Characterization of mitochondrial bioenergetic functionsbetween two forms of Leishmania donovani – a comparativeanalysis

Subhasish Mondal & Jay Jyoti Roy & Tanmoy Bera

Received: 30 May 2014 /Accepted: 21 July 2014 /Published online: 10 August 2014# Springer Science+Business Media New York 2014

Abstract Leishmaniasis is a growing health problem inmanyparts of the world partly due to drug resistance of the parasite.This study reports on the fisibility of studying mitochondrialproperties of two forms of wild-type L. donovani through theuse of selective inhibitors. Amastigote forms of L. donovaniexhibited a wide range of sensitivities to these inhibitors.Mitochondrial complex II inhibitor thenoyltrifluoroacetoneand FoF1-ATP synthase inhibitors oligomycin anddicyclohexylcarbodiimide were refractory to growth inhibi-tion of amastigote forms, whereas they strongly inhibited thegrowth of promastigote forms. This result indicated that com-plex II and FoF1-ATP synthase were not functional inamastigote forms suggesting the presence of attenuated oxi-dative phosphorylation in the mitochondria of amastigoteforms. In contrast, mitochondrial complex I inhibitor rotenoneand complex III inhibitor antimycin A inhibited cellular mul-tiplication and substrate level phosphorylation in amastigoteforms, suggesting the role of complex I and complex III forthe survival of amastigote forms. Further we studied themitochondrial activities of both forms by measuring oxygenconsumption and ATP production. In amastigote form, sub-stantial ATP formation by substrate level phosphorylation wasobserved in NADPH-fumarate, NADH-fumarate, NADPH-pyruvate and NADH-pyruvate redox couples. None of theredox couple generated ATP formation was inhibited byFoF1-ATP synthase inhibitor oligomycin. Therefore, we mayconclude that there are significant differences between thesetwo forms of L. donovani in respect of mitochondrial bioen-ergetics. Our results demonstrated bioenergetic disfunction ofamastigote mitochondria. Therefore, these alterations of met-abolic functions might be a potential chemotherapeutic target.

Keywords Leishmania . Amastigotes . Promastigotes .

Mitochondrial bioenergetics . Oxidative phosphorylation .

Electron transport chain

Introduction

Leishmania donovani is one of the most common speciesresponsible for visceral leishmaniasis (VL) in India, Bangla-desh and Sudan. The pentavalent antimonials are widely usedas intramuscular injection in the treatment of VL, but increasein resistance to this agent led to investigation of new drugs.The risk of human immunodeficiency virus (HIV) co-infection in patients with VL or kala-azar in endemic areasalso has posed a major challenge in control programmes. Newempirical estimates put the number of episodes of clinicalleishmaniasis in the range of approximately eighty thousanddeaths per year (Fuertes et al. 2008). Unfortunately, thesestaggering figures are on the increase, largely as a result ofparasite multidrug resistance (Basu et al. 2008). A number ofstrategies have been proposed to deal with this global healthproblem and one of which is the development of novel drugsfor new parasite targets. In search of new antileishmanial drugtargets, we have focused on the electron transport chain(ETC) of mitochondria of Leishmania parasite. Leishmaniadonovani amastigote mitochondria lack the conventionalcomplex I, II and IV (Chakraborty et al. 2010). Evidencesuggests that amastigote stage can be considered as goldstandard for in vitro and in vivo Leishmania drug discoveryresearch and for evaluation of resistance (Vermeersch et al.2009).

During transition through these different extra- and intra-cellular environments, Leishmania is exposed to many chang-es in their living conditions. These changes include the ele-vated temperature of the mammalian host, toxic oxidantsproduced during phagocytosis by macrophages, acidic pH

S. Mondal : J. J. Roy : T. Bera (*)Division of Medicinal Biochemistry, Department of PharmaceuticalTechnology, Jadavpur University, Kolkata 700032, Indiae-mail: dr.subhasishjprr@gmail.com

J Bioenerg Biomembr (2014) 46:395–402DOI 10.1007/s10863-014-9569-5

and proteases encountered in the macrophage phagolysosome,variations in the availability and types of nutrients, as well asthe availability of oxygen. The mechanism that allows para-sites to withstand these noxious stimuli is probably critical fortheir survival (Pearson and Wilson 1989).

The Leishmania and other trypanosomatids are unique inthat they contain a single mitochondrion per cell which con-volutes and ramifies in situ and occupies around 12 % of thecell volume, and it is associated with the kinetoplast to form akinetoplast-mitochondrial complex (Burn and Krassner1976). Quantitative morphometry of electron micrographs ofLeishmania spp. after 3 h of heat treatment showed that therewas no change in mitochondrial morphology, but after 6 h ofheat treatment the mitochondria had lost their cristae and nolonger possess a clearly defined mitochondrial double mem-brane (Rudzinska et al. 1964). Martin and Mukkada reportedthe evidence for the presence of complex I, II, III and IV in therepiratory chain of L. tropica promastigote (Martin andMukkada 1979). Chakraborty et al. showed the absence ofoxidative phosphorylation in amastigotes of L. donovaniUR6strain and also suggested that energy linked functions inamastigotes might occur through fumarate reduction leadingtoΔpH generation by succinate excretion (Chakraborty et al.2010). Promastigotes are exposed to various concentrations ofreactive oxygen species during phagocytosis by macrophage,which can induce programmed cell death (PCD). Mitochon-drial peroxiredoxin protects L. donovani from PCD.Peroxiredoxin is restricted to the kinetoplast area inpromastigotes, whereas it covers the entire mitochondrion inamastigotes, accompanied by dramatically increased expres-sion of peroxiredoxin (Harder et al. 2006).

In this work we have characterized mitochondrial bioener-getic functions in wild-type L. donovani AG83 promastigotesand amastigotes. We may expect that the exploration of bio-energetic analysis can lead to the identification of tractabletargets for chemotherapy of leishmaniasis.

Materials and methods

Materials

Standard glass wears of Borosil® were used for experimentalpurposes. All chemicals unless otherwise mentioned werepurchased from Sigma-Aldrich (St. Louis, MO).

Parasites and culture conditions

Promastigotes of Leishmania donovani clones, AG83(MHOM/IN/83/AG83) was a VL isolate obtained as a giftfrom Indian Institute of Chemical Biology, Council of Scien-tific and Industrial Research, Kolkata, India. Strain AG83 hasbeen used in India as the reference standard strain of

L. donovani. Parasites were routinely grown as promastigotesin mediumM-199 with 10% heat–inactivated fetal calf serum(FCS) at 24 °C (Chakraborty et al. 2010; Debrabant et al.2004).

Generation of axenic amastigotes

Leishamnia donovani amastigote forms were grown andmaintained as described by Debrabant et al. (Debrabant et al.2004). Axenically grown amastigotes of L. donovani weremaintained at 37 °C in 5 % CO2/air by weekly sub-passagesin MMA/20 at pH 5.5 in petri dishes (Senero and Lemers1997). Under these conditions, promastigotes differentiated toamastigotes within 120 h. Complete promastigote toamastigote differentiation was evidenced by the electron mi-croscopic studies by Gupta N et al. (Gupta et al. 1996) andBrun R et al. (Burn and Krassner 1976). Cultures were main-tained by 1:3 dilutions once in a week. Amastigote multipli-cation rate was found to be 2.86±0.51 (n=12).

Cell viability test

Before starting the experiments viabilities of both amastigoteand promastigote cells were checked by Trypan blue exclu-sion method. It is based on the principle that live cells possessintact cell membranes that exclude the dye whereas dead cellsdo not. Amastigote or promastigote cell suspension (1ml) wassimply mixed with 0.4 % solution of trypan blue (dissolved inphosphate-buffered saline, pH 7.2 and pH 5.5 forpromastigotes and amastigotes, respectively). Sample wasloaded into a hemocytometer and examined immediately un-der a microscope. The number of blue staining cells and thenumber of total cells were counted. Cell viability was ob-served more than 95 % for both types of cells.

Axenic amastigote inhibitor susceptibility assay

Axenic amastigote inhibitor susceptibility assays were carriedout by determining the 50 % inhibitory concentration (IC50)by cell counting method using a hemocytometer. To measurethe IC50 values for different inhibitors, the parasites wereseeded into 96-well plates at a density of 2×105 amastigotes/well in 200 μl culture medium containing 10 μl of differentinhibitors. Parasite multiplication was compared to that ofuntreated control (100 % growth). After 72 h of incuba-tion, cell count was taken microscopically. The resultswere expressed as the percentage of reduction in parasitenumber compared to that of untreated control wells, andthe IC50 was calculated by linear regression analysis(MINITAB V.13.1, PA) or linear interpolation (Huberand Koella 1993). All experiments were repeated threetimes, unless otherwise indicated.

396 J Bioenerg Biomembr (2014) 46:395–402

Preparation of digitonin permeabilized Leishmania cells

Leishmania donovani promastigote and/or amastigote cellswere collected, washed once by buffer A (140 mM NaCl,20 mM KCl, 20 mM Tris, 1 mM EDTA, pH 7.5), and resus-pended in isolation buffer (20mMMOPS-NaOH, 0.3%BSA,350 mM sucrose, 20 mM potassium acetate, 5 mM magne-sium acetate, 1 mMEGTA, pH 7.0). Cells were permeabilizedin a separate tube with digitonin (200 μg or 120 μg/mgprotein) and incubated on ice for 10 min. After incubation,the cells were centrifuged at 6,000 xg for 7 min. Pellets werere-suspended in assay buffer.

ATP measurements

Digitonin permeabilized amastigote cells were exposed tovarious redox couples and an extract was prepared for themeasurements of the effects of redox couples on ATP forma-tion. Cells were washed twice with 140 mM PBS beforeaddition of 200 μg digitonin/mg protein. 0.1 ml digitoninpermeabilized cells (120 μg protein/ml), 2.4 μmolesMg(Ac)2, 1.2 μmoles ADP, 4.8 μmoles inorganic phosphate,0.6 μmole electron donor and 6 μmoles electron acceptorwere added in 0.6 ml assay buffer (KCl, 50 mM; sucrose,300 mM; Tris–HCl, 50 mM; EGTA, 2 mM; pH 7.0) ineppendrop tube (control system). Assay mixture was incubat-ed at 37 °C for 20 min in presence or absence of metabolicinhibitors, followed by addition of 0.14 ml 2.5(N) HClO4 and0.09 ml 8(N) KOH to maintain the pH at 7.0, mixed rapidlyand centrifuged at 12,000 xg at 4 °C for 10 min. 0.1 mlsupernatant, 0.2 mg NADP+, 0.25 μmole glucose, 4 units/mlhexokinase and 2 units/ml glucose-6-phosphate dehydroge-nase were added in 0.28 ml assay buffer (Tris, 50 mM;Mg(Ac)2, 10 mM; EGTA, 5 mM) and was incubated in glasscuvette at a final volume of 0.6 ml for 5 min at 25 °C.Absorbance was recorded at 340 nm (Bergmeyer 1984). Con-trol incubation contained complete system without redox-couple and complete system without ADP.

Measurement of oxygen uptake

Rates of oxygen consumption were measured in digitoninpermeabilization buffer, pH 7.0 (100 mM MOPS, pH 7.0;20 mg/ml protease free bovine serum albumin; 300 mM su-crose, 7 mM dipotassium hydrogen phosphate, 5 mMmagne-sium acetate and 1 mM EGTA) at 25 °C in a water-jacketedDW1 Hansatech Oxygraph+ 1 ml glass chamber (HansatechInstruments Ltd., Norfolk, UK) containing a clark type polar-ographic oxygen electrode. This instrument is totally operatedby computer. The solubility of oxygen in air-saturated, tem-perature equilibrated medium was taken to be 480 ng-atoms/ml at 25 °C and 760 mmHg (Robinson and Cooper 1970).L. donovani promastigote or amastigote cells were treated

with 50 μg digitonin/mg cell protein for 3 min before theaddition of oxidizable substrate and/or electron transport in-hibitor. Digitonin permeabilized cell protein was added at aconcentration of 3 mg/ml in oxygraph glass chamber. Theconcentrations of oxidizable substrates were in 10 μl volume:L-glutamate, 10 mM; succinate, 10 mM; ascorbate, 10 mM;TMPD, 0.4 mM; α-ketoglutarate, 10 mM; DL-malate,10 mM; ADP, 0.1 mM. The concentrations of electron trans-port inhibitors were 40 μM, 2 μg/mg protein, 0.2 mM, 20 μg/mg protein and 2 mM for rotenone, antimycin A, TTFA,oligomycin and cyanide, respectively in 10 μl volume. Sam-ple containing digitonin permeabilized cells without electrondonor and inhibitor was treated as control.

Protein estimation

Total cell protein was determined by the biuret method in thepresence of 0.2 % deoxycholate (Gornall et al. 1949). Onemilligram of protein corresponds to 1.75×108 promastigotecells and 1.14×108 amastigote cells.

Statistical analysis

All experiments were performed in triplicate, with similarresults obtained in at least three separate experiments. Statis-tical significance was determined by Student’s t-test. Signifi-cance was considered as P<0.05. GraphPad Prism 5.01 soft-ware was used for the data analysis purposes.

Results

It appeared from Table 1 that promastigote forms weremore susceptible to electron transport inhibitors than the

Table 1 Evaluation of susceptibilities of wild-type L. donovani axenicpromastigotes and amastigotes to mitochondrial electron transport andATP synthase inhibitors

Compounds tested (IC50±SD, n=3) μMa

Axenic promastigote Axenic amastigote b

Rotenone 0.25±0.032 1.4±0.2

TTFA 0.9×103±120 >4×103 c

Antimycin A 0.30±0.039 0.8±0.2

Oligomycin 11±1.8 >200 c

DCCD 18±2.7 >200 c

a Assays are described inMaterials andMethods. All datas are mean±SDfor three experimentsb The data already published in our previous work (Mondal et al. 2014)c No inhibition was observed at the indicated concentration

J Bioenerg Biomembr (2014) 46:395–402 397

amastigote forms. Mitochondrial complex II inhibitorTTFA (thenoyltrifluoroacetone) and FoF1-ATP synthase(complex V) inhibitors oligomycin and DCCD(dicyclohexylcarbodiimide) were refractory to growthinhibition of amastigote forms, whereas they stronglyinhibited the growth of promastigote forms. Thus, itappeared that complex II and FoF1-ATP synthase ofmitochondrial electron transport chain were functionalin promastigotes. However, when promastigote formswere transformed to amastigote forms, complex II andcomplex V activities were completely lost. Thus, wemay conclude that in mitochondrial electron transportcomplex II and FoF1-ATP synthase were not functionalin amastigotes, suggesting the presence of attenuatedoxidative phosphorylation in the mitochondria ofamastigote forms. Therefore, ATP synthesis in amastigotemitochondria was independent of FoF1-ATP synthase.However, amastigotes were inhibited by rotenone, a com-plex I inhibitor, at higher concentration compared to theirpromastigote forms. Thus, complex I appeared to be func-tional in both forms. Antimycin A, a complex III inhibitor,strongly inhibited the growth of both forms of L. donovanisuggesting that complex III of mitochondrial electrontransport chain was functional.

To study the mitochondrial activity of promastigote andamastigote cells, we measured oxygen consumption and ATPproduction (Tables 2 and 3). For both types of cells digitoninconcentration at 50 μg/mg protein had no detrimental effecton respiratory control ratio (RCR) in presence of α-ketoglutarate as electron donor (Fig. 1). These results showedthat the above mentioned digitonin concentration wouldpermeabilize the plasma membrane of both types of cells.However, at that concentration mitochondrial membrane ofboth types of cells would not be affected, though RCR valuesdiffered due to differences in oxygen uptakes betweenpromastigotes and amastigotes. It appeared from Table 2 that

glucose stimulated 33 % oxygen uptake in promastigote cells,whereas glucose failed to stimulate oxygen uptake inamastigote cells. Here it was worth to note that amastigotecells lost 88 % oxygen uptake rate compared to promastigotecells. This observation was also supported by the function ofmitochondria in digitonin permeabilized cells (Table 3). Stim-ulation of oxygen uptake by L-glutamate, succinate, ascor-bate+TMPD, α-ketoglutarate and DL-malate in digitoninpermeabilized promastigotes were observed, whereasamastigotes were refractory to oxygen uptake stimulation bythe above substrates. Similarly, state 3 respirations in digitoninpermeabilized promastigotes were stimulated, whereas digi-tonin permeabilized amastigotes were refractory to stimula-tion in state 3 respiration. In normal eukaryotic cells themitochondrial oligomycin-sensitive FoF1-ATP synthase uti-lizes proton gradient generated by electron flow from a re-duced substrate to molecular oxygen to synthesize ATP fromADP and Pi. (Rolfe and Brown 1997). FoF1-ATP synthaseinhibitor oligomycin depressed respiration in promastigoteswhich suggested the occurrence of oxidative phosphorylation,whereas oligomycin failed to show any effect on amastigotes.Existence of complex IV in promastigotes is evidenced by theinhibition of oxygen uptake by cyanide. Addition of rotenoneand antimycin A, induced a substantial increase in oxygenconsumption in promastigotes. Data indicated that oxygenconsumption in amastigotes was not affected by rotenone,TTFA, antimycin A, oligomycin, and cyanide.

To determine the effect of mitochondrial effectors on ener-gy transduction, the ATP concentration was measured inpresence of redox couples in digitonin permeabilizedamastigote cells (Table 4). ATP concentration was consider-ably reduced in absence of redox couple or ADP, (data notpresented). Data indicated that FoF1-ATP synthase inhibitoroligomycin did not significantly affected ATP levels. Rote-none strongly reduced ATP levels followed by antimycin A inredox coupled ATP formation, whereas TTFA failed to showany effect. Thus a substrate level phosphorylation, without theinvolvement of FoF1-ATP synthase, might then be used togenerate ATP and rotenone sensitive complex I and antimycinsensitive complex III might have been involved in substratelevel phosphorylation.

Discussion

Previous findings suggests that heat transformed, acidic pHstabilized L. donovani cells down-regulate plasma membraneand mitochondrial electron transport as well as oxygen uptake(Chakraborty et al. 2010), insisted us to explore the nature ofenzymes involved in energy metabolism of L. donovanipromastigotes and amastigotes. Also the preliminary evidencefor dependency of amastigote cells on substrate level phos-phorylation for their survival was published (Mondal et al.

Table 2 Oxygen uptake rates of wild-type Leishmania donovani AG83axenic promastigotes and amastigotes

Addition Rate of oxygen uptake (nmoles/min/mg protein) a

Axenic AG83promastigotes

Axenic AG83amastigotes

Without glucose 14.30±1.80* 2.00±0.27ns

With glucose 19.00±3.20* 2.20±0.36ns

a Rate values represent mean±SD values of four experiments

Oxygen uptake was recorded polarographically as described in Materialsand Methods* P<0.05, significant difference between axenic AG83 promastigotes inpresence and absence of glucosens P>0.05, no significant difference between axenic AG83 amastigotes inpresence and absence of glucose

398 J Bioenerg Biomembr (2014) 46:395–402

Tab

le3

Relativerateof

substrateoxidationby

mito

chondriain

digitoninperm

eabilized

axenicprom

astig

oteandam

astig

otecells

ofwild

-typeLeishm

ania

donovani

AG83

Addition

bRelativerateof

substrateoxidation

a

Endogenous

substrate

L-G

luL-G

lu+ADP

Succi

Succi+ADP

Asc.+

TMPD

Asc.+

TMPD

+ADP

α-K

Gα-K

G+ADP

DL-M

alDL-M

al+ADP

Prom

astig

ote(Pm)

100

Pm+digitonin(digi)

70125

145

110

130

225

275

150

195

138

180

Pm+digi

+rotenone

166

171

320

200

210

Pm+digi

+TTFA

141

98270

192

174

Pm+digi

+antim

ycin

A187

196

277

210

144

Pm+digi

+oligom

ycin

124

111

220

145

142

Pm

+digi

+KCN

3234

3041

41

Amastig

ote(A

m)

12

Am

+digi

79

108

818

1810

126

6

Am

+digi

+rotenone

87

1612

7

Am

+digi

+TTFA

75

1410

6

Am

+digi

+antim

ycin

A8

815

147

Am

+digi

+oligom

ycin

98

1810

6

Am

+digi

+KCN

87

169

6

aRelativerate100hasbeen

considered

forL

.donovanipromastig

oteendogenous

oxygen

uptake

ratetobe

4.5nm

olO2/m

in/m

gcellprotein.Relativeratevalues

representthe

averageof

four

experiments

bL.

donovaniprom

astig

oteor

amastig

otecells

weretreated,whereindicated,with

50μgdigitonin/mgcellproteinfor3minbeforetheadditio

nof

oxidizablesubstrateand/or

electron

transportinhibito

r.The

concentrations

ofoxidizablesubstrates

wereas

follo

ws:L-glutamate-10

mM;succinate-10mM;ascorbate-10mM;T

MPD

-0.4mM;α

-ketoglutarate-10mM;D

L-m

alate-10

mM;A

DP-0.1mM.T

heconcentrations

ofelectron

transportinhibito

rsare40

μM,2

μg/mgprotein,0.2mM,20μg/mgproteinand2mM

forrotenone,antim

ycin

A,T

TFA

,olig

omycin

andKCN,respectively

J Bioenerg Biomembr (2014) 46:395–402 399

2014). Many lower eukaryotes can survive in hypoxic oranaerobic condition via a fermentative pathway that involvesthe use of the reduction of endogenously produced fumarate

as an electron sink. They are highly adapted for prolongedsurvival or even continuous functioning in the absence ofoxygen, whereas many of them are adapted to alternatingperiods in the presence and absence of oxygen (Tielens andvan Hellemond 1998). Some anaerobically functioning eu-karyotes, such as yeast and certain fishes, can survive withoutmitochondrial energy metabolism via cytosolic fermentationsin which NADH/NADPH produced during glycolysis is con-sumed during the reduction of pyruvate to lactate or fumarateto succinate, which are subsequently excreted as end products(Van Hellemond et al. 2003).

Warburg reported that tumor tissues convert glucose tolactate, via the reduction of pyruvate, even in presence ofoxygen, whereas normal tissues use pyruvate, derived fromglycolysis, plus oxygen to produce ATP from oxidative phos-phorylation, in amounts comparable to that derived fromoxidative phosphorylation in normal cells, which werethought to produce 100-fold less ATP from glycolysis relativeto respiration. Thus, cancer was proposed to derive from animpairment of cellular respiration (Warburg 1956). However,studies are rapidly emerging that show active involvement ofmitochondria in malignant transformation. For example, it hasbeen shown that mitochondrial β-F1-ATP synthase was down

Table 4 Relationship betweenredox couple generated ATP for-mation and inhibitors of complexI, II, III and V in wild-type Leish-mania donovani AG83 axenicamastigotes a

a Estimation of redox coupledATP formation in digitoninpermeabilized wild-typeL. donovani AG83 cells in pres-ence or absence of mitochondrialelectron transport inhibitors wascarried out as described in Mate-rials and Methods

Complete system was composedof 120μg digitonin permeabilizedcell (DPC)/ml, 4 mM Mg(Ac)2,2 mM ADP, 8 mM Pi, 1 mMNADPH/NADH and 10 mM fu-marate/pyruvate. Rotenone,TTFA , a n t imy c i n A andoligomycin were added at a con-centration of 40 μM, 200 μM,2 μg/mg DPC, 20 μg/mg DPC,respectively

Addition Rate (nmol/min/mg protein) Relativerate

A. NADPH-fumarate oxidoreductase

DPC + Mg(Ac)2 + ADP + Pi+ NADPH + Fumarate (Complete) 87±9 100

Complete + rotenone 29±5 33

Complete + TTFA 92±12 105

Complete + antimycin A 57±9 65

Complete + oligomycin 77±10 89

B. NADH-fumarate oxidoreductase

DPC + Mg(Ac)2 + ADP + Pi + NADH + Fumarate (Complete) 89±11 100

Complete + rotenone 26±4 29

Complete + TTFA 88±9 98

Complete + antimycin A 48±8 53

Complete + oligomycin 79±7 88

C. NADPH-pyruvate oxidoreductase

DPC + Mg(Ac)2 + ADP + Pi + NADPH + Pyruvate (Complete) 83±10 100

Complete + rotenone 56±9 67

Complete + TTFA 84±11 101

Complete + antimycin A 39±6 47

Complete + oligomycin 66±7 79

D. NADH-pyruvate oxidoreductase

DPC + Mg(Ac)2 + ADP + Pi + NADH + Pyruvate (Complete) 94±9 100

Complete + rotenone 31±5 32

Complete + TTFA 112±11 119

Complete +antimycin A 28±5 29

Complete + oligomycin 91±10 96

Fig. 1 Effect of digitonin concentration on the respiratory control ratio ofL. donovani promastigote and amastigote cells. After digitonin treatment,the cells decreased their rate of oxygen consumption due to dilution ofendogenous substrate concentration. Oxygen consumption was restoredby 10mMα-ketoglutarate in presence of 15mg amastigote cell protein or3 mg promastigote cell protein and the respiratory control ratio wasdetermined upon addition of 0.2 mM ADP. Each point represents theaverage value of three experiments±S. D

400 J Bioenerg Biomembr (2014) 46:395–402

regulated in human liver, colon, renal and breast tumors andsignificantly correlated with disease progress (Cuezva et al.2002, 2004; Dang and Semenza 1999). In addition, reducedexpression of respiratory complexes, including II, III and IV,and deficient complex I, were demonstrated in renal cellcarcinomas (Simonnet et al. 2002, 2003). The attenuatedcapacity for mitochondrial respiration and oxidative phos-phorylation identified in our study corroborates reports dem-onstrating expression of mitochondrial respiratory and oxida-tive phosphorylation enzymes in both amastigote cells and awide spectrum of human tumor. We believe that this is mostlikely a metabolic reflection of overall reduced enzyme activ-ities of respiratory complexes of the mitochondria and FoF1-ATP synthase in both amastigote and cancer cells. It has beensuggested that reduced oxidative phosphorylation is the mech-anism underlying ROS-mediated apoptotic resistance of high-ly glycolytic cancer cells induced by staurosporine(Santamaria et al. 2006). Reduced mitochondrial oxidativephosphorylation limits reverse ATP-synthase supported in-crease in mitochondrial potential and, consequently, reducedROS production, leading to a decrease in apoptotic sensitivity.We would speculate that the more attenuated mitochondrialoxidative phosphorylation of both amastigote and cancer cellswould be more resistant to ROS-mediated apoptotic induc-tion. Therefore, the bioenergetic properties of increased gly-colysis, and attenuated respiration along with oxidative phos-phorylation, identified in this study, provide a metabolic basisfor both tumor and amastigote cell proliferation to resistanceto apoptosis.

In summary, we have demonstrated a unique metabolicphenotype in Leishmania amastigote cell by measuring respi-ration and substrate level phosphorylation (SLP). As thepromastigote forms progress toward amastigote forms, cellsbecome less dependent on oxidative phosphorylation andmore dependent on glycolysis and SLP. The bioenergeticphenotype defined in this study is characterized by an in-creased glycolytic and SLP rates and capacity that are inti-mately linked to an attenuated mitochondrial function. Thesemetabolic alternations may confer on amastigote cells theability to promote growth, survival and invasion within mac-rophage cells. It is evident from Table 3 that oxygen uptakeswere stimulated in presence of antimycin A with respect tocontrol for all types of electron donors except for malate.Antimycin A stimulated state 3 oxygen uptake rate. Thehypothesis behind the fact might be that electron transportchain of promastigote mitochondria became branched andparticipated in oxygen uptake in two or more terminals.Therefore, antimycin A inhibition of one branched pathwaywas likely to stimulate oxygen uptake by the other pathwaywhich was not inhibited by antimycin A. Branched pathwaysare the deviation from the normal electron transport chain ofliver mitochondria. Same observation was also found in Ent-amoeba histolytica (Bera et al. 2006). Our results on bio-

energetic analysis may prove essential for the identificationof tractable targets for therapeutic intervention. SLP, inhibitionof succinate and pyruvate synthesis along with excretioncould be a potential chemotherapeutic strategy for drug de-velopment in leishmaniasis.

The differences between parasite and host energy metabo-lism described in our work hold great promise as targets forchemotherapy. For example, most of the act iveantileishmanial compounds tested on promastigotes failed toinhibit amastigotes (Mattock and Peters 1975; Peters et al.1980). If an agent that can specifically inhibit the amastigotefumarate reductase and pyruvate reductase is found, it isexpected to be extremely effective and selective as anantileishmanial agent.

Acknowledgments Dr. Subhasish Mondal was awarded Research As-sociateship from Indian Council of Medical Research, New Delhi, Indiato carry out this research work. Mr. Jay Jyoti Roy was awarded “RajivGandhi National Fellowship” from University Grants Commission, NewDelhi, India which is also gratefully acknowledged.

Conflict of interest The authors have declared that no competinginterests exist.

References

Basu JM, Mukherjee A, Banerjee R, Saha M, Singh S, Naskar K et al(2008) Inhibition of ABC transporters abolishes antimony resistancein Leishmania infection. Antimicrob Agents Chemother 52(3):1080–1093

Bera T, Nandi N, Sudhahar D, Akbar MA, Sen A, Das P (2006)Preliminary evidence on existence of transplasma membrane elec-tron transport in Entamoeba histolytica trophozoites: a key mecha-nism for maintaining optimal redox balance. J Bioenerg Biomembr38(5–6):299–308

Bergmeyer HU (1984) Ed., Vol 6, pp. 163–172, VCH, Weinhiem, W.Germany-Deerfield.

Burn R, Krassner SM (1976) Qualitative ultrastructural investigations ofmitochondrial development in Leishmania donovani during trans-formation. J Protozool 23(4):493–497

Chakraborty B, Biswas S, Mondal S, Bera T (2010) Stage specificdevelopmental changes in the mitochondrial and surface membraneassociated redox system of Leishmania donovani promastigote andamastigote. Biochem Mosc 75(4):494–504

Cuezva JM, Krajewska M, de Heredia ML, Krajewski S, Santamaria G,KimH et al (2002) The bioenergetic signature of cancer: a marker oftumor progression. Cancer Res 62(22):6674–6681

Cuezva JM, Chen G, Alonso AM, Isidoro A, Misek DE, Hanash SM et al(2004) The bioenergetic signature of lung adenocarcinomas is amolecular marker of cancer diagnosis and prognosis.Carcinogenesis 25(7):1157–1163

Dang CV, Semenza GL (1999) Oncogenic alterations of metabolism.Trends Biochem Sci 24(2):68–72

Debrabant A, Joshi MB, Pimenta PF, Dwyer D (2004) Generation ofLeishmania donovani axenic amastigotes: their growth and biolog-ical characteristics. Int J Parasitol 34(2):205–217

Fuertes MA, Nguewa PA, Castilla J, Alonso C, Pérez JM (2008)Anticancer compounds as leishmanicidal drugs : challenges in che-motherapy and future perspectives. Curr Med Chem 15(5):433–439

J Bioenerg Biomembr (2014) 46:395–402 401

Gornall AG, Bardawill CJ, David MM (1949) Determination of serumproteins by means of the biuret reaction. J Biol Chem 177(2):751–766

Gupta N, Mittal N, Goyal N, Maitra SC, Rastogi AK (1996)Ultrastructural characterization of serially passaged amastigote likeforms of Leishmania donovani. Trop Med 38(2):39–50

Harder S, Bente M, Isermann K, Bruchhaus I (2006) Expression of amitochondrial peroxiredoxin prevents programmed cell death inLeishmania donovani. Eukaryot Cell 5(5):861–870

Huber W, Koella JC (1993) A comparison of three methods of estimatingEC50 in studies of drug resistance of malaria parasites. Acta Trop55(4):257–261

Martin E, Mukkada AJ (1979) Identification of the terminal respiratorychain in kinetoplast. mitochondrial complexes of Leishmaniatropica promastigotes. J Biol Chem 254(23):12192–12198

Mattock NM, Peters W (1975) The experimental chemotherapy of leish-maniasis. II. The activity in tissue culture of some antiparasitic andantimicrobial compounds in clinical use. Ann Trop Med Parasitol69(3):359–371

Mondal S, Roy JJ, Bera T (2014) Generation of adenosine tri-phosphatein Leishmania donovani amastigote forms. Acta Parasitol 59(1):11–16

Pearson, R.D., Wilson, M.E. (1989). Parasitic Infections in theCompromised Host (Walzer PD, Genta RM, eds.) Marcell Dekker,Inc., New York, p 31–81.

Peters W, Trotter ER, Robinson BL (1980) The experimental chemother-apy of leishmaniasis, VII. Drug responses of L. major andL. mexicana amazonensis, with an analysis of promising chemicalleads to new antileishmanial agents. Ann Trop Med Parasitol 74(3):321–335

Robinson J, Cooper JM (1970) Method of determining oxygen concen-trations in biological media, suitable for calibration of the oxygenelectrode. Anal Biochem 33(2):390–399

Rolfe DF, Brown GC (1997) Cellular energy utilization and molecularorigin of standard metabolic rate in mammals. Physiol Rev 77(3):731–758

Rudzinska MA, Alesandro PAD, Trager W (1964) The fine structure ofLeishmania donovani and the role of the kinetoplast in theleishmani–leptomonad transformation. J Protozool 11:166–191

Santamaria G, Martinez-Diez M, Fabregat I, Cuezva JM (2006) Efficientexecution of cell death in non-glycolytic cells requires the genera-tion of ROS controlled by the activity of mitochondrial H+-ATPsynthase. Carcinogenesis 27(5):925–935

Senero D, Lemers JL (1997) Axenically cultured amastigote forms as anin vitro model for investigation of antileishmanial agents.Antimicrob Agents Chemother 41(5):972–976

Simonnet H, Alazard N, Pfeiffer K, Gallou C, Beroud C, Demont J et al(2002) Low mitochondrial respiratory chain content correlates withtumor aggressiveness in renal cell carcinoma. Carcinogenesis 23(5):759–768

Simonnet H, Demont J, Pfeiffer K, Guenaneche L, Bouvier R, Brandt Uet al (2003) Mitochondrial complex I is deficient in renaloncocytomas. Carcinogenesis 24(9):1461–1466

Tielens AG, van Hellemond JJ (1998) The electron transport chain inanaerobically functioning eukaryotes. Biochim Biophys Acta1365(1–2):71–78

Van Hellemond JJ, Van der Klei A, van Weelden SW, Tielens AG (2003)Biochemical and evolutionary aspects of anaerobically functioningmitochondria. Philos Trans RSoc LondBBiol Sci 358(1429):205–213

VermeerschM, da Luz RI, Toté K, Timmermans JP, Cos P,Maes L (2009)In vitro susceptibilities of Leishmania donovani promastigote andamastigote stages to antileishmanial reference drugs : practical rel-evance of stagespecific differences. Antimicrob Agents Chemother53(9):3855–3859

Warburg O (1956) On respiratory impairment in cancer cells. Science124(3215):269–270

402 J Bioenerg Biomembr (2014) 46:395–402