoglutarate dehydrogenase, pyruvate dehydrogenase, and branched-chain 2-oxoacid dehydrogenase, donate electrons to complex I through the formation of NADH (20). They have a common E3 subunit (dihydrolipoamide dehydrogenase) that transfers electrons from the dihydrolipoate of the E2 subunit to FAD and NAD⁺ . Production of O₂^•− and H₂O₂ is at the flavin in the matrix space (20), but this has not been tested in any trypanosomatid. Electrons from NADH enter the respiratory chain in the NADH-binding site, which was found in early work as a site of O₂^•− production (24). Attempts to detect the formation of ROS by complex I using dihydroethidium and flow cytometry detected some activity when the (NADH: ubiquinone oxidoreductase accessory subunit) of T. brucei procyclic forms was downregulated by RNA interference (RNAi) (153). However, this production was attributed to other metabolic processes because there were no alterations in cell growth, membrane potential, or NADH dehydrogenase activity, and it was concluded that complex I has no important function in energy metabolism of T. brucei procyclic forms (153). Similar results were found in T. cruzi epimastigotes (28).

T. brucei procyclic forms have a second NADH dehydrogenase that has flavin as a cofactor (60). The purified enzyme was able to generate O₂^•−, as detected by reduction of acetylated cytochrome c (59). Respiratory inhibitors were also used to produce ROS in isolated mitochondria (59). Mitochondrial membranes of T. brucei procyclic forms were also able to generate O₂^•− and H₂O₂ in the presence of NADH and their inhibition by fumarate suggested that fumarate reductase, which is able to produce succinate from fumarate using NADH, was the source of ROS (146). Similar results were obtained with T. cruzi epimastigotes (28).

In mammalian mitochondria, glycerol-3-phosphate dehydrogenase generates O₂^•− (20). This enzyme, which is FAD dependent, is a very important component of a shuttle between the glycosomes and mitochondria of the bloodstream forms of T. brucei and is important to maintain the glycosomal redox balance. However, there are no reports of its O₂^•− production activity in trypanosomes. Succinate dehydrogenase, or complex II, was able to generate O₂^•− in T. cruzi epimastigotes (135), but only in the presence of inhibitors. Finally, complex III or ubiquinol:cytochrome c oxidoreductase is the most important source of ROS in mammalian mitochondria (20), but mechanistic studies on generation of ROS by this complex in trypanosomes are lacking (143). Figure 3 shows a scheme of the potential or demonstrated O₂^•−-generating sites associated with the respiratory chain of trypanosomatids.

FIG. 3.

Potential sites of O₂^•− formation in the RC of trypanosomatids. Electrons that enter the RC at complex I, type II NADH-DH, and complex II, reduce UQ. G3PDH, which regenerates glycosomal G3P back to DHAP, also provides electrons to UQ. From UQ, electrons are transferred to complex III, cytochrome c (cyt c), and finally to complex IV, where they reduce O₂ to H₂O. Proton (H⁺) transfer through complexes III and IV generates an electrochemical gradient that is used to produce ATP from ADP and inorganic phosphate by complex V (ATP synthase). In Trypanosoma brucei bloodstream forms, the RC includes G3PDH and the TAO. Complexes II and III, UQ, and FR are potential sites for O₂^•− formation. DH, dehydrogenase; DHAP, dihydroxyacetone phosphate; FR, fumarate reductase; G3P, glycerol-3-phosphate; G3PDH, glycerol-3-phosphate dehydrogenase; O₂^•−, superoxide anion; RC, respiratory chain; TAO, trypanosome alternative oxidase; UQ, ubiquinone. Modified from the study by Tomás and Castro (143). Color images are available online.

Defenses Against ROS in Trypanosomatids

Trypanosomatids live in very different environments and their protection against the toxicity of ROS depends on those environmental conditions (91). The vector forms have stages exposed to low oxygen levels, such as the intestine of the vectors, and these forms were found lacking enzymes required for decomposition of H₂O₂ (19, 39, 40, 42, 45–47). Trypanosomatids lack genes for thioredoxin reductase, selenocysteine-dependent glutathione peroxidase (GSHPx), catalase, and glutathione reductase (79, 89). However, some defense mechanisms are present and we will describe them in more detail.

Superoxide Dismutases

Superoxide dismutases (SODs) catalyze the dismutation of O₂^•− to H₂O₂ and O₂. T. brucei possesses four iron-dependent SODs: in the cytosol, SODB1; glycosomes, SODB2; and mitochondria, SODA and SODC (56, 157), where they are involved in dismutation of locally generated O₂^•−. Although some of the potential sources of O₂^•− in the mitochondria of trypanosomatids are known (Fig. 3), the cytosolic and glycosomal sources have not been identified. T. cruzi recombinant SODs have been characterized (81, 100, 141). These enzymes can be inactivated by peroxynitrite and one of them, TcSODA, has been crystallized (98). It has been proposed that SOD inhibitors, such as the iron chelator N1,N6-bis(2,3-dihydroxybenzoyl)-1,6-diaminohexane (bis-C6-DHB), could be trypanocidal agents (102). This proposal assumes that O₂^•− is the toxic species. Another possibility is that H₂O₂, the product of the SOD reaction, and the hydroxyl radical that can be produced by the Fenton reaction could be more damaging than O₂^•−. In this case, SOD inhibition would be protective if H₂O₂ is not degraded. In this regard, a hydroxyphthalazine SOD inhibitor was reported to decrease the parasitemia levels of T. cruzi in infected mice (109). The susceptibility of T. cruzi to benznidazole and crystal violet, but not to nifurtimox, increases after overexpression of cytosolic/glycosomal SOD (SODB1) (141). The reason for these effects is unknown.

As indicated above, the mitochondria of trypanosomatids possess two iron-dependent SODs (SODA and SODC) (56, 157), but they are not individually essential in T. brucei bloodstream forms, as examined by RNAi, although downregulation of SODA led to higher sensitivity to the ROS generator, paraquat (157). Overexpression of SODA also made Leishmania chagasi more resistant to paraquat (116). The mitochondrial SODA from Leishmania amazonensis has been suggested to initiate ROS-mediated signaling required for differentiation and protection of parasites against oxidative stress (103).

The Trypanothione System and Peroxidases

While intracellular thiol homeostasis in mammals is maintained by glutathione/glutathione reductases and thioredoxin/thioredoxin reductases, in trypanosomatids, thiol homeostasis is maintained by the trypanothione [T(SH)₂]/trypanothione disulfide [T(S)₂] reductase couple (79).

Glutathione and spermidine combine to synthesize T(SH)₂ [N¹,N⁸-bis(glutathionyl)spermidine] (Fig. 4). In a first step, an ATP-using reaction combines glutathione (GSH) and spermidine to form monoglutathionylspermidine (glutathionyl spermidine [GSH-SPD]). A second molecule of GSH is then added to generate T(SH)₂ with the consumption of another ATP. Both reactions are catalyzed by trypanothione synthetase (TryS) (34, 112, 113) (Fig. 4). Oxidation of T(SH)₂ results in T(S)₂. T(S)₂ is reduced back to T(SH)₂ by a trypanothione disulfide reductase (TryR) (62, 86) in an NADPH-dependent reaction (Fig. 5).

FIG. 4.

T(SH)₂ synthesis occurs by condensation of spermidine with glutathione to form N¹-glutathionyl spermidine. Addition of a second glutathione leads to the formation of T(SH)₂. Both reactions consume ATP and are catalyzed by T(SH)₂ synthase. T(SH)₂, trypanothione.

FIG. 5.

Redox metabolism in trypanosomatids. Reduction of T(S)₂ to T(SH)₂ is catalyzed by TryR with conversion of NADPH into NADP⁺. Oxidation of T(SH)₂ to T(S)₂ is coupled to the reduction of TryX(S)₂, oxidized glutathione (GSSG), and dhA or oxidized Cc. These compounds are generated by the action of peroxidases catalyzing the decomposition of H₂O₂ (APx/CcPx, GSHPxs I and III, and TryXPx) and/or hydroperoxides (ROOH; GSHPxs I and II, and TryXPx, also called Prx). APx, ascorbate peroxidase; Cc, cytochrome c; CcPx, cytochrome c peroxidase; dhA, dehydroascorbate; GSHPx, glutathione peroxidase; GSSG, glutathione disulfide; H₂O₂, hydrogen peroxide; Prx, peroxiredoxin; TryR, trypanothione disulfide reductase; TryX, tryparedoxin; TryXPx, tryparedoxin peroxidase; TryX(S)₂, tryparedoxin disulfide; T(S)₂, trypanothione disulfide. Color images are available online.

TryR has homology to glutathione disulfide (GSSG) reductase and is specific for T(S)₂. The enzyme was crystallized alone or bound to substrates or inhibitors (14, 82, 90, 92, 163). Several inhibitors have been tested, such as polyamine derivatives (15, 94, 107, 108), nitrofurans and naphthoquinones (2, 86), dibenzazepines (67), crystal violet (104), phenothiazines and related tricyclics (13, 32, 71, 87), bisbenzylisoquinoline alkaloids (63), acridines (18), diphenylsulfide derivatives (9), Mannich bases (93), natural products (35), and ajoene (64), as well as some terpyridine–platinum complexes (17). These compounds inhibit the activity of the enzyme and parasite growth in vitro or in vivo. The enzyme is cytosolic, but there is some evidence of its presence in the mitochondria and glycosomes (79).

T(SH)₂ oxidation leads to reduction of intermediates (dehydroascorbate or oxidized cytochrome c, GSSG, or tryparedoxin disulfide); several peroxidases (ascorbate/cytochrome c peroxidase, cysteine-dependent GSHPx, and tryparedoxin peroxidase (TryXPx), respectively) use these intermediates to degrade H₂O₂ or organic hydroperoxides (Fig. 5).

The ascorbate peroxidase (APx) activity was first found in extracts of T. cruzi epimastigotes (41), preceding its discovery in plants (69). The enzyme belongs to Class I of the peroxidase–catalase superfamily, contains heme, and uses ascorbate or reduced cytochrome c (Cc) as the substrate to degrade H₂O₂, but is not active on organic hydroperoxides (19, 41, 139, 156) (Fig. 5). This APx belongs to the hybrid-type group A of APx-CcPx (cytochrome c peroxidase).

The T. cruzi APx colocalizes with BiP, an endoplasmic reticulum marker (156). Others have localized it to glycosomes (41), where ascorbate synthesis takes place (95). More recent work, using electron microscopy, has found the T. cruzi enzyme in the mitochondria and outer surface of amastigotes and trypomastigotes (78). The Leishmania major APx was found in the inner mitochondrial membrane (52). The recombinant enzyme from T. cruzi was studied in detail (78, 158). There is no correlation between its expression and metacyclogenesis or virulence (123), and the enzyme is not essential for viability in the mammalian host or to maintain the chronic infection (139). However, TcAPx null mutants are less infective in vitro and are more sensitive to exogenous H₂O₂ (139). T. cruzi strains resistant to benznidazole have higher expression of TcAPx (106), and as the L. major enzyme (1, 84, 161), TcAPx has cytochrome c peroxidase activity (78). The L. major APx was crystallized (83). Overexpression of this mitochondrially located enzyme protects L. major from oxidative stress (53). The enzyme is not present in T. brucei, Trypanosoma congolense, and Trypanosoma vivax. Other characteristics of the trypanosomatid peroxidases have also been reviewed (30, 121).

Cysteine-dependent GSHPxs, with some exceptions, are not able to decompose H₂O₂ (19, 23). In contrast to the mammalian homologs, these enzymes have cysteine instead of selenocysteine in their active site. GSHPx I from T. cruzi is localized in the glycosomes and cytosol and can use reduced tryparedoxin [TryX(SH)₂] or GSH as a substrate (155, 159). The crystal structure of this enzyme has been obtained (117). GSHPx II from T. cruzi is present in the endoplasmic reticulum and can use only GSH (158). The T. brucei GSHPxs I and II localize predominantly in the cytosol (57). GSHPx III localizes to the mitochondria of T. brucei and shows strong preference for reduced tryparedoxin or thioredoxin (74). The peroxidases from T. cruzi can degrade organic hydroperoxides, but not H₂O₂ (155, 158). The GSHPxs I and III from T. brucei, in contrast, decompose H₂O₂ (74, 133).

TryXPxs, also known as peroxiredoxins, are in the family of 2-cysteine peroxiredoxins and use tryparedoxin to decompose H₂O₂ (70, 96, 154). Two isoforms exist, one mitochondrial and the other cytosolic (159). They can degrade peroxynitrite (120, 124, 142, 144). The TryXPx crystal structure has been determined (126). These peroxiredoxins play an important role in the defense mechanism against macrophage-generated nitrosative and oxidative stresses (4). The peroxidase activity of these enzymes depends on T(SH)₂ (Fig. 5) (123). The mitochondria of T. brucei also have a TryXPx (or peroxiredoxin). Interestingly, the mitochondrial TryXPx of Leishmania infantum (140) and the cytosolic TryXPx of T. cruzi (125) also function as chaperones. Recent work using a tryparedoxin-coupled biosensor revealed that T. brucei harbors a mitochondrial T(SH)₂/T(S)₂ system (57). However, synthesis of T(SH)₂ and reduction of T(S)₂ occur in the cytosol (112, 137), and it was suggested that trypanosomes might possess an exchanger of T(S)₂ and T(SH)₂ and an oxidoreductase to transfer reducing equivalents from T(SH)₂ onto the mitochondrial peroxidases (57) (Fig. 6).

FIG. 6.

H₂O₂ metabolism in mitochondria of trypanosomes. RC-generated O₂^•− is dismutated to H₂O₂ via SODs A and C. H₂O₂ is metabolized by APx/CcPx (absent in Trypanosoma brucei), GSHPx III, and TryXPx (or peroxiredoxin), with concomitant oxidation of ascorbate or cytochrome c (A/Ccr), GSH, and tryparedoxin [TryX(SH)₂], respectively. These compounds are reduced by T(SH)₂. There is probably a [T(S)₂)/(T(SH)₂] exchanger, and T(SH)₂ is regenerated by cytosolic T(SH)₂ reductase using NADPH. Ccr, reduced cytochrome c; GSH, glutathione; SOD, superoxide dismutase. Color images are available online.

Transformation of epimastigotes of T. cruzi into metacyclic trypomastigotes is accompanied by increased expression of several antioxidant enzymes, such as APx, TryXPx, tryparedoxin, TryS, and iron SOD (8). It was proposed that the change indicates preparation of the infective forms for potential damage generated by the respiratory burst of mammalian phagocytic cells (8).

In conclusion, past (143) and current evidence indicates that mitochondria of trypanosomatids have enzymatic defenses against ROS, such as SOD (SODA and SODC), APxCcpx (Leishmania spp.), GSHPx III (T. brucei), and TryXPx (T. cruzi, T. brucei, and Leishmania spp.) (Fig. 6).

Other Thiols

T. cruzi possesses T(SH)₂ analogs derived from spermine or other polyamines such as homotrypanothione, N¹,N¹²-bis(glutathionyl)spermine, N¹-glutathionyl-N⁸-acetylspermidine, and N¹-glutathionyl-N¹²-acetylspermine (7, 111). These compounds are the result of condensation of GSH with cadaverin, spermine, N-acetyl spermine, and N¹- and N⁸-acetylspermine catalyzed by TryS (7), but their importance has not been elucidated. Ovothiol (N¹-methyl-4-mercaptohistidine) (6) also occurs in different life cycle stages of T. cruzi and Leishmania spp. (138), but its relevance is not known. The ovothiol biosynthetic enzyme, 5-histidylcysteine sulfoxide synthase, was characterized in T. cruzi (21).

Mitochondrial Ca²⁺ Transport

The mitochondrial Ca²⁺ uniporter (MCU) was discovered 60 years ago as a result of studies with rat kidney mitochondria (38, 147). When MCU was found to be absent in Saccharomyces cerevisiae mitochondria (25), it was thought that this transporter was not present in nonanimal species (26, 101). However, an MCU was described in T. cruzi (48, 49). T. cruzi mitochondrial Ca²⁺ transport was found to have the same properties of the animal MCU: electrogenicity, low affinity for Ca²⁺, high capacity, and inhibition by ruthenium red (48, 49).

The MCU was later found in several Leishmania spp. (12, 149, 151) and T. brucei (150, 152, 160). Interestingly, it was found that mitochondria of the bloodstream form of T. brucei were also able to transport Ca²⁺, although they lack a conventional respiratory chain and oxidative phosphorylation (150). These mitochondria use the ATP synthase, working in reverse, as an ATPase to generate the electrochemical gradient needed to transport Ca²⁺, and this transport is inhibited by oligomycin (150).

The lack of an MCU in yeast (25) and its presence in trypanosomes (48) and mammals (90) led to the comparison of the mitochondrial proteomes of yeast, trypanosomes, and humans to identify mitochondrial proteins present in trypanosomes and humans and absent in yeast, resulting in first, the identification of the modulator of MCU in mammals, the mitochondrial calcium uptake 1 (MICU1) (118), and later, of the MCU pore subunit (11, 37, 44).

Several components, in addition to the pore-forming subunit MCU, form part of the MCU complex (11, 37, 51), including the MCU paralog, MCUb, which is a dominant negative subunit (130); the mitochondrial calcium uniporter regulator 1 (MCUR1), which is a scaffolding subunit (97); the essential mitochondrial regulator (EMRE) (132); and MICU1 (118), 2 (MICU2), and 3 (MICU3) (128) (Fig. 7A).

FIG. 7.

MCU complex organization. (A) In animals, the MCU complex is constituted by the pore-forming subunit MCU and regulator proteins MCUb, MICU1, MICU2, EMRE, and MCUR1. (B) The MCU complex in trypanosomes comprises the pore-forming subunits MCU, MCUb, MCUc, and MCUd, probably forming a hetero-hexamer, and Ca²⁺-sensing subunits MICU1 and MICU2. Black balls, Ca²⁺ ions; dark orange circular sectors: EF hand domains, S letters enclosed in the circle are cysteine residues that can form a disulfide bridge. Ca²⁺, calcium ion; EMRE, essential mitochondrial regulator; IMS, inter membrane space; MCU, mitochondrial calcium uniporter; MCUR1, mitochondrial calcium uniporter regulator 1; MICU, mitochondrial calcium uptake. Reprinted from the study by Docampo et al. (51) with permission from Elsevier. Color images are available online.

The trypanosome MCU complex has a different composition (Fig. 7B) and characteristics that differ from those of the mammalian complex. (i) The MCU subunit is essential for T. brucei growth and infectivity in vivo (77), while MCU knockout mice survive with only alterations in skeletal muscle function under strenuous exercise (115). (ii) There are paralogs of the trypanosome MCU, named MCUb, MCUc, and MCUd, which are necessary for mitochondrial Ca²⁺ transport. MCUc and MCUd are only present in trypanosomatids (75). These two subunits, together with MCU and MCUb, form hetero-hexameric complexes in membranes (75). In contrast, the recombinant MCUs described in fungi (10, 58, 105, 162) and zebra fish (10) form homotetramers, while the recombinant protein of Caenorhabditis elegans forms homopentamers (110). These differences were considered as evidence of parallel evolution of the uniporter in animals and trypanosomes (127). (iii) While in vertebrate cells, a fundamental function of mitochondrial Ca²⁺ transport is regulation of oxidative phosphorylation (27), T. brucei bloodstream forms are the only cells that have a functional MCU complex, but do not have a conventional respiratory chain and oxidative phosphorylation, and its function in these stages warrants further exploration (150). (iv) The trypanosome MCU interacts with the ATP synthase subunit c (76). This interaction could have potential implications for the understanding of mitochondrial physiology. The biological significance of this interaction is evidenced by the formation of a megacomplex by the ATP synthase, the ADP/ATP translocator (adenine nucleotide translocase [ANT]), the inorganic phosphate carrier (PiC), and the MCU complex. The ATP synthase uses ADP, transported by the ADP/ATP translocator, and P_i, transported by the PiC, to synthesize ATP, and this activity is stimulated by Ca²⁺ transported by the MCU complex. (v) The inositol 1,4,5-trisphosphate receptor (IP₃R) of trypanosomes localizes to the acidocalcisome (33, 77), the most important Ca²⁺ store in trypanosomes (50). This IP₃R is essential in the bloodstream forms of T. brucei (77). There are also membrane contact sites between acidocalcisomes and mitochondria (131). Ca²⁺ release from acidocalcisomes mediated by the IP₃R can be taken up by the mitochondria to stimulate mitochondrial bioenergetics of T. cruzi (33).

Mitochondrial Ca²⁺ release in mammalian cells is by either an H⁺- or Na⁺-coupled antiporter. The molecular identity of the Na⁺-coupled antiporter was recently discovered (114), but trypanosomes lack homolog genes. However, a Ca²⁺/H⁺ antiporter was proposed to be present in T. cruzi mitochondria based on their response to addition of Ca²⁺ and EGTA (49). A protein named Letm1 was proposed to act as a Ca²⁺/H⁺ antiporter in the mitochondria of mammalian cells (85, 145). Studies in T. brucei suggested that TbLetm1 is involved in maintaining mitochondrial volume via K⁺/H⁺ exchange across the inner membrane (72). However, recent work has shown that T. cruzi Letm1 is important for Ca²⁺ uptake and release (54). Both mitochondrial Ca²⁺ influx and efflux are reduced in TcLetm1 knockdown trypanosomes. These changes lead to Ca²⁺ overload and increased autophagy, as well as reduced invasion of host cells and intracellular replication (54).

Mitochondrial Ca²⁺ Overload, ROS Generation, and Cell Death in Trypanosomes

Early work on T. cruzi (42, 43) described that treatment of different life cycle stages of the parasite with naphthoquinones that generate ROS resulted in cell death and morphological changes that are now considered typical of mammalian apoptosis, such as mitochondrial disruption, chromatin condensation, and plasma membrane blebbing. Later reports in the mid 1990s suggested that T. cruzi epimastigotes can undergo apoptosis under starvation or after complement exposure (5), and this led to subsequent studies centered on apoptosis in parasitic protozoa (136). It has been indicated that robust experimental evidence demonstrating regulated or programmed cell death in parasitic protozoa is still lacking (129). However, some characteristic features of apoptosis such as release of cytochrome c from the mitochondria, DNA fragmentation, and exposure of phosphatidylserine in the plasma membrane have been observed in T. cruzi subjected to different stresses (55, 80, 88, 119, 122). Their relevance as markers of apoptosis in protozoa has been extensively discussed (129).

Trypanosomatids lack homologs to proteins involved in apoptosis in animal cells, such as the TNF-related family of receptors, Bcl-2 family members, and caspases (136). However, metacaspases and cathepsin-like proteases (88), as well as nucleases such as endonuclease G (Endo G) (66) and TaD (65), have been shown to play roles in some of the apoptosis-like phenotypic changes that accompany cell death in different trypanosomatids. What appears to be clear is that there is a mitochondrial pathway of cell death in trypanosomatids. For example, the interplay between mitochondrial Ca²⁺ overload, ROS generation, and mitochondrial dysfunction upon complement-dependent cell death has been described in T. cruzi (80). A similar interplay between Ca²⁺ overload, ROS generation, and mitochondrial alteration upon overexpression of MCU in T. brucei has been reported (77). In contrast to mitochondrial Ca²⁺ overload, decreased mitochondrial Ca²⁺ uptake leads to increase in the AMP/ATP ratio and autophagy, suggesting that a delicate bioenergetic balance is required to keep the cells alive (77) (Fig. 8).

FIG. 8.

Consequences of decreased and increased mitochondrial Ca²⁺ uptake. Reduced Ca²⁺ uptake leads to decreased ATP synthesis, resulting in a higher AMP/ATP ratio and autophagy, while increased Ca²⁺ uptake leads to mitochondrial Ca²⁺ overload, ROS generation, and cell death. Background shows a procyclic form of Trypanosoma brucei. ROS, reactive oxygen species. Color images are available online.

Opening of the Mitochondrial Permeability Transition Pore and Cell Death in Trypanosomes

The mitochondrial permeability transition pore (mPTP) is an alternative mechanism to the mitochondrial (intrinsic) pathway of apoptosis and a mediator of cell death mechanisms in mammals (29). The mPTP is a nonselective channel of high conductance located at contact sites between the inner and outer mitochondrial membranes. Several proteins have been shown to be components of this channel, including voltage-dependent anion channels (VDAC), ANT, PiC, cyclophilin D (CypD), and other proteins such as dimers of the ATP synthase (68). Opening of the mPTP is increased by Ca²⁺ overload, thiol oxidation, pyridine nucleotide oxidation, oxidative stress, alkalinization, or low transmembrane potential. Cyclosporin A, which binds to CypD, inhibits its opening. Opening of this pore determines mitochondrial dysfunction and cell death by either apoptosis or necrosis (61). The intramitochondrial Ca²⁺ plays an important role in control of the redox balance that affects the opening of the PTP (148).

It has been suggested that an mPTP may be formed in T. cruzi based on the loss of mitochondrial membrane potential and cell viability when a CypD homolog (TcPyD22) is overexpressed and H₂O₂ is present. This mechanism is sensitive to cyclosporin A (22, 23).

The c-ring of the ATP synthase (3, 16) or a channel inside ATP synthase dimers has been suggested as forming the pore (16, 68) of the mPTP. However, ATP synthases devoid of c subunits maintain mPTP formation characteristics (73), suggesting that the c-ring is not the channel. The interaction between the MCU and the c subunit of the ATP synthase described above suggests a role for the MCU complex in formation of the mPTP, which needs to be investigated.

Conclusions

The mitochondria of trypanosomatids have mechanisms for ROS generation and defenses against their toxicity, and Ca²⁺ overload has a role in ROS generation and cell death. However, there are still several gaps in our knowledge of these mechanisms and roles. The mitochondrial sites for ROS generation have not been characterized in detail, and while defenses against ROS production depend on the T(SH)₂ system, it is not known whether the T(SH)₂/T(S)₂ couple has access to the mitochondrial matrix or they exchange with their cytosolic pools. It is not yet clear whether opening of an mPTP occurs in these mitochondria, and which is its composition.

Abbreviations Used

ANT

adenine nucleotide translocase

APx

ascorbate peroxidase

Ca²⁺

calcium ion

CcPx

cytochrome c peroxidase

Ccr

reduced cytochrome c

CypD

cyclophilin D

DH

dehydrogenase

dhA

dehydroascorbate

DHAP

dihydroxyacetone phosphate

DIC

differential interference contrast

EMRE

essential mitochondrial regulator

FR

fumarate reductase

G3P

glycerol-3-phosphate

G3PDH

glycerol-3-phosphate dehydrogenase

GSH

glutathione

GSHPx

glutathione peroxidase

GSSG

glutathione disulfide

H₂O₂

hydrogen peroxide

IMS

intermembrane space;

IP₃R

inositol 1,4,5-trisphosphate receptor

MCU

mitochondrial calcium uniporter

MCUR1

mitochondrial calcium uniporter regulator 1

MICU

mitochondrial calcium uptake

mPTP

mitochondrial permeability transition pore

MT

MitoTracker

O₂^•−

superoxide anion

PiC

inorganic phosphate carrier

Prx

peroxiredoxin

RC

respiratory chain

RNAi

RNA interference

ROS

reactive oxygen species

SOD

superoxide dismutase

TryR

trypanothione disulfide reductase

TryS

trypanothione synthetase

TryX

tryparedoxin

TryXPx

tryparedoxin peroxidase

TryX(S)₂

tryparedoxin disulfide

T(S)₂

trypanothione disulfide

T(SH)₂

trypanothione

UQ

ubiquinone

Authors' Contributions

R.D. was involved in conceptualization, writing—draft, and writing—review and editing; and A.E.V. was involved in conceptualization, writing—draft, and writing—review and editing.

Author Disclosure Statement

The authors declare no conflicts of interest.

Funding Information

Work in the authors' laboratories is funded by the U.S. National Institutes of Health (grant AI108222 to R.D.) and the Brazilian State Agency Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP; grant 2017/17728-8 to A.E.V.).