Trypanosoma cruzi tryparedoxin II interacts with different peroxiredoxins under physiological and oxidative stress conditions.

Trypanosoma cruzi, the etiologic agent of Chagas disease, has to cope with reactive oxygen and nitrogen species during its life cycle in order to ensure its survival and infection. The parasite detoxifies these species through a series of pathways centered on trypanothione that depend on glutathione or low molecular mass dithiol proteins such as tryparedoxins. These proteins transfer reducing equivalents to peroxidases, including mitochondrial and cytosolic peroxiredoxins, TcMPx and TcCPx, respectively. In T. cruzi two tryparedoxins have been identified, TXNI and TXNII with different intracellular locations. TXNI is a cytosolic protein while TXNII due to a C-terminal hydrophobic tail is anchored in the outer membrane of the mitochondrion, endoplasmic reticulum and glycosomes. TXNs have been suggested to be involved in a majority of biological processes ranging from redox mechanisms to protein translation. Herein, a comparison of the TXNII interactomes under physiological and oxidative stress conditions was examined. Under physiological conditions, apart from the proteins with unknown biological process annotation, the majority of the identified proteins are related to cell redox homeostasis and biosynthetic processes, while under oxidative stress conditions, are involved in stress response, cell redox homeostasis, arginine biosynthesis and microtubule based process. Interestingly, although TXNII interacts with both peroxiredoxins under physiological conditions, upon oxidative stress, TcMPx interaction prevails. The relevance of the interactions is discussed opening a new perspective of TXNII functions.

Trypanosoma cruzi mitochondrial tryparedoxin peroxidase is located throughout the cell and its pull down provides one step towards the understanding of its mechanism of action.

Trypanosoma cruzi depends on the effectiveness of redox metabolism to survive and ensure infection in the host. Homeostasis of redox metabolism in T. cruzi is achieved by the actions of several proteins that differ in many aspects from host proteins. Although extensive research has been performed examining hydroperoxide cytosolic antioxidant defense centered on trypanothione, the mechanisms of mitochondrial antioxidant defense are not yet known. The aim of this study was to elucidate the partners of TcMPx antioxidant pathway and to determine the influence of the cellular context (physiological versus oxidative stress). Through co-precipitation coupled with a mass spectrometry approach, a variety of proteins were detected under physiological and oxidative stress conditions. Interestingly, functional category analysis of the proteins identified under physiological conditions showed that they were involved in the stress response, oxidoreduction, thiol transfer, and metabolic processes; this profile is distinct under oxidative stress conditions likely due to structural alterations. Our findings help to elucidate the reactions involving TcMPx and most importantly also reveal that this protein is present throughout the cell and that its interaction partners change following oxidative stress exposure. The involvement and significance of the proteins found to interact with TcMPx and other possible functions for this protein are discussed widening our knowledge regarding T. cruzi mitochondrial antioxidant defenses.

Release of the cytosolic tryparedoxin peroxidase into the incubation medium and a different profile of cytosolic and mitochondrial peroxiredoxin expression in H2O2-treated Trypanosoma cruzi tissue culture-derived trypomastigotes.

Trypanosoma cruzi antioxidant enzymes are among the factors that guarantee parasite survival and maintain infection, enabling T. cruzi to cope with oxidative stress. Herein, the expression of cytosolic (TcCPx) and mitochondrial (TcMPx) tryparedoxin peroxidases was evaluated in tissue culture-derived trypomastigotes upon incubation with different concentrations of H(2)O(2). TcCPx expression slightly increased (5.4%) in cells submitted to 10 µM H(2)O(2) treatment when compared to the control, but decreased when higher H(2)O(2) concentrations (20-50 µM) were employed. Under these conditions, TcMPx expression increased (∼53%) with 10 µM-treatment compared to the control, followed by a reduction that reached ∼46% of the control when using the highest concentration tested. Interestingly, in the supernatant of the incubations, TcCPx, but not TcMPx, was detected, and its levels increased concomitantly with its decreased expression in the intracellular compartment. Our data show that peroxiredoxins in the tissue culture-derived trypomastigote can be modulated under oxidative stress and are present in higher amounts when compared to the epimastigote stage of T. cruzi. Additionally, due to the different expression patterns observed upon H(2)O(2)-treatment, each peroxiredoxin may play a distinct role in protecting the parasite under oxidative stress conditions.

The influence of low oxygen on macrophage response to Leishmania infection.

Hypoxia (low oxygen tension) is a common feature of inflamed and infected tissues. The influence of hypoxia on macrophage responses to micro-organisms has only recently been studied. This study demonstrates that hypoxia induced macrophages to control Leishmania amazonensis, an intracellular parasite that causes cutaneous and cutaneous metastatic lesions. The mechanisms that contribute to the control of macrophages against L. amazonensis infection under a hypoxic microenvironment are not known. Nitric oxide, TNF-α, IL-10 or IL-12 is not responsible for the decrease in parasitism under hypoxia. Live L. amazonensis entry or exocytosis of internalized particles as well as energetic metabolism was not impaired in infected macrophages; no apoptosis-like death was detected in intracellular parasites. Reactive oxygen species (ROS) is likely to be involved, because treatment with antioxidants N-acetylcysteine (NAC) and ebselen inhibits the leishmanicidal effect of macrophages under hypoxia. Leishmania amazonensis infection induces macrophages to express hypoxia-inducible factor-1 (HIF-1α) and -2 (HIF-2α). Data indicate that hypoxia affects the microbial activities and protein expression of macrophages leading to a different phenotype from that of the normoxic counterpart and that it plays a role in modulating Leishmania infection.

Mechanism of Trypanosoma cruzi death induced by Cratylia mollis seed lectin.

Incubation of T. cruzi epimastigotes with the lectin Cramoll 1,4 in Ca(2+) containing medium led to agglutination and inhibition of cell proliferation. The lectin (50 microg/ml) induced plasma membrane permeabilization followed by Ca(2+) influx and mitochondrial Ca(2+) accumulation, a result that resembles the classical effect of digitonin. Cramoll 1,4 stimulated (five-fold) mitochondrial reactive oxygen species (ROS) production, significantly decreased the electrical mitochondrial membrane potential (Delta Psi(m)) and impaired ADP phosphorylation. The rate of uncoupled respiration in epimastigotes was not affected by Cramoll 1,4 plus Ca(2+) treatment, but oligomycin-induced resting respiration was 65% higher in treated cells than in controls. Experiments using T. cruzi mitochondrial fractions showed that, in contrast to digitonin, the lectin significantly decreased Delta Psi(m) by a mechanism sensitive to EGTA. In agreement with the results showing plasma membrane permeabilization and impairment of oxidative phosphorylation by the lectin, fluorescence microscopy experiments using propidium iodide revealed that Cramoll 1,4 induced epimastigotes death by necrosis.

Distinct phosphatase activity profiles in two strains of Trypanosoma cruzi.

Phosphorylation of parasite proteins plays a key role in the process of cell invasion by Trypanosoma cruzi, the etiologic agent of Chagas' disease. In this sense, characterization of parasite kinases and phosphatases could open new possibilities for the rational design of chemotherapeutic agents for the treatment of Chagas' disease. In this work, we analyzed phosphatase activities in T. cruzi homogenates from 2 strains belonging to different lineages and with different resistance to oxidative stress. Tulahuen 2 cells (Lineage I) showed higher phosphatase activities and specificity constants when compared to the Y strain (Lineage II). Tulahuen 2 had an optimum phosphatase activity at pH 4.0 and the Y strain at pH 7.0. In both cases, neutral­basic, but not acid, phosphatase activities were increased in the presence of Mg2+. Although calcium had an inhibitory effect at a pH of 7.0 and 8.0 in the Y strain, this inhibition was restricted to pH 8.0 in the other strain. Different substrates and acid phosphotyrosine and alkaline phosphatase inhibitors exhibited distinct effects on the phosphatase activity of both strains. Our results provide a better understanding of T. cruzi phosphatases and reinforce the notion of heterogeneity among T. cruzi populations.

Trypanosoma cruzi strains, Tulahuen 2 and Y, besides the difference in resistance to oxidative stress, display differential glucose-6-phosphate and 6-phosphogluconate dehydrogenases activities.

The drugs currently available for Chagas'disease treatment are unsatisfactory due to limited efficacy and toxic side effects, making the search for more specific pharmacological agents a priority. The components of the Trypanosoma cruzi trypanothione-dependent antioxidant system have been pointed out as potential chemotherapeutic targets for the development of more specific drugs. To work properly, this system must have a current supply of NADPH, provided by glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD). Here, we compare two T. cruzi strains, Tulahuen 2 and Y, regarding growth rate, cytosolic tryparedoxin peroxidase (TcCPX) concentration and pentose phosphate pathway dehydrogenases activities. Tulahuen 2 cells show higher values as compared to the Y strain when the following parameters are compared: TcCPX concentration, resistance to H2O2, growth index and G6PD activity. Different patterns of G6PD and 6PGD activities were observed among strains along the growth curve and when cells were challenged with H2O2. These data reinforce the heterogeneity within T. cruzi populations and also the importance of G6PD in protecting the parasite against reactive oxygen species.

Characterization of the intracellular Ca(2+) pools involved in the calcium homeostasis in Herpetomonas sp. promastigotes.

Trypanosomatids of the genus Herpetomonas comprises monoxenic parasites of insects that present pro- and opisthomastigotes forms in their life cycles. In this study, we investigated the Ca(2+) transport and the mitochondrial bioenergetic of digitonin-permeabilized Herpetomonas sp. promastigotes. The response of promastigotes mitochondrial membrane potential to ADP, oligomycin, Ca(2+), and antimycin A indicates that these mitochondria behave similarly to vertebrate and Trypanosoma cruzi mitochondria regarding the properties of their electrochemical proton gradient. Ca(2+) transport by permeabilized cells appears to be performed mainly by the mitochondria. Unlike T. cruzi, it was not possible to observe Ca(2+) release from Herpetomonas sp. mitochondria, probably due to the simultaneous Ca(2+) uptake by the endoplasmic reticulum. In addition, a vanadate-sensitive Ca(2+) transport system, attributed to the endoplasmic reticulum, was also detected. Nigericin (1 microM), FCCP (1 microM), or bafilomycin A(1) (5 microM) had no effect on the vanadate-sensitive Ca(2+) transport. These data suggest the absence of a Ca(2+) transport mediated by a Ca(2+)/H(+) antiport. No evidence of a third Ca(2+) compartment with the characteristics of the acidocalcisomes described by A. E. Vercesi et al. (1994, Biochem. J. 304, 227-233) was observed. Thapsigargin and IP(3) were not able to affect the vanadate-sensitive Ca(2+) transport. Ruthenium red was able to inhibit the Ca(2+) uniport of mitochondria, inducing a slow mitochondrial Ca(2+) efflux, compatible with the presence of a Ca(2+)/H(+) antiport. Moreover, this efflux was not stimulated by the addition of NaCl, which suggests the absence of a Ca(2+)/Na(+) antiport in mitochondria.

Peroxynitrite affects Ca2+ transport in Trypanosoma cruzi.

Macrophages play an important role against Trypanosoma cruzi infection, via superoxide, nitric oxide, and peroxynitrite production. Peroxynitrite has been shown to be highly cytotoxic against Trypanosoma cruzi epimastigotes. Calcium is involved in many vital functions of the parasites, being its intracellular concentration governed by several transport systems, involving mitochondrial and non-mitochondrial compartments. In this paper, we report the effect of peroxynitrite on the calcium uptake systems, as studied by digitonin-permeabilized trypanosomes in the presence of arsenazo III. Peroxynitrite, at biologically relevant concentrations produced within phagosomes (250-750 microM), inhibited calcium uptake in a dose-dependent manner. Peroxynitrite decreased the mitochondrial membrane potential obtained in the presence of tetramethyl-p-phenylenediamine (TMPD)/ascorbate. In addition, a decrease of the non-mitochondrial Ca(2+)-uptake, concomitant with the inactivation of a Ca(2+)-dependent ATPase activity, was observed. HPLC analyses of the cellular adenine nucleotide pool showed a time-dependent decrease of ATP content and energy charge of the parasite; however this drop in ATP levels was significantly delayed with respect to decrease of the ATP-dependent Ca(2+)-transport. We conclude that the disruption of calcium homeostasis by peroxynitrite may contribute to the observed cytotoxic effects of macrophages against T. cruzi.

Ca2+-independent permeabilization of the inner mitochondrial membrane by peroxynitrite is mediated by membrane protein thiol cross-linking and lipid peroxidation.

Peroxynitrite anion, the reaction product of superoxide and nitric oxide, is a potent biological oxidant, which inactivates mammalian heart mitochondrial NADH-coenzyme Q reductase (complex I), succinate dehydrogenase (complex II), and ATPase, without affecting cytochrome c oxidase (complex IV). In this paper, we evaluated the effect of peroxynitrite on mitochondrial membrane integrity and permeability under low calcium concentration. Phosphate buffer was used in most of our experiments since Hepes, Tris, mannitol, and sucrose were found to inhibit the oxidative chemistry of peroxynitrite. Peroxynitrite (0.1-1.0 mM) caused a dose-dependent decrease in the ability of mitochondria to build up a membrane potential when N,N,N',N'-tetramethyl-p-phenylenediamine/ascorbate were used as substrate. Elimination of the membrane potential was accompanied by penetration of the osmotic support (KCl/NaCl) into the matrix as judged by the parallel occurrence of mitochondrial swelling. This swelling was partially inhibited by dithiothreitol (DTT) or butylated hydroxytoluene (BHT) and was insensitive to ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, ADP, and cyclosporin A. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized membrane proteins indicated that alterations in membrane permeability were associated with the production of protein aggregates due to membrane protein thiol cross-linking. The protective effect of DTT on both mitochondrial swelling and protein polymerization suggests the involvement of disulfide bonds in the membrane permeabilization process. In addition, the increase in thiobarbituric acid-reactive substances and the partial inhibitory effect of BHT indicate the occurrence of lipid peroxidation. These results support the idea that under our experimental conditions peroxynitrite causes mitochondrial structural and functional alterations by Ca2+-independent mechanisms through lipid peroxidation and protein sulfhydryl oxidation.

Disruption of Ca2+ homeostasis in Trypanosoma cruzi by crystal violet.

We have demonstrated previously that crystal violet induces a rapid, dose-related collapse of the inner mitochondrial membrane potential of Trypanosoma cruzi epimastigotes. In this work, we show that crystal violet-induced dissipation of the membrane potential was accompanied by an efflux of Ca2+ from the mitochondria. In addition, crystal violet inhibited the ATP-dependent, oligomycin-, and antimycin A-insensitive Ca2+ uptake by digitonin-permeabilized epimastigotes. Crystal violet also induced Ca2+ release from the mitochondria and endoplasmic reticulum of digitonin-permeabilized trypomastigotes. Furthermore, crystal violet inhibited Ca2+ uptake and the (Ca(2+)-Mg2+)-ATPase of a highly enriched plasma membrane fraction of epimastigotes, thus indicating an inhibition of other calcium transport mechanisms of the cells. Disruption of Ca2+ homeostasis by crystal violet may be a key process leading to trypanosome cell injury by this drug.

Evidence for free radical formation during horseradish peroxidase-catalyzed N-demethylation of crystal violet.

Crystal violet (gentian violet) can undergo an oxidative metabolism, catalyzed by horseradish peroxidase, resulting in formaldehyde formation. The N-demethylation reaction was strongly inhibited by reduced glutathione. Evidence for the formation of a crystal violet radical during the horseradish peroxidase catalyzed reaction was the detection of thiyl and ascorbate radicals from glutathione and ascorbate, respectively. The concentration of radicals from both compounds was significantly increased in the presence of crystal violet. Oxygen uptake was stimulated when glutathione was present in the system and this oxygen uptake was dependent on the dye and enzyme concentration. Oxygen uptake did not occur when ascorbate, instead of glutathione, was present in the system. However, when glutathione was present, ascorbate totally inhibited the glutathione-stimulated oxygen uptake in the crystal violet/horseradish peroxidase/hydrogen peroxide system. Although a weak ESR spectrum from a crystal violet-derived free radical was detected when the dye reacted with H2O2 and horseradish peroxidase, using the fast flow technique, this spectrum could not be interpreted.

A calmodulin-activated (Ca(2+)-Mg2+)-ATPase is involved in Ca2+ transport by plasma membrane vesicles from Trypanosoma cruzi.

High-affinity Ca(2+)-activated ATPases that do not show any demonstrable dependence on Mg2+ have been reported in the plasma membranes of different trypanosomatids, and it has been suggested [McLaughlin (1985) Mol. Biochem. Parasitol. 15, 189-201; Ghosh, Ray, Sarkar & Bhaduri (1990) J. Biol. Chem. 265, 11345-11351] that these enzymes may have a role in Ca2+ transport by the plasma membrane and in the regulation of intracellular Ca2+ in these parasites. In this report we investigated Ca2+ transport by Trypanosoma cruzi plasma membrane vesicles using Arsenazo III as a Ca2+ indicator. These vesicles accumulated Ca2+ upon addition of ATP only when Mg2+ was present and released it in response to the Ca2+ ionophore A23187, but were insensitive to inositol 1,4,5-trisphosphate. Ca2+ transport was insensitive to antimycin A, oligomycin and carbonyl cyanide p-trifluorophenylhydrazone, ruling out any mitochondrial contamination. Staurosporine and phorbol myristate acetate had no effect on this activity, while low concentrations of vanadate (10 microM) completely inhibited it. In addition, we describe a high-affinity vanadate-sensitive (Ca(2+)-Mg2+)-ATPase in the highly enriched plasma membrane fraction of T. cruzi. Kinetic studies indicated that the apparent Km for free Ca2+ was 0.3 microM. On the other hand, Ca(2+)-ATPase activity and Ca2+ transport were both stimulated by bovine brain calmodulin and by endogenous calmodulin purified from these cells. In addition, trifluoperazine and calmidazolium, at concentrations in the range in which they normally exert anti-calmodulin effects, inhibited the calmodulin-stimulated Ca(2+)-ATPase activity. These observations support the notion that a Mg(2+)-dependent plasma membrane Ca2+ pump is present in these parasites.

Digitonin permeabilization does not affect mitochondrial function and allows the determination of the mitochondrial membrane potential of Trypanosoma cruzi in situ.

Digitonin can be used to permeabilize selectively the plasma membrane of Trypanosoma cruzi epimastigotes without significantly affecting the functional integrity of mitochondria. Addition of digitonin at concentrations close to 64 microM caused decrease in the rate of basal respiration of epimastigotes similar to that caused by oligomycin. A further addition of carbonyl cyanide p-trifluorophenylhydrazone (FCCP) brought respiration to the same rate observed prior to the inclusion of digitonin or oligomycin. This suggests that like oligomycin, digitonin is shifting respiration to a nonphosphorylating state probably by depleting the cells from adenine nucleotides due to permeabilization of the plasma membrane. The use of low concentrations of digitonin allowed the quantitative determination of the mitochondrial membrane potential of these cells in situ using safranine O. The response of epimastigotes mitochondrial membrane potential to phosphate, FCCP, valinomycin, nigericin, ADP, and Ca2+ indicates that these mitochondria behave similarly to vertebrate mitochondria regarding the properties of their electrochemical proton gradient. In addition, T. cruzi mitochondria are able to build up and retain a membrane potential of a value comparable to that of mammalian mitochondria. The trypanocidal drug crystal violet, as well as other cationic drugs such as dequalinium, induced a rapid dose-related collapse of the inner mitochondrial membrane potential.

Ca2+ transport in digitonin-permeabilized trypanosomatids.

The use of digitonin to permeabilize Leishmania mexicana mexicana, Leishmania agamae, and Crithidia fasciculata plasma membranes enabled us to study Ca2+ transport in situ. The present results show that the mitochondria of these trypanosomatids are able to build up and retain a membrane potential as indicated by a tetraphenylphosphonium-sensitive electrode. Ca2+ uptake caused membrane depolarization compatible with the existence of an electrogenically mediated Ca2+ transport mechanism in these mitochondria. Ca2+ uptake was partially inhibited by ruthenium red, almost totally inhibited by carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and stimulated by inorganic phosphate. Large amounts of Ca2+ were retained by C. fasciculata mitochondria even after addition of thiols and NAD(P)H oxidants such as t-butylhydroperoxide and diamide. In contrast, Ca2+ was not retained in the matrix of Leishmania sp. mitochondria for long periods of time. In addition to the mitochondrial Ca2+ uptake, a vanadate-sensitive Ca2(+)-transporting system was also detectable in these trypanosomatids.

The mitochondrion of Trypanosoma cruzi is a target of crystal violet toxicity.

The first morphological alteration observed in Trypanosoma cruzi different stages upon incubation with crystal violet was mitochondrial swelling. The use of digitonin to solubilize T. cruzi plasma membrane allowed the demonstration of an uncoupling action of crystal violet on epimastigote mitochondria in situ. Low concentrations of crystal violet (20-50 microM) or carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP; 0.5 microM) uncoupled the respiratory control mechanism. The inhibition of State 3 respiration by oligomycin was released by crystal violet or FFCCP. Crystal violet released respiratory control, and enhanced ATPase activity of digitonin-permeabilized epimastigotes. Higher concentrations of crystal violet inhibited mitochondrial respiration. The uncoupled effect of crystal violet was stimulated by inorganic phosphate. In addition, crystal violet inhibited endongenous and glucose-stimulated respiration of the intact epimastigotes, and inhibited the Mg2+-ATPase in the epimastigote mitochondrial fractions. The inhibition of this Mg2+-ATPase increased up to pH 9.0 and decreased with increasing protein concentration. These data indicate that the T. cruzi mitochondrion is apparently the main target of crystal violet toxicity.

Prevention of Chagas' disease resulting from blood transfusion by treatment of blood: toxicity and mode of action of gentian violet.

Blood transfusion is the second most important mechanism of transmission of Chagas' disease. Gentian violet, a cationic dye, is currently used in blood banks in endemic areas in attempts to eliminate such transmission. A photodynamic action of gentian violet has been demonstrated in Trypanosoma cruzi. Visible light causes photoreduction of gentian violet to a carbon-centered radical. Under aerobic conditions this free radical autooxidizes generating superoxide anion whose dismutation yields hydrogen peroxide. This photodynamic action of gentian violet is thus probably mediated by the oxygen reduction products. Since irradiation with visible light in the presence of sodium ascorbate reduces the effective dose and time of contact of the dye with T. cruzi-infected blood, a possible application of these findings can be envisaged. In addition to this photodynamic action, an uncoupling effect of gentian violet on mitochondrial oxidative phosphorylation has been described in rat liver and T. cruzi mitochondria. Gentian violet released respiratory control, hindered ATP synthesis, enhanced ATPase activity, released the inhibition of State 3 respiration by oligomycin, and produced swelling of isolated rat liver mitochondria or T. cruzi mitochondria in situ. Taken together, these results indicate that the T. cruzi mitochondrion is the main target of gentian violet toxicity in the dark.

Crystal violet as an uncoupler of oxidative phosphorylation in rat liver mitochondria.

Crystal violet exhibited characteristics of an uncoupler of oxidative phosphorylation, i.e. it released respiratory control, hindered ATP synthesis, enhanced ATPase activity, and produced swelling of isolated rat liver mitochondria. Maximal stimulation of respiration, ATPase activity, and swelling was observed at a concentration of 40 microM. The inhibition of State 3 respiration by oligomycin was released by crystal violet. High concentrations of crystal violet inhibited mitochondrial respiration. The uncoupling effect of crystal violet required inorganic phosphate and was abolished by N-ethylmaleimide. The adenine nucleotides ADP and ATP protected mitochondria from uncoupling by the dye. The dye taken up by mitochondria was released into the incubation medium on induction of uncoupling. In the absence of phosphate, the dye did not cause uncoupling, but its retention was much greater than in the presence of phosphate. Crystal violet is suggested to induce uncoupling by acting on the membrane, rather than by its electrophoretic transfer into the mitochondria.