Molecular and Cellular Biochemistry 289: 83–90, 2006.DOI: 10.1007/s11010-006-9151-5 c�Springer 2006

Chronic heat-shock treatment drivendifferentiation induces apoptosis in Leishmaniadonovani

Puneet Raina and Sukhbir KaurParasitology Laboratory, Department of Zoology, Panjab University, Chandigarh-160014, India

Received 26 October 2005; accepted 10 February 2006

Abstract

The present study investigates the role of apoptosis in the regulation of cell numbers of Leishmania donovani during the in vitrodifferentiation of promastigote stage to amastigote stage in axenic conditions. We report that apoptosis is induced in Leishmaniadonovani due to chronic heat-shock treatment of 37 ◦C that also mediates the differentiation of promastigotes to amastigotes.This is characterized by the fragmentation of DNA, blebbing in the parasite cell membrane, nuclear condensation, formationof preapoptotic bodies and involvement of Ca++ in the apoptotic process. The flowcytometric analysis shows an early andsteep rise in percentage apoptotic nuclei till 48-hour stage of differentiation and then a gradual decline, suggesting synergisticaction of Ca++ ATPase and probably Hsp70. Hsp70 might be rescuing cells from apoptosis in the death signaling pathway.Incubation of the culture with Ca++ chelator EGTA (1 mM) brings down the percentage of apoptotic nuclei considerablyshowing thereby that calcium is needed for the process of cell death here that occurs by apoptosis. The survival of the infectiveindividuals appears to be decided by the parasite in the early stages of its differentiation. Our studies show the potential ofthe physiological temperature of 37 ◦C in inducing apoptosis in Leishmania donovani and the therapeutic use it can be putto. (Mol Cell Biochem 289: 83–90, 2006)

Key words: Leishmania donovani, heat-shock, apoptosis, axenic culture

Introduction

Leishmania, an intramacrophage obligate parasitic proto-zoan causes leishmaniasis which is a group of diseasesencompassing myriad of clinical manifestations rangingfrom self-healing cutaneous ulcers to a severe disease withmassive tissue destruction and even death [1]. The causativespecies, coupled with the way the host’s immune systemreplies, dictates the intensity of the disease [2–4]. Visceralleishmaniasis(VL), caused by Leishmania donovani, is atropical disease, endemic in 88 countries, of which 72 areclassed as developing countries, including 13 of the leastdeveloped countries with 90% of VL cases occurring in just

Address for offprints: S. Kaur, Department of Zoology, Panjab University, Chandigarh-160014, India (E-mail: puzoology@yahoo.com)

five countries-Bangladesh, India, Nepal, Sudan and Brazil[5]. Disability-adjusted life years (DALYs) lost due to leish-maniasis are close to 2.4 million, there are 1.0–1.5 millioncases of cutaneous leishmaniasis (CL) and 500,000 cases ofVL each year, and a population of 350 million is at risk [6].Leishmania have a digenetic life cycle. They occur in themidgut of the sandfly vector, extracellularly as a motile flag-ellated and elongated promastigote and reside intracellularlyin mammalian macrophages as an immotile and roundedamastigote.

Apoptosis is a ubiquitous or universal phenomenon in de-velopmental, differential and homoeotic pathways. Observa-tion of programmed cell death in D. discoideum [7],E. coli

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[8] and T. thermophila [9] proved that the regulated celldeath is not strictly a multicellular phenomenon. In fact activeprogrammed cell death has been found to be of paramountimportance towards maintaining cell numbers in parasites.Detection of Programmed Cell Death (PCD) in Leishma-nia amazonensis [10] opened up a whole new perspectiveof apoptosis in trypanosomatids. This study was targeted atdetection and documentation of programmed cell death as aphenomenon in the in vitro culture of Leishmania donovaniat the critically important physiological temperature of 37 ◦Cat which promastigotes differentiate into amastigotes in thehuman host.

Materials and methods

Parasite culture

Leishmania donovani promastigotes (strain MHOM/IN/80/Dd8) were maintained in modified NNN (Novy, McNeal and Nicolle) medium with Minimum Essential Medium(MEM) as overlay at 22±1 ◦C in the BOD-incubator [11]. Fortheir conversion process to axenic amastigotes, promastig-otes were cultured in the same medium and incubated at37 ± 0.5 ◦C in presence of 5% CO2 and 90–95% humid-ity. Since heat-shock is an inducer of apoptosis [10, 12], thepresent study was based on giving a chronic heat-shock treat-ment to the promastigotes maintained at 22 ± 1 ◦C, by trans-ferring 48-hour old promastigote culture to CO2-incubatorat 37 ± 0.5 ◦C, 5% CO2 concentration and a humidity of90–95%. The parasites were harvested every 24 h till 96 h.Promastigotes not supplemented with MEM for ten dayswere employed as positive control, since media withdrawalgenerated stress is a proven apoptosis inducer. Promastigotessupplemented with MEM every 48 h were used as a negativecontrol.

DNA fragmentation assay

DNA was isolated from parasites cultured at different inter-vals of chronic heat-shock treatment by phenol-chloroform-isoamyl alcohol method [13], run on 0.8% agarose gel andvisualised under UV transilluminator (Hoefer).

Scanning electron microscopy

The cells were processed by the method of Zhao et al. [14]with minor modifications. Parasites at various durations ofchronic heat-shock were harvested by washing thrice in Phos-phate Buffered Saline (PBS) at 3000 rpm for 10 min. Thepellet was resuspended in 2.5% buffered-glutaraldehyde and

washed thrice with PBS as before. Stubs were viewed underthe Scanning Electron Microscope (JEOL Z 601).

Transmission electron microscopy

Parasites at various durations of chronic heat-shock were har-vested from cultures and processed for transmission elec-tron microscopy by the standard protocol. The grids werefixed in the specimen holder and examined under Zeiss EM906.

Flow cytometry

The percentage of apoptotic nuclei was studied by flow cy-tometry following the method of Ormerod [15] with minormodifications. Briefly, suspension of single cells was pre-pared by washing the parasite in PBS and fixing in ice-cold 70% ethanol at 4 ◦C for 30 min. Cells were harvestedby centrifugation and the pellet was resuspended in PBScontaining DNAse-free RNAse and Propidium Iodide (PI).The contents were incubated at 37 ◦C for 30 min. The PIfluorescence of individual nuclei was measured by FAC-Scan flow cytometer (Becton and Dickinson, USA) usingCellQuest

R©software. A suspension of 10 mm diameter cal-

ibration beads was run and FSC peak was adjusted aroundchannel 600 of 1024 linear scale. The FSC threshold was pro-gressively increased until unwanted signals in 1–200 chan-nels of red fluorescence were less than 1%. Apoptotic cell nu-clei were easily distinguishable from the debris by high FSCvalue due to condensation of nuclear chromatin in apoptoticcells.

To study the involvement of Ca++ in the process of apopto-sis, the parasite culture was incubated in 1 mM final concen-tration of EGTA [(ethyleneglycol-bis (β-aminoethyl ether)]in addition to the normal medium and processed as in caseof flow cytometry.

Statistical analysis

The results were analysed by Student’s t-test.

Results

DNA fragmentation assay

DNA samples isolated by phenol-chloroform-isoamyl alco-hol method were run on a 0.8% agarose gel. No fragmenta-tion was observed in normal promastigotes while a markedlydamaged DNA showing typical DNA ladder was seen in

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Fig. 1. DNA fragmentation assay of chronic heat-shock treated Leishmaniadonovani. (Lane 1: Negative Control, Lanes 2–5: Parasite harvested at 24,48, 72 and 96 h of chronic heat-shock treatment, Lanes 6 and 7: Positivecontrol).

positive control and parasites subjected to chronic heat-shocktreatment of various durations. A fair amount of backgroundsmearing was observable in the gels (Fig. 1).

Scanning electron microscopy (SEM)

Analysis of positive control, negative control and parasitessubjected to chronic heat-shock treatment of 24, 48, 72 and

96 h. showed membrane blebbing characteristic of apoptosis(Fig. 2).

Transmission electron microscopy (TEM)

The transmission electron microscopy of the parasitesshowed initiation of condensation of the chromatin in theearly stages of chronic heat-shock treatment pointing towardsapoptosis. An increasing condensation was observed in thelater stages. The normal promastigotes had peripherally ar-ranged chromatin. The nuclear condensation proceeded to agreater extent in the later stages. Vesicular preapoptotic bod-ies were seen in the later stages followed by their extrusionand nuclear fragmentation (Fig. 3).

Flow cytometry

The percentage of apoptotic nuclei was significantly less innormal promastigotes when compared with promastigotesstarved of MEM for ten days (i.e., positive control) whichhave a high percentage of apoptotic cells. We also found thatthe percentage of apoptotic cells was maximum in parasitessubjected to 48 h of chronic heat-shock treatment and thenit showed a downward trend. Incubation with 1 mM EGTAshowed a lesser percentage of apoptotic cells as compared tothe cultures of same duration of chronic heat-shock treatedparasites (Fig. 4). The percentage apoptotic nuclei at variousdurations of heat-shock, with and without EGTA, was com-pared and all the comparisons were found to be significant atp < 0.0025.

Discussion

This study was aimed at determining whether a molecular in-sult precipitated by chronic heat-shock treatment of Leishma-nia donovani promastigotes with 37 ◦C temperature, whichis necessary for its differentiation, is involved in inducingapoptosis in the organism during this developmental phe-nomenon, and whether Ca++ has a role in it. We were alsointerested in finding out whether apoptosis in Leishmaniadonovani bears any similarity to metazoan apoptosis. Thetemperature of 37 ◦C is crucial due to two reasons. First, it isthe same temperature as is encountered by the parasite in thehuman host. Secondly, thymocytes exposed to a temperatureof 43 ◦C die by necrosis if maintained at that temperature, butshow apoptosis if the temperature is brought down to 37 ◦Csoon enough [16]. We demonstrated apoptosis in Leishma-nia donovani during its various stages of differentiation frompromastigotes to amastigotes. The programmed cell death inthis case resembled that in metazoans with regard to DNA

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Fig. 2. Scanning Electron Micrographs of Leishmania donovani: (A) Typical normal promastigote (B) Promastigote on media withdrawal undergoing stress-induced apoptosis (C) Parasite undergoing apoptosis during differentiation into amastigote; note that the parasite is rounding off while the flagellum still persists(D) Completely differentiated amastigote showing morphological features of apoptosis.

fragmentation, nuclear condensation, membrane blebbing,formation of preapoptotic bodies and involvement of Ca++

in the process was found. Apoptosis has been shown to occurin lower multicellulars like C. elegans [17] and Drosophila[18]. A paradigm shift in the views about this highly regu-lated cellular process was seen with the observation of thesame in unicellular organisms like Dictyostelium discoideum[7], E. coli [8], Peridinium gatunense [19], Tetrahymena [9,20], yeast [21–23], Blastocystis hominis [24] etc. Apoptosisin Trypanosoma [25–27] and Leishmania [10, 28–30] have fi-nally proved that unicellular organisms also exhibit apoptosis.

Cell damage is already shown to be associated with disreg-ulation of Ca++ homoeostasis [31]. Increase in intracelularCa++ concentration due to redistribution of internal Ca++

pools during heat-shock [32] and inhibition of Ca++ uptakeby the parasite’s mitochondria and endoplasmic reticulumwhich sensitizes the cell to heat-shock that is abrogated bythe use of Fura-2 suggest that intracellular Ca++ uptake poolsare a culprit in heat induced death of promastigotes [10].

Against this backdrop, we wanted to study programmedcell death in Leishmania donovani during its life cycle by giv-

ing an in vitro culture of this parasite the thermal conditionsit faces in its definitive host i.e., man. Apoptosis occurring inthe parasite resembled in some respects to the one describedin the multicellulars as to the molecular characterisation ofthe process. DNA fragmentation found in the parasites hasalready been documented in other cases of apoptosis too[9, 10, 18, 21]. Certain amount of smearing encountered ingels is not an extraordinary phenomenon as DNA smearingin absence of DNA ladders is known in L. amazonensis [10]yeast [22] and some metazoan cell types [33]. Membraneblebbing in the parasite, observable in scanning electron mi-croscopy (SEM), compared favourably with the similar mor-phology in apoptotic thymocytes [34].

Flowcytometrically, we found that normal promastigotesdo show a very less number of apoptotic cells which is ex-plainable since apoptotic cells have been found to be gen-erated after every cycle of cell division in cell cultures asin lymphocytes [35]. Promastigotes starved of MEM for tendays were found to have a high percentage of apoptotic nu-clei. Parasites subjected to chronic heat-shock showed anincrease in apoptotic nuclei till 48 h. and then a decline till

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Fig. 3. Transmission Electron Micrographs (A) Normal promastigote of Leishmania donovani (B) Early stage of promastigote differentiation showing com-paction of chromatin (C) and (D) Formation and extrusion of vesicular preapoptotic bodies containing condensed portions of chromatin. (E) Necrotic cellsshowing organellar destruction (F) Normal amastigotes. (fl-flagellum, n-nucleus, nc-nucleolus, nm-nuclear membrane, pap-preapoptotic bodies).

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Fig. 4. Percentage apoptotic nuclei in Leishmania donovani at various dura-tions of chronic heat-shock treatment. Results are expressed as mean ± S.D.of three experiments. The change in percentage apoptotic nuclei at differ-ent levels of heat shock was compared, with and without incubation with1 mM EGTA and found to be significant at all levels by Student’s t-test(p < 0.0025). (NC-Negative Control, PC-Positive Control).

96 h. The decrease after 48-hours. stage onwards was ratherpuzzling and we suspected calcium to play a role in this, asCa++ has been proposed to be an appropriate candidate for asecond messenger during the morphogenetic transformationof L. donovani and an estimation of intracellular free calciumconcentration at different intervals during the conversion ofpromastigotes to axenic amastigotes has revealed an increaseduring the conversion [36]. Ca++-modulated apoptosis hasalso been found to occur in Leishmania (Leishmania) ama-zonensis [10]. We, therefore, were interested in investigat-ing whether Ca++ ions play any role in apoptosis duringthe differentiation of promastigotes to axenic amastigotes.Incubation with 1 mM EGTA, a calcium chelator, lowered thepercentage of apoptotic cells as compared to heat-shockedstages incubated without EGTA of the same duration. Thedecrease in percentage apoptotic nuclei beyond 48 h. is prob-ably due to synergistic effects of the activation of Ca++ AT-Pase and EGTA. Prasad et al. [36] have already documentedthat there is a steep decrease in Ca++ concentration duringthe conversion of promastigotes to amastigotes, and we havefound that incubation with EGTA is decreasing the percent-age apoptotic nuclei. It is safe to deduce that concentration ofCa++ ions, which is initially increasing when the promastig-ote culture is kept under conditions conducive for differentia-tion, is also driving apoptosis in promastigotes differentiatinginto amastigotes. But since very high levels of Ca++ are toxic,

Ca++ATPase comes into action and lowers the Ca++ levelsto a threshold value. This decrease in Ca++ levels may be re-sponsible for the decrease in the percentage apoptotic nuclei.The discovery of Ca++-mediated cell death pathway in T.brucei brucei [25] and the occurrence of calcium-mediatedactivation of adenylate cyclase in Trypanosoma brucei [37]and potentially advocates involvement of Ca++-pathway inthe process. Coupled with it may be the role of Hsp70 whichis known to be released in response to the heat-shock as it isa potent inhibitor of apoptosis. Samali and Cotter [38] havereported that an increase in cellular levels of Hsp 70 eitherby a mild heat shock treatment or by stable transfection in-creases the resistance of U937 and Wehi-s cells to apoptoticcell death. Further investigations about the role of Hsps inLeishmania need to be carried out.

The ultrastructural details of the process analyzed by TEM(Transmission Electron Microscope) led us to believe beyonddoubt that the proceedings are emanating from nuclear direc-tives. Formation of preapoptotic bodies and preservation ofcell-organellar morphology is a pointer towards the fact thatliving parasites are undergoing this process and that thereis no involvement of post-mortem activation of Ca++ de-pendent endonucleases-a fact appreciated earlier by Moreiraet al. [10].

L. donovani populations are largely clonal [39] and thus itis logical for an altruistic mechanism like apoptosis to pro-mote and maintain genetic stability within the clonal popu-lation [29]. Similar findings have been reported in yeast [21]and mammalian immune system [40]. Since L. donovani andthe related parasites live in insect gut or macrophages, it be-comes highly imperative for the parasite to strictly regulatethe cell numbers or else cause the death of insect vector or themacrophages. Further, competition among the individuals ofparasites also leads to this altruistic phenomenon.

In protozoan populations PCD only occurs in forms per-forming, or unable to perform, a specific function [10]. Rapidsuicide in a damaged cell in case of a unicellular organismspares nutrient resources for the surrounding cells, usuallyits own clone. Therefore its suicide will confer an advantageto its clonal relatives, and thus ensure the continuity of thegenome of the damaged individual. The host-parasite inter-actions may have exerted selective pressures on the cell deathphenotype of kinetoplastid parasites resulting in the more re-cent emergence of apoptotic machinery through a process ofconvergent evolution [29].

Since parasites also show apoptosis in the gut of the vec-tor as in the case of Plasmodium berghei [41], PCD pathwaycould be involved in vivo in regulating the parasite popu-lation, triggered by limited resources or host growth factorsand cytokines. Welburn et al. [25] have cloned and sequenced22 differentially displayed genes which represent the first en-dogenous genes to be identified and implicated in cell death intrypanosomatids. The recruitment of cysteine proteases and

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mitochondria to the cell death machinery has been reportedby Arnoult et al. [29] in L. major, and PhiPhiLux (PPL)-cleavage activity has been documented by Lee et al. [30] inL. donovani.

Apoptosis in intracellular protozoan parasites seems tohave arisen as a two pronged fork in the welfare of the para-site. One, it is undergoing apoptosis so as to remain in just thesufficient numbers that does not overburden the resources ofthe invaded cell to such an extent that it kills itself along withthe parasite. Two, the parasite as a genotypic command, soto say, is eliminating the weaker clonal relatives so that onlythe fittest individuals survive to reproduce and mount furtherbenign attacks on related host cells so as to remain propagat-ing. That PCD is not a process of in vitro culture practiceswas proved by Lee et al. [30] when they found apoptosisoccurring in freshly isolated parasite as well.

The studies, which prove that a parasite does show apop-tosis either in vivo or in vitro, answer but one part of a bigquestion. A more important question that follows is how thesurviving cells avoid apoptosis. There are a lot of apoptoticagents in vivo, namely lectins, dietary components, oxida-tive stresses, immunosurveillance, heat-shock and the like.Despite such a milieu of apoptosis inducers, the infectiveparasite survives and proliferates successfully. The mecha-nism of this evasion is a real challenge for the molecularbiologists. Any drug-targetting has to take this phenomenoninto account to mount a successful attack on this disease.It is thus very important to decipher the biochemical path-ways leading to cell death in single celled organisms and thegenes regulating this process. Many groups around the worldare pursuing this line of action. It is important to determinewhether these organisms share some or all of the effectorsand regulators common to multicellular PCD or have evolvedtheir own divergent pathways. In case the latter is true, wehave an exciting prospect of utilising the process of cell sui-cide in single celled organisms as a therapeutic strategy toselectively activate PCD in parasites.

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