Transbilayer translocation of membrane phosphatidylserine and its role in macrophage invasion in Leishmania promastigotes

Molecular & Biochemical Parasitology 128 (2003) 1–9

Transbilayer translocation of membranephosphatidylserine and its role in macrophage

invasion inLeishmania promastigotes�

Amit Tripathi, C.M. Gupta∗Division of Molecular and Structural Biology, Central Drug Research Institute, Lucknow 226 001, India

Received 5 November 2002; received in revised form 22 January 2003; accepted 30 January 2003

Abstract

Infectivity of Leishmania promastigotes has been shown to be growth cycle-dependent and restricted to the stationary phase. Byusing annexin V–FITC binding and procoagulant activity measurement assays, we show here that the promastigotes in the stationaryphase contain significantly higher amounts of phosphatidylserine (PS) on their surface as compared to the log phase promastigotes. Wealso demonstrate that the infectivity of the promastigotes is determined by the presence of PS on their surface. In addition, by usingNBD-labelled phospholipids, we show that the promastigote plasma membrane contains ATP-dependent out-to-in and ATP-independentin-to-out PS translocases which regulate the PS localisation in two-halves of the membrane bilayer, and that the greater amounts of externalPS observed in the stationary phase promastigotes is perhaps due to the slower ATP-dependent out-to-in PS movements in these cells, ascompared to the log phase promastigotes.© 2003 Elsevier Science B.V. All rights reserved.

Keywords: Leishmania promastigotes; Infectivity; Phosphatidylserine; Plasma membrane; Transbilayer movements

1. Introduction

Leishmania parasite is a protozoan organism, whichcauses the dreaded disease ‘kala-azar’. The promastigoteform of this parasite upon transfer by the infected vectorto the vertebrate host invades its mononuclear macrophageswherein its flagellated, motile and spindle-shape body istransformed into a nonmotile and spherically-shaped form,called amastigote[1]. The invasion is a receptor-mediatedprocess and may involve several macrophage receptors,namely, the mannosyl fucosyl receptor, CR3 receptor, re-

Abbreviations: PC, phosphatidylcholine; PE, phosphatidylethanol-amine; PS, phosphatidylserine; NBD,N-(7-nitro-2,1,3-benzoxadiazol-4-yl)amino; BSA, bovine serum albumin; C6-NBD-, 1-palmitoyl-2-[6-N-(7-nitro-2,1,3-benzoxadiazol-4-yl)amino] caproyl; C12-NBD-, 1-palmi-toyl-2-[12-N-(7-nitro-2,1,3-benzoxadiazol-4-yl)amino] dodecanoyl; RVV,Russel’s viper venom; DMEM, Dulbecco’s Modified Eagle Medium

� This is a communication no. 6085 from the Central Drug ResearchInstitute, Lucknow (India).

∗ Corresponding author. Tel.:+91-522-2223286/2210932;fax: +91-522-2223405/2223938.

E-mail addresses: [email protected], [email protected](C.M. Gupta).

ceptors for glycosylated molecules, and fibronectin receptor[2]. Two surface molecules of promastigotes, namely gp63,a glycoprotein, and LPG, a lipophosphoglycan, have beensuggested as the ligands for attachment of these macrophagereceptors[3]. However, recent studies[4–6] have revealedthat phosphatidylinositol-anchored surface proteins, likegp63 and LPG, may not be essential for entry and survivalof Leishmania promastigotes in the host macrophages.

Phosphatidylserine (PS) is one of the phospholipid con-stituents of the promastigote plasma membrane[1]. Specificreceptors for this phospholipid exist on the mononuclearmacrophage surface[7]. Earlier studies have shown that themacrophages scavenge the aged cells from the circulationthrough these receptors[7]. Furthermore, several investiga-tors have reported that exposure of PS on the surface of theaged or apoptotic cells is the prerequisite for their uptakeby the macrophages[8–15]. SinceLeishmania amastigoteshave recently been shown to invade the fresh macrophagesby exposing their PS on the cell surface[16], we consid-ered it of interest to study the mechanisms that regulate thetransbilayer movements of PS in theLeishmania promastig-otes and also the effect of PS exposure on the promastigoteinfectivity.

0166-6851/03/$ – see front matter © 2003 Elsevier Science B.V. All rights reserved.doi:10.1016/S0166-6851(03)00024-0

2 A. Tripathi, C.M. Gupta / Molecular & Biochemical Parasitology 128 (2003) 1–9

2. Materials and methods

2.1. Materials

Sodium azide, sodium orthovanadate, bovine serum albu-min (BSA) fatty acid-free, 2-deoxyglucose, Russel’s vipervenom (RVV), annexin V, annexin V–FITC conjugate, Tri-ton X-100 and ATP estimation kit were purchased fromSigma Chemical Company (St. Louis, USA). Pre-coatedaluminium-backed silica gel G-60 F254 TLC plates werebought from E. Merck. 1-Palmitoyl-2-[6-N-(7-nitro-2,1,3-benzoxadiazol-4-yl) amino] caproyl-sn-glycero-3-phospho-choline (C6-NBD-PC), 1-palmitoyl-2-[6-N-(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]caproyl-sn-glycero-3-phospho-ethanolamine (C6-NBD-PE), 1-palmitoyl-2-[6-N-(7-nitro-2,1,3-benzoxadiazol-4-yl) amino] caproyl-sn-glycero-3-ph-osphoserine (C6-NBD-PS), 1-palmitoyl-2-[12-N-(7-nitro-2,1,3-benzoxadiazol-4-yl) amino] dodecanoyl-sn-glycero-3-phosphocholine (C12-NBD-PC) and 1-palmitoyl-2-[12-N-(7-nitro-2,1,3-benzoxadiazol-4-yl) amino] dodeca noyl-sn-gly-cero-3-phosphoserine (C12-NBD-PS) were from Avanti Po-lar Lipids. Dulbecco’s Modified Eagle Medium (DMEM)was bought from Gibco-BRL. Purity of the lipids was ex-amined by thin-layer chromatography and were found to be>95% pure.

2.2. Parasite culture

Promastigotes ofLeishmania tropica (MHOM/IN/1978/UR6) were cultured and maintained in Brain heart Infusionagar as described earlier[17].

2.3. Promastigote binding with annexin V–FITC conjugate

Promastigotes from mid-log phase (harvested after 2 daysof growth in L-15 medium[18]) as well as early station-ary phase (harvested after 7 days of growth in L-15) werecentrifuged at 2500× g for 10 min at 4◦C. The cells werewashed with annexin V-binding buffer (100 mM HEPES,150 mM NaCl, 5 mM KCl, 5 mM CaCl2 and 1 mM MgCl2;pH 7.4) under identical conditions. The washed cells wereresuspended in the same buffer and incubated with annexinV–FITC (1�g annexin V/4× 106 cells) at 4◦C for 25 min.To it was added propidium iodide (10�g in 10�l distilledwater) and the mixture incubated for 5 min at 4◦C. The cellswere washed at 2500× g for 1 min at 4◦C and resuspendedto a density of 4×106 cells/ml. Data were collected in a BDFACS calibur and analysed by Cell quest. A total of 20,000events were harvested from each sample. The control cellswere incubated in annexin V-binding buffer alone withoutannexin V–FITC, under identical conditions.

2.4. RVV assay

Promastigotes from mid-log phase or early stationaryphase were washed with 150 mM phosphate-buffered saline

(pH 7.2) and the washed cells were resuspended in thesame buffer. For analysing the effect of these promastigoteson the RVV-induced coagulation, 100�l of citrated humanblood plasma, prepared from fresh human blood, and 10 ngof RVV in 10�l distilled water, after 2 min incubationat 37◦C, were added to 100�l promastigote suspensionand 100�l of 25 mM CaCl2. The kinetics of coagulationwas monitored by measuring absorbance at 600 nm in aBeckman DU-640 UV/visible spectrophotometer.

2.5. Effect of annexin V binding onpromastigote infectivity

Mid-log phase and early stationary phase promastig-otes were separately washed with the annexin V-bindingbuffer and resuspended in the same buffer at a cell densityof about 106 cells/ml. The cell suspensions were incu-bated with or without annexin V (1�g/4× 106 promastig-otes) at 26◦C for 30 min. Macrophages (J774 A.1 cellline) grown on coverslips inside 6-well plate in DMEMat 37◦C for 12 h were challenged with these promastig-otes at a macrophage-to-promastigote ratio of 1:10. Theincubation continued for another 2 h at 37◦C. After wash-ing with DMEM and resuspension in the same media,the infected macrophages were further grown for 24 h at37◦C. The infection was monitored by washing the cellswith phosphate-buffered saline, fixing the washed cells inmethanol, and then staining the fixed cells with Giemsafor 30 min at 26◦C. After washing with distilled water anddrying at room temperature, the coverslips were mountedon slides containing Mowiol (Calbiochem) and then ob-served under Nikon microscope at 100× magnificationusing paraffin oil. The infectivity index was calculated bymultiplying the percentage of infected macrophages by theaverage number of amastigotes/infected cell.

2.6. Out-to-in movements of NBD-labelled phospholipids

Mid-log phase promastigotes (7.2 × 109) were washedwith 10 ml of HEPES-buffered saline (20 mM HEPES,137 mM NaCl and 3 mM KCl; pH 7.4) and resuspendedin 10 ml of the same buffer. After incubation at 0◦C for10 min, 25�g of NBD-labelled phospholipid in ethanol(1�g/�l) was added to the cell suspension under rapidmixing. It was incubated at 0◦C for 40 min. The cellswere collected and washed, and finally resuspended in 3 mlof HEPES-buffered saline. The cell suspension was incu-bated at 26◦C. Measured aliquots (0.5 ml) were withdrawnat different time intervals from this cell suspension, andadded to 0.5 ml of BSA solution at 0◦C. Three percentBSA solution with 5 min incubation and 10% BSA solu-tion with 10 min incubation were used for back-exchangingthe membrane-associated C6-NBD and C12-NBD phospho-lipids, respectively. The cell suspension was centrifugedat 3000× g for 3 min and the supernatant was carefullycollected. The pellet was solubilised in 1 ml of 1% Triton

A. Tripathi, C.M. Gupta / Molecular & Biochemical Parasitology 128 (2003) 1–9 3

X-100 by vigorous vortexing. The NBD fluorescence wasmeasured both in the supernatant and Triton X-100 ex-tract. The fluorescence measurements were carried outon Perkin-Elmer LS-5B Luminescence spectrometer at anemission wavelength of 524 nm, slit width 10 nm (excitation470 nm, slit width 20 nm) at 25◦C.

2.7. In-to-out movements of NBD-labelledphospholipids

In-to-out movements of NBD-labelled phospholipidswere measured essentially in the same manner as describedabove for the out-to-in movements, except that the in-cubations of the promastigotes after their labelling withNBD-labelled phospholipids were carried out in the pres-ence of 6 ml of 1.5% BSA at 0◦C rather than at 26◦C inthe absence of BSA. Aliquots of 1.0 ml were withdrawnat different time intervals and centrifuged at 3000× g for3 min. The supernatants were carefully collected and thepellet was solubilised by vortexing it in 1.0 ml of 1% TritonX-100 for 3 min. NBD fluorescence was measured in boththe supernatant and Triton X-100 extract.

2.8. Treatment of mid-log phase promastigotes withsodium orthovanadate

Cells (about 2.4 × 109) were treated with sodium ortho-vanadate (20 mM) in HEPES-buffered saline (2.0 ml) for20 min at 0◦C, as described[19]. After completing the treat-ment, the cells were washed with HEPES-buffered salinecontaining 20 mM sodium orthovanadate and resuspendedin the same buffer for further experiments.

2.9. ATP-depletion

Mid-log phase promastigotes (about 2.4× 109 cells) sus-pended in HEPES-buffered saline (4.0 ml) were treated with2-deoxyglucose (50 mM) and sodium azide (20 mM) for 2 hunder ice-cold condition. The treated cells were washed withthe same buffer two times. The control cells were incubatedfor 2 h under identical conditions in HEPES-buffered salineonly.

2.10. ATP estimation

An aliquot of ATP-depleted cells was treated with 10 vol.of distilled water containing 0.1% digitonin under vigor-ous vortexing at 0◦C for 2 min to lyse the cells. The lysatewas centrifuged at 13,000× g for 3 min and the supernatantcarefully collected. After diluting it by 10-fold with the as-say mix buffer, a measured aliquot was withdrawn and to itwere added luciferin and luciferase for ATP estimation. Lu-minous intensity was recorded on a Bio-Orbit Luminome-ter. At least 90% of the total cellular ATP could be depletedunder our experimental conditions.

2.11. In-to-out translocation of C6-NBD phospholipidsin presence of C12-NBD-phospholipids

To the washed cells (7.2 × 109) suspended in 10 ml ofHEPES-buffered saline was added 25�g of C6-NBD-phos-pholipid in 25�l of ethanol at 0◦C. The mixture was incu-bated for 30 min with intermittent mixing. The cells werewashed and then divided into two equal portions. To one ofthese portions was added 50�g of C12-NBD-phospholipidin 50�l of ethanol. The remaining portion was treated with50�l of ethanol only. Both of these portions were incubatedat 0◦C for 15 min and washed two times at 0◦C. The washedcells were resuspended in 6.0 ml of 1.5% BSA and incu-bated at 0◦C. Measured aliquots (1.0 ml) at different timeintervals were withdrawn and the supernatant carefully col-lected after pelleting the cells at 3000× g for 3 min. Thecell pellets were solubilised in 1% Triton X-100 (1.0 ml),and NBD-fluorescence recorded in both the supernatant andthe Triton X-100 extract.

2.12. Out-to-in translocation of C6-NBD-phospholipidsin presence of C12-NBD-phospholipids

The procedure used here was similar to that used abovefor the in-to-out translocation of C6-NBD-phospholipids inpresence of C12-NBD-phospholipids with the exception thatincubation was carried out at 26◦C in absence of BSA.Aliquots of 0.5 ml were withdrawn from 3 ml of final cellsuspension in HEPES-buffered saline. These aliquots wereadded to 0.5 ml of 3% BSA solution at 0◦C. After 5 minincubation at the same temperature, the mixture was cen-trifuged at 3000× g for 3 min. The supernatants were care-fully collected and the pellets solubilised in 1.0% TritonX-100 (1.0 ml). NBD fluorescence was measured in both thesupernatants and Triton X-100 extracts.

2.13. Cell viability

Viability of Leishmania promastigotes was examined byusing the Trypan Blue exclusion assay[20]. Not more than5% cells were rendered nonviable, as compared to the con-trol cells, under any of the above described experimentalconditions.

3. Results

3.1. Detection of PS on the promastigote surface

PS is a procoagulant membrane phospholipid, which innormal mammalian cells is located exclusively in the in-ner surface of the plasma membrane bilayer[21]. Exposureof this phospholipid on the cell surface has been measuredby a number of techniques including flow cytometry usingFITC-labelled annexin V as a ligand[22,23] and measure-ment of the procoagulant activity of cells in the RVV assay


Fig. 1. (A) Flow cytomerty of annexin V–FITC bound promastigotes.Curve (a): log phase cells without any treatment, curve (b): annexinV–FITC treated log phase cells, and curve (c): annexin V–FITC treatedstationary phase cells. (B) Procoagulant activity of phosphatidylserine inpromastigotes in presence of Russel’s viper venom. Key: (�) log phasecells, and (�) stationary phase cells.

[24]. Fig. 1shows that the binding of annexin V–FITC con-jugate with the stationary phase promastigotes was higherthan that with the log phase cells, indicating the presenceof PS on the surface of the stationary phase promastigotes.This is further confirmed by our finding that unlike the logphase promastigotes, the stationary phase cells exhibited sig-nificant procoagulant activity as judged from the decreasedclotting time in the RVV assay with an increase in the num-ber of the stationary phase promastigotes (Fig. 1B).

3.2. Effect of masking of the cell surface PS on thepromastigote infectivity

PS on the promastigote surface was masked by separatelyincubating the log and stationary phase promastigotes withannexin V. The effect of PS masking on the infectivity wasanalysed by infecting J774 A.1 macrophages with annexinV-bound as well as unbound promastigotes and then cal-culating the infectivity index, as given inSection 2. Fig. 2shows that the infectivity index of the stationary phase pro-mastigotes significantly decreased (P < 0.05) by preincu-bating the cells with annexin V. In contrast, no change in the

Fig. 2. Effect of annexin V binding on the promastigote infectivity. (A)Log phase cells and (B) stationary phase cells. Percentage infectivityindex = (infectivity index of annexin V treated cells)/(infectivity indexof annexin V untreated cells)× 100. Infectivity index was calculatedas given inSection 2. Values shown are means of three independentexperiments±S.D. Statistical significance (P-value) was calculated byusing Student’st-test.

infectivity index was observed with the log phase promastig-otes, under identical conditions. This is consistent with theearlier report[16].

3.3. Incorporation and redistribution of exogenousphospholipids in plasma membranes of Leishmaniapromastigotes

Diacylglycerophospholipids containing NBD-group at thetail end of their C-2 acyl chain have been widely used tostudy the transbilayer phospholipid movements in a vari-ety of cells[25–28]. We employed C6-NBD and C12-NBDanalogs of PC, PE and PS as the fluorescent probes to anal-yse the transbilayer movements of these lipids in plasmamembranes of the log phaseLeishmania promastigotes. TheNBD-lipids were incorporated in the membranes by incubat-ing the cells with these lipids for 40 min at 0◦C. Appropri-ate controls using the vesicles that were formed of the lipidsextracted from theLeishmania promastigotes were taken.The rates of the NBD-lipids uptake by the cells were simi-lar to those observed with the vesicles (data not shown). Toanalyse whether the exogenously incorporated lipids werelocated on the outer surface of the promastigotes or weredistributed also to the internal compartments including theinner surface of the plasma membrane bilayer, we subjectedthe NBD-lipids incorporated cells to the BSA back-exchange[29] as described inSection 2. As may be seen inFig. 3,an incubation of the C6-NBD-lipid-labelled promastigotesfor 20 min at 0◦C with 1.5% BSA was sufficient to almostcompletely back-exchange the cell-associated NBD fluores-cence. However, no back-exchange of the C12-NBD-lipidsoccurred from the labelled cells in identical conditions. Ahigher concentration (5%) of BSA was required to affectthe back-exchange of these lipids from the cells (data notshown).

These results clearly indicated that the exogenously sup-plied NBD-lipids could readily be incorporated and redis-tributed in Leishmania promastigotes. This incorporation


Fig. 3. Incorporation and redistribution of C6-NBD-lipids in surface mem-branes ofLeishmania promastigotes at 0◦C. (A) C6-NBD-PC incorpo-rated cells prior to BSA back-exchange, and (B) the same cells after20 min of BSA back-exchange. Please note that 20 min incubation with1.5% BSA, removed most of the cell-associated NBD fluorescence.

and redistribution could not be attributed solely to the en-docytosis of the NBD-lipids followed by their redistributionin the plasma membrane, because only a small area of theLeishmania promastigote plasma membrane, the flagellarpocket membrane, is active in endocytosis and exocytosis[30]. To further rule out the above possibility, we examinedthe incorporation and redistribution of the C6-NBD-lipids inpromastigotes that were subjected to ATP-depletion. CellularATP was depleted (>90%) as described inSection 2, and thedepleted cells incubated with various NBD-lipids for 40 minat 0◦C. The incorporation and redistribution remained un-altered even under these conditions (data not shown). Sinceendocytosis has been reported to be dependent of the cellu-lar ATP status[31], it was inferred that the incorporation andredistribution of the NBD-lipids in the promastigote plasmamembrane can not be exclusively attributed to endocytosis.

3.3.1. Out-to-in movements of exogenous phospholipidsin plasma membranes of Leishmania promastigotes

To examine the presence of any energy-dependent phos-pholipid translocation system(s) in the promastigote plasma

Fig. 4. Time-dependent inward movements of C6-NBD-PC (�),C6-NBD-PE (�), and C6-NBD-PS (�) in Leishmania promastigoteplasma membrane at 26◦C. Values shown are means of two independentexperiments with a maximum variation of about 2%.

membrane, we raised the temperature of the cells, after in-corporation and redistribution of the various C6-NBD-lipidsin their plasma membranes, from 0 to 26◦C, which wasthe optimum temperature for growth and propagation ofthe Leishmania promastigotes. Measured aliquots with-drawn at different time periods from the cell suspension at26◦C were incubated with 1.5% BSA for 5 min at 0◦C forback-exchanging the external NBD-lipids. Most of the cells(>95%) remained viable, as judged by the Trypan Blueexclusion assay[20], at 26◦C under our experimental con-ditions. Also the back-exchanged NBD-lipids were found toremain largely intact at 26◦C as ascertained by extractingthese lipids from the BSA solution using Bligh–Dyer ex-traction procedure[32] followed by analysis of the extractedlipids by TLC.

Fig. 4 shows that the accessibility of the externalNBD-lipids to BSA in the promastigote surface decreasedwith time at 26◦C. Whereas this decrease was quite signif-icant (>25%) at 60 min in the case of the C6-NBD-PS andC6-NBD-PC-labelled cells, only a small (<10%) decreasewas observed with the C6-NBD-PE-labelled cells. To fur-ther confirm whether the observed decrease was due toenergy-dependent out-to-in translocation of the NBD-lipids,we examined the accessibility of these lipids to 1.5% BSAin cells that were depleted of ATP (>90%) or treated withorthovanadate[19], as given inSection 2. The extent ofaccessibility of C6-NBD-PC and C6-NBD-PS to BSA wasconsiderably greater in the ATP-depleted or orthovanadatetreated cells, as compared to the cells that received notreatment (Fig. 5).

These results indicated that the promastigote plasmamembrane contained an ATP-dependent inward pump,which rapidly translocated PC and PS from the outer to theinner monolayer. However, it was not clear whether boththe PC and PS were translocated by the same translocator


Fig. 5. Inhibition by cellular ATP-depletion and sodium orthovanadate ofthe inward movements of C6-NBD-PC and C6-NBD-PS in Leishmaniapromastigote plasma membrane at 26◦C. Key: (�) C6-NBD-PC in normalcells, (�) C6-NBD-PS in normal cells, (�) C6-NBD-PC in ATP-depletedcells, (×) C6-NBD-PS in ATP-depleted cells, (�) C6-NBD-PC in vanadatetreated cells, and (�) C6-NBD-PS in vanadate treated cells. Values shownare means of two independent experiments with a maximum variation of2.5%.

or required two separate translocation systems specific tothese lipids. To discriminate between these two possibilities,we competed the inward movements of C6-NBD-PS withC12-NBD-PC and C6-NBD-PC with C12-NBD-PS. As onlythe C6-NBD-lipids and not the C12-NBD-lipids could beback-exchanged by incubating with1.5% BSA for 5 min at0◦C, this strategy enabled us to analyse the movements ofone phospholipid species in presence of another. Results ofthese experiments given inFig. 6 revealed that the inwardmovement of C6-NBD-PS was reduced by the presenceof C12-NBD-PC, presumably due to high affinity of thetranslocator for the longer fatty acyl chain-containing phos-pholipids[25,26]. In contrast to C12-NBD-PC, the presenceof C12-NBD-PS exerted a remarkable influence on theinward movements of C6-NBD-PC. Instead of its transloca-tion from the outer to the inner monolayer, this lipid in thepresence of C12-NBD-PS appeared to migrate from innerto the outer monolayer. This finding may be attributed tothe high preference of the translocator to C12-NBD-lipidsover the C6-NBD-lipids [25,26] as well as to the intrinsicpreference of PS to localize in the inner monolayer[21].

3.3.2. In-to-out movements of exogenous phospholipidsin plasma membranes of Leishmania promastigotes

The in-to-out phospholipid movements were induced byincubating the NBD-lipids-incorporated cells with BSA, asreported earlier[33]. The cells were incubated with 1.5%BSA at 0◦C for variable time periods, and then the NBDfluorescence measured in both the Triton X-100 extract andthe cell pellet.Fig. 7 shows that the rate of the outward mi-gration of the PC was greater than that observed with the PEand PS. These outward movements of NBD-phospholipidswere not inhibited by depleting the cells of ATP (>90%)or by orthovanadate treatment (data shown), suggestingthat some ATP-independent mechanism may be involvedin these movements. To further examine the specificity of

Fig. 6. Inward movements of C6-NBD-PC and C6-NBD-PS in Leish-mania promastigote plasma membrane at 26◦C in presence ofC12-NBD-phospholipids. (A) C6-NBD-PS movement in absence (�) andpresence (�) of C12-NBD-PC; (B) C6-NBD-PC movement in absence(�) and presence (�) of C12-NBD-PS. Values shown are means of twoindependent experiments with a maximum variation of about 4%.

this in-to-out phospholipid translocator, we competed theoutward movements of C6-NBD-PC with C12-NBD-PSand of C6-NBD-PS with C12-NBD-PC. Fig. 8 shows thatthe in-to-out movements of C6-NBD-PS were inhibitedby the presence of C12-NBD-PC, whereas no effect ofC12-NBD-PS was observed on the outward translocation ofC6-NBD-PC.

4. Discussion

Earlier studies have shown that infectivity ofLeishma-nia promastigotes is growth cycle-dependent and restrictedto the stationary phase[34]. The present study demon-strates that the higher infectivity of the stationary phasepromastigotes, as compared to the log phase promastigotes,is primarily due to exposure of the procoagulant membranephospholipid, PS, on their surface, which may help pro-mastigote internalisation by the macrophages through thePS receptor[7]. This is consistent with a recent study whichshowed that exposure of PS on the surface ofLeishmania


Fig. 7. Time-dependent outward movements of various NBD-labelledphospholipids inLeishmania promastigote plasma membrane at 0◦C.C6-NBD-phospholipids were incorporated and redistributed in promastig-otes at 0◦C as given inSection 2. The cells were divided into two equalportions. While one portion was incubated at 0◦C in presence of 1.5%BSA, the second portion was incubated in buffer only, under identicalconditions. Open symbols, incubations in buffer only; solid symbols, in-cubations in presence of BSA. Inverted triangles, C6-NBD-PC; circles,C6-NBD-PE; squares, C6-NBD-PS. Values shown are means of two in-dependent experiments with a maximum variation of about 4%.

amastigotes is essential for their internalisation and subse-quent survival within the host macrophages[16].

PS distribution in plasma membranes of a variety of cellsis known to be regulated by an energy-dependent out-to-inaminophospholipid pump[25–28]. Since log phase pro-mastigotes have been shown to have virtually no PS in theirplasma membrane, it was of interest to study the mecha-nisms that regulate the PS distribution in two-halves of thepromastigote plasma membrane bilayer. Results presentedhere clearly demonstrate that as in other cells, the log phasepromastigotes contain an energy-dependent out-to-in andenergy-independent in-to-out phospholipid translocationsystems which perhaps determine the transbilayer phos-pholipid distributions in the parasite surface membrane.To examine whether the PS externalisation in the station-ary phase promastigotes is due to altered rates of the PStransbilayer movements in these cells, we analysed the PSmovements in both the mid-log phase and early stationaryphase promastigotes under identical conditions. Whereasthe ATP-dependent out-to-in PS movement in the stationaryphase appeared to be little slower (P = 0.05 at 60 min timepoint) than that observed with the log phase promastigotes(Fig. 9), no differences in the in-to-out PS movements wereseen under our experimental conditions (data not shown).From these findings, we infer that the energy-dependentout-to-in PS translocation activity in theLeishmania pro-mastigotes is reduced when these cells enter their stationaryphase. As in-to-out PS movements essentially remain unal-tered in the stationary phase promastigotes compared to thelog phase promastigotes, it is suggested that the observed

Fig. 8. Outward movements of C6-NBD-PC and C6-NBD-PS inLeishmania promastigote plasma membrane at 0◦C in presence ofC12-NBD-phospholipids. (A) C6-NBD-PC movement in absence (�) andpresence (�) of C12-NBD-PS; and (B) C6-NBD-PS movement in absence(�) and presence (�) of C12-NBD-PC. Values shown are means of twoindependent experiments with a maximum variation of about 3%.

reduced activity of the out-to-in PS translocase is perhapsresponsible for the exposure of PS on surface of the sta-tionary phase promastigotes. It is, however, not yet clear asto how this parasite inactivates the out-to-in phospholipidtranslocation system upon entering the stationary phase.

Fig. 9. Out-to-in movements of C6-NBD-PS in plasma membranes ofmid-log phase (�) and early stationary phase (�) promastigotes. Valuesshown are means of three independent experiments±S.D.


PE is the major phospholipid constituent in surface mem-branes of theLeishmania parasite[1]. Since this lipid istranslocated across the promastigote surface membrane pri-marily through some energy-independent mechanisms, wespeculated that PE in this membrane could be symmetri-cally distributed in two-halves of the membrane bilayer. Toexamine the validity of this speculation, we labelled the pro-mastigotes with fluorescamine, isolated the surface mem-branes from the labelled cells, followed by estimation of theamounts of labelled and unlabelled PE, using published pro-cedures[35,36]. Results of these studies (data not shown)revealed that about 45% of the total surface membrane PEis localised in the outer monolayer.

This study shows that unlike the other plasma membranes[25–28], only PS and not PE is asymmetrically distributedin the plasma membranes of theLeishmania promastigotes.This PS asymmetry in the parasite cells appears to play acrucial role in controlling the promastigote infectivity, asexternalisation of PS in the stationary phase promastigotesrenders these parasites infective to the host macrophages.Since localisation of PS in the two monolayers of the pro-mastigote plasma membrane bilayer is controlled by trans-bilayer PS movements across the membrane, we concludethatLeishmania promastigotes may regulate their infectivityby regulating the ATP-dependent out-to-in PS translocaseactivity.

Acknowledgements

We thank Dr. Anu Puri for supplying us some of the flu-orescently labelled phospholipid samples. One of us (AT) isgrateful to the Council of Scientific and Industrial Research,New Delhi (India) for award of the research fellowship.

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Transbilayer translocation of membrane phosphatidylserine and its role in macrophage invasion in Leishmania promastigotes