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Molecular & Biochemical Parasitology 152 (2007) 192–202

Mitochondrial superoxide mediates heat-induced apoptotic-likedeath in Leishmania infantum

Juan F. Alzate a,b,c, Andres A. Arias a, David Moreno-Mateos a,Alberto Alvarez-Barrientos d, Antonio Jimenez-Ruiz a,∗

a Departamento de Bioquımica y Biologıa Molecular, Campus Universitario, Universidad de Alcala, 28871 Alcala de Henares, Madrid, Spainb Escuela de Bacteriologıa, Universidad de Antioquia, Medellın, Colombia

c Programa de Estudio y control de enfermedades Tropicales, PECET, Universidad de Antioquia, Medellın, Colombiad Fundacion Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain

Received 6 September 2006; received in revised form 22 December 2006; accepted 4 January 2007Available online 9 January 2007

Abstract

Previous studies have shown that heat stress triggers a process of programmed cell death in Leishmania infantum promastigotes that resemblesapoptosis in higher eukaryotes. Even though this cell death process takes about 40 h to be completed, several early changes in the heat-stressed cellscan be observed. Hyperpolarization of the parasite mitochondrion is the earliest event detected, which correlates with an increase in respiration ratesand a concomitant increase in superoxide radical production. Induction of oxidative stress seems to mediate the heat-induced cell death process,as indicated by the partial prevention of parasite death observed when cell cultures are supplemented with N-acetyl-cysteine or glutathione. Theseantioxidants are able to diminish the concentration of superoxide radical but they do not prevent mitochondrial hyperpolarization. Treatment ofthe heat stressed parasites with the inhibitors of the mitochondrial respiration TTFA, antimycin A and KCN significantly decreases the productionof superoxide radicals, which confirms the mitochondrial origin of this reactive oxygen species.© 2007 Elsevier B.V. All rights reserved.

Keywords: Cellular respiration; Mitochondria; Oxidative stress; Superoxide radical; Heat shock; Programmed cell death

1. Introduction

During mitochondrial respiration, several reactive oxygenspecies (ROS), mainly superoxide anion (O2

•−), are formed asby-products, [1]. Mitochondrial respiratory chain complexes Iand III constitute the main source of superoxide in mammals,which is then converted to hydrogen peroxide (H2O2) by spon-taneous dismutation or by superoxide dismutase (SOD) [2,3].Low concentrations of ROS are then usually present in the cellsand seem to be implicated in physiological processes. How-ever, overproduction of these toxic oxygen intermediates may

Abbreviations: CMFDA, chloro-methyl-fluorescein-diacetate; DHE, dihy-droethidium; GSH, glutathione; H2DCFDA, 2′,7′-dichlorodihydrofluoresceindiacetate; PI, propidium iodide; NAC, N-acetyl cysteine; TTFA, thenoyltriflu-oroacetone; SOD, superoxide dismutase; ROS, reactive oxygen species; PCD,programmed cell death

∗ Corresponding author. Tel.: +34 91 885 5109; fax: +34 91 885 4585.E-mail address: antonio.jimenez@uah.es (A. Jimenez-Ruiz).

induce damage to proteins, lipids and DNA, which eventuallyleads to programmed cell death (PCD) or necrosis [4–7]. Try-panosomatids, the causative agents of African sleeping sickness(Trypanosoma brucei gambiense and T.B. rhodesiense), Naganacattle disease (T. congolense, T.b. brucei), South-American Cha-gas’ disease (T. cruzi) and the different forms of Leishmaniasis,lack a typical rotenone-sensitive complex I in their electrontransfer chain but, despite this significant difference, respirationin these parasites also leads to the production of ROS [8].

In higher eukaryotes, enzymes such as superoxide dismu-tase [9], catalase [10] and the selenium-containing enzymeglutathione peroxidase [11] constitute an effective systemto eliminate these by-products generated during oxidativephosphorylation. Superoxide dismutase is responsible for thedismutation of O2

•− into H2O2 and O2, and catalase and glu-tathione peroxidase are responsible for the removal of H2O2 byconverting it into water and molecular oxygen. The latter twoenzymes are not found in Trypanosomatids, instead, they con-tain three different classes of peroxidases that use trypanothione

0166-6851/$ – see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.molbiopara.2007.01.006

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as the electron donor [12]. Even though the activity of theseperoxidases is lower than that observed in glutathione peroxi-dase, their high concentration (5% of the total soluble protein insome cases [13,14]) allows an efficient control of the oxidativestress associated to the activity of the parasites. On the otherhand, as described in higher eukaryotes, superoxide anion elim-ination in these parasites is based on the activity of SOD, forwhich two different subtypes have been described (SODA andSODB). Overexpression of these SOD in L. chagasi parasitesconferred enhanced protection against the free radical generat-ing agents paraquat and nitroprusside [15], whereas, parasitescontaining a single allele knock out of one of the SODB genesexhibited an important reduction in growth in the presence ofparaquat and a significant decrease in survival within humanmacrophages [16]. Similar results have been described for L.tropica [17]. Because, ROS generation is one of the microbici-dal strategies of the macrophage [18], it was not surprising thatresistance to oxidative stress correlated with the ability of theparasite to infect and proliferate within cell culture macrophages[15,19].

Recent results from our laboratory indicate that a mild heatshock (38 ◦C) induces a process of PCD in Leishmania infan-tum promastigotes similar to apoptosis from higher eukaryotes[20]. This effect may be relevant during infection because inthis process the parasites are transferred from a temperatureof 25–27 ◦C to 37–38 ◦C (depending on the vertebrate host).It has been postulated that the apoptotic-like death of some par-asites during infection, because of its similarity with apoptosisof the host’s cells, could be involved in restricting the immuneresponse of the host [20]. In an attempt to define the cascade ofprocesses that lead to heat-induced cell death, we searched forearly changes in the parasites during heat exposure. Among thevery first alterations, we have found a fast increase in oxygenconsumption by the parasites and an early hyperpolarization oftheir single mitochondrion. The results presented in this paperindicate that the early increase in mitochondrial activity is asso-ciated to a rapid increase in O2

•− intracellular concentrationand that this increase is directly involved in the process of celldeath.

2. Materials and methods

2.1. Chemical and reagents

Dihydroethidium (DHE), chloro-methyl-fluorescein-diace-tate (CMFDA) and 2′,7′-dichlorodihydrofluorescein diacetate(H2DCFDA), were obtained from INVITROGEN MolecularProbes (Leiden, The Netherlands). Other reagents of the highestquality were purchased from Sigma (St. Louis, MO) or Merck(Darmstadt, Germany).

2.2. Cells and culture conditions and heat shock stress

L. infantum promastigotes (M/CAN/ES/96/BCN150 MON-1), kindly provided by Dr. Alonso (CBMSO-UniversidadAutonoma Madrid, Spain), were grown in RPMI-1640 medium(Gibco, Paisley, UK) supplemented with 10% heat-inactivated

foetal calf serum (FCS), antibiotics, and 2 mM glutamine at26 ◦C. Transfected strains were selected at 20 �g/mL Genetecin(PROMEGA CAT# 11811-031) in RPMI/10%FCS and thenmaintained at 100 �g/mL. In the assays with the transfectedstrains, the parasites were maintained 1 week without genetecinto avoid interference of this antibiotic with the death process.The absence of any effect on the expression level of the trans-genes was confirmed. Heat treatment of the promastigotes wasperformed during logarithmic growth phase at a concentration of1.5 × 106 parasites/mL at 38 ◦C. Late stationary promastigoteswere obtained after incubation of the parasites for more that 10days with a starting inoculum of 1 × 106 parasites/mL. In theseconditions, saturation of the culture was achieved after 4 days.

2.3. Mitochondrial transmembrane potential (�Ψm)

Parasites in RPMI medium/10%FCS at 1.5 × 106 parasites/mL were incubated with 50 nM tetramethylrhodamin methylesther perchlorate (TMRM, Sigma CAT# T5428) 15 min priorto their analysis in the cytometer. TMRM loading was carriedout at 26 ◦C in control parasites and at 38 ◦C in heat-stressedparasites and they were kept at these temperatures until theiranalysis in the cytometer. TMRM concentration was adjusted tomeasure either hyperpolarization or depolarization of the para-site mitochondrion without the need of probe removal. Possiblechanges in the solubility or fluorescence of the dye due to vari-ations in temperature were discarded by using the uncouplercarbonyl cyanide p-trifluoromethoxyphenylhydrazone (100 �MFCCP) at both temperatures.

2.4. Measure of respiration rates

The overall oxygen consumption by the parasites was mea-sured using the oxygraph (Hansatech-Instruments). Briefly,logarithmic promastigotes were centrifuged and resuspended inPBS 10 mM glucose at a final concentration of 10 × 106/mL.Parasite suspension of 1 mL was transferred to the oxygraphchamber and the oxygen consumption rate was measured dur-ing at least 5 min for every treatment. Respiration rates of controlparasites cultured at 26 ◦C were measured. To analyze the effectof HS, the temperature of these parasites was raised from 26 ◦Cto 38 ◦C with a constant flow of water at 38 ◦C over the exteriorcompartment of the measuring chamber. As a control, oxy-gen consumption was completely inhibited by the addition ofpotassium cyanide (KCN) 3 mM at both temperatures (26 ◦C or38 ◦C).

2.5. ROS production detection and antioxidants treatment

Before each experiment the parasites were preloaded 45 minprior to the analysis with dihydroethidium (DHE) or 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) at 1 �M andfluorescence emission was measured at the indicated times usinga FACScalibur flow cytometer (Beckton-Dickinson, BD). Glu-tathione (GSH) and N-acetyl-cysteine (NAC) were dissolved insterile water and added to 1 mM final concentration 1 h prior tothe heat challenge.

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The analysis of the production of superoxide under oxidativephosphorylation uncoupling conditions was carried out with theionophores FCCP and CCCP: parasites were loaded with theDHE probe as described before, 35 min later the ionophoresFCCP or CCCP were added at the concentrations indicated infigures; then the DHE fluorescence was measured with the flowcytometer after 60 min of incubation at 26 ◦C or 38 ◦C.

For the induction of oxidative stress with menadione (dis-solved 50 mM in ethanol), this compound was supplemented ata concentration of 10 �M to the parasite culture and incubatedfor the time indicated on each experiment.

2.6. Intracellular thiol levels measures

Intracellular thiol levels were measured with the thiol reactiveprobe chloro-methyl-fluorescein-diacetate (CMFDA) [21,22].After 1 h of treatment in the different conditions, aliquots of500 �L of the treated parasites (26 ◦C, 26 ◦C + Menadione or38 ◦C) were supplemented with 100 nM of the probe CMFDA,incubated for 30 min at 26 ◦C and protected from light. Then, theparasites were centrifuged at 800 × g for 5 min, the medium withthe probe was removed and the parasites were resuspended in500 �L of PBS. Subsequently, the parasites were re-centrifuged,the PBS was removed and the parasites were resuspended in300 �L of PBS and then immediately analyzed with the flowcytometer.

2.7. DNA content analysis by flow cytometry

One and a half million parasites were centrifuged at 1000 × gfor 5 min, the pellet was resuspended in 100 �L of PBS, 600 �Lof −20 ◦C cold 70% ethanol were added and then parasiteswere incubated on ice for 30 min. After incubation, the para-sites were washed with 1 mL of PBS-EDTA 50 mM, pelletedat 1000 × g and then resuspended in 400 �L of PBS/50 mMEDTA/50 �g/mL RNase and incubated for 30 min at 37 ◦C.Then, propidium iodide (PI) was added to a final concentra-tion of 5 �g/mL and the parasites were immediately analyzedfor PI fluorescence.

2.8. Measure of parasite death after HS challenge

DNA content of the parasites and plasma membrane per-meability to propidium iodide (PI) were analyzed as describedpreviously [20].

2.9. Inhibition of cellular respiration

Exponentially growing parasites were pre-incubated withKCN 10 �M, antimycin A 10 �M or thenoyltrifluoroacetone(TTFA) 500 �M during 30 min before the heat treatment.

2.10. Flow cytometry data acquisition and analysis

Parasites treated with the procedures described above wereanalyzed in a FACScalibur flow cytometer (BD, san Jose, CA)equipped with a 488 nm Ar-laser. All the analyses and graphicalrepresentations were carried out with the package CELLQUESTPRO (BD) version 4.

2.11. Fluorescence microscopy

Logarithmically growing parasites were labelled with 1 �MDHE in RPMI as described above, then incubated at 38 ◦C or26 ◦C during 1 h and labelled with 10 �g/mL Hoechst 33342.One drop of each cell culture was deposited on a glass slide,covered with a coverslip and immediately photographed in afluorescent microscope (NIKON Diaphot 300). Excitation wasdone with an UV-filtered Hg lamp.

2.12. Determination of SOD activity

Either logarithmic or late stationary parasites were lysedin 100 �L of ice cold lysis buffer (20 mM HEPES, pH 7.2,1 mM EGTA, 210 mM manitol, 70 mM sucrose) at a concentra-tion of 160 × 106 parasites/mL. After sonication, lysates werecentrifuged at 1500 × g for 5 min at 4 ◦C. SOD activity wasdetermined in the supernatants as indicated by the kit manufac-turer (superoxide dismutase assay kit; Cayman Chemical).

Fig. 1. Analysis of mitochondrial membrane potential (�Ψm) and oxygen consumption in heat-treated parasites. (A) Relative fluorescence due to TMRM of L.infantum promastigotes. Parasites grown at 26 ◦C were exposed to 38 ◦C for 90 min. As a control, parasites were treated with the uncoupler FCCP (100 �M) bothat 26 ◦C and 38 ◦C during the same period of time. Fluorescence was analyzed by flow cytometry (n = 3). (B) Oxygen consumption of L. infantum promastigotesincubated at 26 ◦C and 38 ◦C. Respiration of the parasites was abolished at both temperatures by addition of KCN at 3 mM (26 ◦C + KCN and 38 ◦C + KCN) (n = 5).The asterisks indicate a significant difference between mean values of fluorescence (A) or oxygen consumption (B) of the parasites incubated at 38 ◦C and at 26 ◦C.p-Values are indicated under each panel. Error bars represent standard deviation.

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2.13. Statistics

All the analyses were carried out with a minimum of threeindependent experiments. Statistical significance of the dif-ferences between treatments were evaluated by a two-tailedunpaired t-test.

3. Results

3.1. Heat shock induces a rapid mitochondrialhyperpolarization and increases O2 consumption rates

The highly sensitive probe TMRM was used to measurechanges in the mitochondrial transmembrane potential (�Ψm)of logarithmically growing L. infantum promastigotes exposedto 38 ◦C. Previous studies in our group have already shown theability of this probe to specifically detect changes in this poten-tial in Leishmania parasites [20]. The results presented in Fig. 1Aindicate a rapid increase in �Ψm when the parasites are incu-bated at 38 ◦C, as shown by the significant and sustained increasein TMRM-derived fluorescence observed after 30 min of heatexposure. The uncoupler FCCP (100 �M) was added as a con-trol both at 26 ◦C and 38 ◦C. The similar pattern of fluorescencein the presence of the uncoupler at both temperatures discardsany possible effect of the temperature in the fluorescence of theTMRM-labelled parasites.

To assess whether the early mitochondrial hyperpolarizationcould be due to an increase in mitochondrial activity, oxygenconsumption of the promastigotes was measured under basaland heat-stressing conditions (26 ◦C and 38 ◦C, respectively).As shown in Fig. 1B, promastigotes double their oxygen con-sumption rates early after transferring to 38 ◦C, which suggestthat hyperpolarization could be the consequence of an increasedflow of electrons across the respiratory chain when the para-sites are exposed to 38 ◦C. As expected, oxygen consumption atboth 26 ◦C and 38 ◦C is almost abolished when the parasites areincubated with the respiratory chain complex IV inhibitor KCN.

3.2. HS induces oxidative stress in Leishmaniapromastigotes

As indicated before, it has been described that the electrontransport that occurs during mitochondrial oxidative phospho-rylation generates a significant amount of ROS. Accordingly,it would not be surprising that the described increase in mito-chondrial respiration rates and mitochondrial hyperpolarizationwere associated to higher rates of ROS production and, conse-quently, to higher concentration of this species into the cell. Theprobes dihydroethidium (DHE) [23] and dihydrodichlorofluo-rescein (H2DCFDA) [24] were used to detect changes in theintracellular concentration of O2

•− and peroxides, respectively.Both of them were used at 1 �M concentration and the parasiteswere exposed to them 45 min prior to any change in temper-ature conditions. In both cases, the probe is modified by thepresence of its specific ROS and transforms in a fluorescentmolecule that accumulates inside the cell. Accordingly, a pro-gressive increase in fluorescence is expected with time, even in

control conditions, due to the physiological production of ROSby the mitochondrion. As shown in Fig. 2A, an increase in DHE-derived fluorescence is observed immediately after incubationof the parasites at 38 ◦C when compared with control parasitesat 26 ◦C. Differences in the intensity of fluorescence are statisti-cally significant after 20 min of HS treatment and continue to beso even after 16 h of heat treatment, when the presence of deadparasites is already evident (data not shown). Pre-incubation of

Fig. 2. (A) Analysis of O2•− intracellular concentration in heat-treated par-

asites. L. infantum parasites were incubated at the temperatures indicated infigure (26 ◦C and 38 ◦C). Parasites incubated at 38 ◦C were also supplementedwith GSH 1 mM (38 ◦C + GSH) or NAC 1 mM (38 ◦C + NAC) 1 h before heattreatment. DHE-derived fluorescence was evaluated by flow cytometry at thetime points indicated in figure. Error bars represent standard deviation. Aster-isks indicate a significant difference between mean values of fluorescence ofthe parasites incubated at 38 ◦C and at 26 ◦C. *p < 0.05; **p < 0.001; n = 3. (B)Analysis of intracellular peroxide concentration in heat-treated parasites. L.infantum parasites were incubated at the temperatures indicated in figure (26 ◦Cand 38 ◦C). H2DCFDA-derived fluorescence was evaluated by flow cytometryat the time points indicated in figure. Error bars represent standard deviation(n = 3). (C) Analysis of mitochondrial membrane potential (�Ψm) in the pres-ence of the antioxidants GSH and NAC. L. infantum parasites were incubated atthe temperatures indicated in the figure (26 ◦C and 38 ◦C). Parasites incubatedat 38 ◦C were also supplemented with GSH 1 mM (38 ◦C + GSH) or NAC 1 mM(38 ◦C + NAC) 1 h before heat treatment. Relative fluorescence due to TMRMwas evaluated by flow cytometry at 30 and 120 min of treatment. Error bars repre-sent standard deviation. Asterisks indicate a significant difference between meanvalues of fluorescence of the parasites incubated at 38 ◦C (38 ◦C; 38 ◦C + GSH;38 ◦C + NAC) and at 26 ◦C. *p < 0.05; n = 3.

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Fig. 3. Intracellular thiol levels in heat-treated parasites. (A) L. infantum promastigotes were incubated at 26 ◦C either in the presence or in the absence of 10 �Mmenadione. The oxidation rate of the DHE was measured by flow cytometry during the first 90 min of treatment. (B) Cellular levels of thiols were estimated withthe probe CMFDA. The fluorescence corresponding to this probe was measured in parasites incubated for 1 h in the following conditons: 26 ◦C; 26 ◦C with 10 �Mmenadione (26 ◦C + menadione) and 38 ◦C. Error bars represent standard deviation. Asterisks indicate a significant difference between mean values of fluorescence(n = 3).

heat-treated parasites with the antioxidants Glutathione (GSH)or N-acetyl-cysteine (NAC) restores the fluorescent signal tothe basal levels observed in control (26 ◦C) parasites (Fig. 2A).Unexpectedly, H2DCFDA-derived fluorescence does not showany increase in heat-stressed parasites (Fig. 2B), which indicatesthat peroxide concentration is not affected during the heat shock.

Since treatment of the parasites with the antioxidants GSHand NAC restores O2

•− concentration of heat-stressed parasitesto basal levels, mitochondrial transmembrane potential of theheat-treated parasites in the presence of GSH and NAC wasevaluated, in an attempt to determine whether mitochondrialhyperpolarization could be a consequence of O2

•− accumu-lation. The results presented in Fig. 2C demonstrate that themitochondrial hyperpolarization associated to HS is maintainedin the presence of the antioxidants, which indicates that theobserved alterations in the mitochondria are not a consequenceof ROS production.

Increased cellular levels of ROS can lead the cell to amore oxidized state termed oxidative stress, characterized bythe oxidation of several intracellular molecules and/or groups,such as the intracellular thiol groups. In order to determinewhether the increased production of superoxide radical underHS induces oxidative stress in the promastigotes, we treated theparasites with chloro-methyl-fluorescein-diacetate (CMFDA), acell-permeant thiol-reactive probe [21,22]. This molecule reactswith thiol groups, labelling the cell with a green fluorescencethat is proportional to the thiol levels of the cell, allowing us tocompare the thiol levels at basal conditions (26 ◦C) or after heatstress (38 ◦C). Menadione, a well-known inducer of oxidativestress, was used as a positive control. The results presented inFig. 3A demonstrate that menadione induces an accelerated oxi-dation of the probe DHE when compared to the control parasites,as an indication of its ability to increase the intracellular con-centration of O2

•−. As expected, treatment of the parasites withmenadione for 1 h induced a significant reduction in the fluores-cent signal associated to CMFDA (Fig. 3B), as an indication ofthe decrease in the concentration of thiol groups. A significantreduction of the fluorescence was also observed in the parasitesafter incubation at 38 ◦C for 1 h, which demonstrates a reductionin the thiol levels in the heat-treated cells that is indicative ofoxidative stress (Fig. 3B).

Fig. 4. Analysis of O2•− intracellular concentration in heat-treated para-

sites exposed to respiratory chain inhibitors. L. infantum promastigotes werepreloaded with Dihydroethidium 1 �M (DHE) as described in materials andmethods, exposed at the temperatures indicated in figure and treated with therespiratory chain inhibitors: (A) TTFA (Complex II); (B) antimycin A (ComplexIII) and (C) KCN (Complex IV). Fluorescence was evaluated by flow cytometryat the time points shown in each figure. Error bars represent standard deviation.Asterisks indicate a significant difference between mean values of fluorescenceof the parasites incubated at 38 ◦C in the absence or in the presence of respiratorychain inhibitors. The p-value is indicated in each panel (n = 3).

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Fig. 5. Superoxide intracellular distribution in heat-treated parasites. Parasites were preloaded with DHE 1 �M, then exposed for 1 h to 26 ◦C or 38 ◦C, stainedwith Hoechst33342 and observed at the microscope at a 1000× magnification. (A) Phase-contrast image of the parasites incubated at both temperatures. (B) Thesame parasites were observed with a 330–380 nm excitation filter and a 435 nm barrier filter for Hoechst33342 staining. (C) The same parasites observed with a450–490 nm excitation filter and a 520 nm barrier filter for 2-hydroxyethidium staining.

3.3. Inhibitors of cellular respiration diminish the O2•−

production

In order to determine the participation of the mitochondrialrespiratory complexes in the HS-induced O2

•− production inLeishmania promastigotes, specific inhibitors of the electrontransfer chain were added to the parasite cell cultures beforethe HS challenge, and O2

•− production was then analyzed. Asshown in Fig. 4A, a significant reduction in O2

•− productioncan be observed when the promastigotes are incubated with theinhibitor of the complex II TTFA. Similarly, treatment of theparasites either with the inhibitor of complex III, antimycin A(Fig. 4B), or with the inhibitor of complex IV, cyanide (Fig. 4C),significantly decreases the steady state concentration of O2

•−under HS conditions. Rotenone, the usual inhibitor of complexI, was not tested because of the differences already described inthe complex I of the respiratory chain in Leishmania parasites[8]. The results obtained with the inhibitors of complex II, III andIV indicate that a partial blockage of the electron transport sig-nificantly diminishes the rate of heat-induced O2

•− production.It must be pointed out that the concentrations of these inhibitorshad to be adjusted to diminish but not abolish mitochondrial O2consumption because a strong inhibition of respiration duringthe heat treatment caused a massive death of the parasites aftera few minutes. The significant inhibition of O2

•− productionobserved when the parasites are treated with respiratory chaininhibitors confirms the major role of mitochondrial respirationon the production of this radical under HS conditions.

Mitochondrial localization of the O2•− production was

further confirmed by fluorescence microscopic analysis of heat-treated parasites stained with DHE and Hoechst 33342. A clear

blue signal due to Hoechst labelling of DNA (Fig. 5B; 26 ◦C and38 ◦C) can be observed both at 26 ◦C and 38 ◦C in the nuclei andthe kinetoplast, the two DNA-containing structures present inthe parasites. The kinetoplast is a structure composed by a cate-nated network of DNA maxicircles and minicircles located intothe mitochondrion, close to the base of the flagellum that consti-

Fig. 6. Effect of mitochondrial uncouplers on the O2•− production. Promastig-

otes were preloaded with 1 �M DHE and then supplemented with the uncouplingagents FCCP or CCCP at the concentrations indicated in figure. After 60 minof incubation at 26 ◦C or 38 ◦C, the DHE oxidation was measured by flowcytometry. Control parasites without uncouplers were also analyzed at 26 ◦Cand 38 ◦C. Bars represent the mean of DHE fluorescence. Error bars representstandard deviation. Asterisks indicate a significant difference of the mean val-ues of fluorescence vs. the fluorescence of the control parasites incubated at thesame temperature (26 ◦C or 38 ◦C) (n = 3).

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Fig. 7. Effect of the antioxidants GSH and NAC on the parasite heat-induced cell death. L. infantum parasites were supplemented with GSH 1 mM or NAC 1 mMprior to the heat-treatment and compared with heat-treated non-supplemented parasites. Heat treatment was extended for 24 h. Apoptotic parasites were identifiedeither by their low �Ψm detected by low TMRM-derived fluorescence (A) or by the presence of degraded DNA (B). The histogram (C) shows the percentage ofpositive parasites for both death-related criteria: low TMRM staining (low �Ψm) and DNA degradation (Hypodiploids). Error bars represent standard deviation.Comparison between the antioxidant-supplemented and the non-supplemented parasites was done by a two-tailed unpaired t-test. *p < 0.0001; n = 6.

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tutes the genome of this organelle. On the other hand, only DNAfrom the kinetoplast is labelled with the 2-hydroxyethidium gen-erated after oxidation of DHE (Fig. 5C; 38 ◦C). This labelling isobserved in the parasites treated at 38 ◦C but not in those treatedat 26 ◦C. The labelling of DNA from kinetoplast but not from thenucleus that is specifically observed in the heat-treated parasitesindicates that heat-induced production of O2

•− must be mainlylocated in the mitochondrial matrix.

3.4. Heat shock induced superoxide formation isindependent of �Ψm

It is generally accepted that mitochondrial hyperpolariza-tion in mammals leads to an increase in the O2

•− productionand, consequently, the use of mitochondrial uncouplers usuallydecreases ROS formation in these cells [1]. The results pre-sented demonstrate that HS induces in Leishmania both events:mitochondrial hyperpolarization and mitochondrial ROS pro-duction. To test whether the heat-induced superoxide formation

is dependent on the �Ψm, we measured the DHE oxidation ratesat 26 ◦C and at 38 ◦C under uncoupling conditions (Fig. 6). Ifthe observed increase in O2

•− production at 38 ◦C were dueto the increase in �Ψm, DHE oxidation should be equivalentat both temperatures in the presence of mitochondrial uncou-plers. Unexpectedly, the already described increase in DHEoxidation under heat-shock conditions is maintained even inthe presence of the uncouplers FCCP or CCCP, which indicatesthat heat-induced O2

•− production is not due to the increasein �Ψm.

3.5. Antioxidants prevent heat-induced apoptotic-like death

The evidence that O2•− concentration in the heat-stressed

parasites is considerably higher than that of control para-sites led us to test the possible role of oxidative stress in theinduction of the already described heat-induced apoptotic-likedeath that occurs after a prolonged exposition of the parasitesto 38 ◦C (24 h) [20]. To address this question, we analyzed

Fig. 8. Comparison of the heat-shock effect in logarithmic and late stationary parasites. (A) SOD activity was analyzed in logarithmic and late stationary L. infantumpromastigotes. Error bars represent standard deviation. The asterisk indicates a significant difference of the mean values of SOD activity between late stationary andlogarithmic parasites. p < 0.0001. Error bars represent standard deviation (n = 6). (B) Logarithmic and late stationary L. infantum promastigotes were incubated at38 ◦C for 24 h. Dead parasites are identified by their positive PI staining. Fluorescence was analyzed by flow cytometry. The asterisk indicates a significant difference ofthe mean values of PI-derived fluorescence between logarithmic and late stationary parasites (p < 0.0001). Error bars represent standard deviation (n = 6). (C) Relativefluorescence due to TMRM of L. infantum promastigotes. Parasites grown at 26 ◦C were exposed to 38 ◦C for 90 min. Fluorescence was analyzed by flow cytometry.The asterisks indicate a significant difference between mean values of fluorescence of the logarithmic parasites incubated at 38 ◦C and at 26 ◦C (p < 0.0001). Thesquares indicate a significant difference between mean values of fluorescence of the late stationary parasites incubated at 38 ◦C and at 26 ◦C (p < 0.0001). Error barsrepresent standard deviation (n = 9). (D) Effect of acidification of the culture media in heat-induced cell death of logarithmic L. infantum promastigotes. Exponentiallygrowing promastigotes were transferred to fresh media at pH 5.4 and maintained in that media for 12 h prior to heat shock at 38 ◦C. Control parasites were incubatedat the same temperatures but in fresh media at pH 7.2. The asterisk indicates a significant difference between mean values of fluorescence of the parasites incubatedat 38 ◦C and at 26 ◦C (p < 0.005). Error bars represent standard deviation (n = 3).

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whether the observed antioxidant-mediated reduction in O2•−

concentrations is able to protect the promastigotes against heat-induced apoptotic-like death. Cell death was quantified eitherby analyzing the percentage of parasites that have lost theirmitochondrial transmembrane potential (low �Ψm) or the per-centage of parasites that have partially degraded their DNA(Hypodiploids), both processes already described as character-istic of an apoptotic-like death in Leishmania parasites [20].As shown in Fig. 7A and C, after 24 h of heat treatment, thepercentage of parasites with low �Ψm decreases from 85% inheat-stressed control parasites to less than 60% in heat-stressedparasites supplemented with GSH or NAC. A similar reductionfrom 80% to less than 50% is observed when the percentage ofhypodiploid cells is used as the criteria for cell death (Fig. 7Band C). These results indicate that oxidative stress is a major,although probably not exclusive, determinant in HS-inducedapoptotic-like death in L. infantum.

3.6. Stationary promastigotes express higher levels of SODand are more resistant to heat-induced death

It has been previously reported that late stationary promastig-otes express higher levels of SOD than those growing at thelogarithmic phase [15]. Higher levels of SOD activity shoulddecrease the steady state concentration of O2

•− inside the para-sites and, according to the results shown above, these parasitesshould be more resistant to heat-induced cell death. The resultsshown in Fig. 8 indicate that SOD activity in late stationary par-asites is 10-fold higher that that observed in logarithmic ones(Fig. 8A) which, as expected, correlates with a higher resistanceto heat-induced death as revealed by a decrease in the numberof PI positive cells (Fig. 8B). It must be pointed out that deathcaused by incubation at 38 ◦C is diminished but not abolishedin late stationary parasites.

We have already shown that mitochondrial hyperpolarizationat 38 ◦C does not depend on O2

•− concentrations. Consistently,the higher level of SOD activity in late stationary promastigotesdoes not affect the heat-induced hyperpolarization of the innermitochondrial membrane, as revealed by a similar increase inthe relative TMRM-derived fluorescence in both promastigotepopulations (Fig. 8C).

To test whether acidic conditions, associated to both latestationary promastigotes and promastigotes invading the hostmacrophages, could affect the heat-induced cell death process,we analyzed the percentage of PI positive cells after heat shock oflogarithmic parasites maintained for 12 h at pH 5.4. The resultsshown in Fig. 8D indicate that parasites incubated at acidicconditions are slightly more prone to death after heat shock, pre-sumably as a consequence of the addition of the two stressingstimuli.

4. Discussion

Trypanosomatids are among the present-day nucleated cellsthat earlier diverged during evolution from the branch that hasled to higher eukaryotes. This is probably the reason why they

differ significantly from most eukaryotic cells in many molec-ular aspects, while simultaneously retaining features usuallyconsidered characteristic of plants, yeasts or even bacteria.The organization of their genes in large clusters of repeatedsequences whose transcription begins in a few specific positionsin every chromosome, the lack of RNA polymerase II promotersor the fact that all their mRNAs contain the same sequence addedto the coding region by a trans-splicing event are some examplesof their special peculiarities. Their single large mitochondrionand their atypical respiratory chain, which lacks a typical com-plex I and oxidizes NADH by an alterative route [8], by no meansescape from this pattern of molecular and cellular uniqueness.However, as in higher eukaryotic cells, ROS production dur-ing electron transfer has been demonstrated in these parasites[8]. The low concentration of ROS generated during normalmetabolism of eukaryotic cells is involved in several cellularprocesses such as cell signaling [25,26]. However, overproduc-tion of such respiration by-products may lead to the modificationof macromolecules inside the cells, which eventually causes celldeath by PCD or necrosis.

Our results indicate that both O2•− anion and peroxides are

produced in Leishmania promastigotes under in vitro standardgrowth conditions, as revealed by the constant degradation of thespecific probes DHE and H2DCFDA, respectively. This basalROS production seems to be perfectly controlled by scavengersystems in the cell, which maintain the steady-state concentra-tion of these species at safe and constant levels.

Parasites subjected to 38 ◦C are still able to control peroxideconcentrations in the cell during a period of several hours, butO2

•− concentration rapidly increases after the change in tem-perature, probably as an indication of an overproduction of thistoxic radical that cannot be degraded at a sufficient rate underthese conditions. It would be expected that some of the O2

•−produced was dismutated, either enzymatically or chemically, toperoxide in this cells. However, we are not able to detect a sig-nificant increase in the concentration of peroxides, which mightbe a consequence of the high peroxiredoxin activity alreadydescribed for these parasites [27]. It has been estimated thatin the trypanosomatid Crithidia fasiculata, peroxidases accountfor 5% of the total soluble protein [13,14].

Simultaneously, the single mitochondrion in the heat-stressedparasites seems to be working at a higher rate as indicated byboth its higher rate of O2 consumption and its observed hyper-polarization. To test whether this higher mitochondrial activitymight be responsible for the increased levels of O2

•− pro-duction, several respiratory chain inhibitors were tested underheat-stressing conditions. Our results demonstrate that three ofthese inhibitors: TTFA, antimycin A and cyanide are able tolower O2

•− intracellular concentrations under heat-shock condi-tions, as an indication of the mitochondrial origin of this reactivespecies. Traditionally, in mammalian models of mitochondrialROS generation, the production of these oxidant is not revertedby the use of inhibitors of complexes III and IV because normallythese treatments result in the reverse transfer of electrons fromcomplex II to I and a concomitant generation of more super-oxide radical. As indicated before, complex I in Leishmaniadiffers from that present in mammalians, which might explain

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why the use of inhibitors of complex III and IV does not increaseO2

•− production. In fact, our evidences indicate that incubationof the promastigotes with inhibitors of complexes II, III or IVsignificantly decreases the steady state concentration of O2

•−.Our results indicate that any decrease in the flow of electronsthrough the mitochondrial complexes decreases the superoxideproduction, suggesting a direct relationship between respirationrates and ROS generation. A decrease in mitochondrial super-oxide generation after treatment with antimycin A and KCNhas also been proved to occur in some specific cases in mam-mals. When porcine renal tubular cells or human lung cancercells were, respectively, treated with cisplatin or bortezomib (aproteasome inhibitor), the researchers observed a mitochondrialhyperpolarization accompanied of ROS production. Under thesecircumstances, treatment of the cells with antimycin A loweredthe ROS production [28,29]. Furthermore, the treatment of theporcine tubular cells with KCN also led to a decline in ROS pro-duction [28]. The evidences presented by these authors support,like our results, that the decrease in the electron transport ratesin the respiratory chain diminishes O2

•− formation, probably asa consequence a decrease in electron leaking.

The microscopic analysis of heat-stressed parasites stainedwith DHE revealed that the 2-Hydroxyethidium produced afteroxidation of this probe with O2

•− is mainly located inside thesingle mitochondrion of the parasite, as indicated by the fluo-rescence associated with the DNA in the kinetoplast but not inthe nucleus. This result confirms that the single mitochondrionis probably the source of most of the O2

•− generated during theheat treatment of the parasites.

In mammalian cells, it has been described that hyperpolar-ization of the inner mitochondrial membrane leads to O2

•−production [1]. Consequently, treatment of mammalian cellswith mitochondrial uncouplers usually ablates O2

•− formation[30]. Surprisingly, our results demonstrate that mitochondrialhyperpolarization is not responsible for the increase in superox-ide generation under heat stress conditions, as indicated by thefact that this increase is not abolished when �Ψm is decreasedas a consequence of the presence of the uncoupling agents FCCPor CCCP. This result, together with the increase in O2 con-sumption rates observed at 38 ◦C, suggests that incubation ofthe parasites at this temperature increases the electron transferrate along the mitochondrial complexes, which probably aug-ments the process of electron leaking associated to the movementof electrons through the respiratory complexes and, in conse-quence, increases the rate of O2

•− generation.The molecular response to heat shock has been extensively

studied in Leishmania. It has been demonstrated that upon heattreatment the translational machinery is mainly oriented to thesynthesis of HSP70 and HSP83 [31], although some other heatshock proteins are also translated [32]. However, despite thepreferential translation of the HSP70 and HSP83, the steadystate level of these proteins is not significantly affected afterthe heat treatment as a consequence of their high concentrationsunder normal growing conditions [33]. Accordingly, becausethe concentration of HSPs does not change significantly duringheat shock in these parasites, it is not expected that HSPs maybe involved in the process of ROS generation at 38 ◦C.

Published results have already shown that Leishmania pro-mastigotes die after a mild heat-shock treatment (38 ◦C)following a process similar to the apoptotic death in highereukaryotes [20]. The data presented in this paper demonstratethat treatment of the parasites at 38 ◦C causes a rapid increasein the O2

•− anion concentration and a subsequent induction ofoxidative stress, which suggests that this reactive species mightbe involved in the apoptotic cell death process already described.Our data indicating that GSH or NAC are able to maintain a lowO2

•− concentration both in control and in heat-stressed parasitesand that these antioxidants simultaneously reduce the percent-age of dead parasites after HS strongly support this hypothesis.The effect of these two antioxidants could be due to two differentmechanisms: first, both molecules can enter in the biosyntheticpathway of trypanothione, the major intracellular thiol in Leish-mania; consequently, they are able to increase the intracellularlevels of this molecule, rendering the cell more resistant tooxidative stress; second, both GSH and NAC are direct scav-engers of the superoxide radical, and hence, are able to reduceits intracellular levels.

The relationship between HS and oxidative stress in Leish-mania parasites has already been suggested by Miller et al. [34],who showed that HS can induce resistance to H2O2-mediateddeath. It is also interesting to point out that over-expressionof SOD in promastigotes increases their ability to infectmacrophages in vitro [16]. It is tempting to speculate that, inthese genetically modified parasites, over-expression of SODmight exert a protective effect during in vitro macrophage infec-tion not only by its action against the ROS cocktail delivered bythe infected macrophages (as it had been proposed previously)but also against the endogenous O2

•− generated during the heatshock associated to infection. In fact, due to the short life ofthis radical and to its low capacity to diffuse through the cellmembranes, the most likely explanation of the SOD-inducedinfectivity would be the ability of this enzyme to eliminate theintracellular O2

•− generated in the parasite’s mitochondrionafter the heat shock associated to infection. It must be pointedout that even though over-expression of SOD increases the infec-tivity of the parasites, wild type parasites are obviously able toinfect the vertebrate host. This means that the enzymes presentin wild type parasites are able to protect a significant proportionof the population against the restrictive conditions found in thenew host. In fact, the results presented in this paper, together withthose reported by other authors [15], clearly demonstrate that latestationary parasites, which are similar to the metacyclic infec-tive forms, express a ten fold higher SOD activity that correlateswith a higher resistance to heat stress. However, a significantproportion of this parasite population is still clearly suscepti-ble to heat-induced death. In this context, we have postulatedbefore that the co-existence of dying and surviving parasites dur-ing infection might be relevant in order to escape the immunesystem of the vertebrate, possibly because the parasites dyingby an apoptotic-like pattern present molecular signals to themacrophages that would impair their activation [20]. Obviously,the transformation of promastigotes in amastigotes that occursinto the host macrophages during the first hours of infection,presumably due to a combination of high temperature, low pH

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and probably some other signals, is associated to an adapta-tion to the new temperature, which now becomes the optimalgrowing temperature for the differentiated parasite. This pro-cess of adaptation is not easily reproducible in the laboratory,where acidification of the media must be accompanied by slowincreases in temperature in order to obtain replicating axenicamastigotes.

Acknowledgements

We thank Isabel Trabado and Gema Cervantes for their assis-tance with cell culture and flow cytometry data acquisition. Wethank Dr. Gago for his helpful discussions. We also thank GemaPerez for her useful suggestions about SOD experiments. Thiswork was supported by grants PI021052 from Instituto de SaludCarlos III and SAF2001-0989 from Ministerio de Ciencia yTecnologıa.

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