FEBS Letters 583 (2009) 3209–3214

journal homepage: www.FEBSLetters .org

Membrane binding requirements for the cytolytic activityof Leishmania amazonensis leishporin

Thiago Castro-Gomes a, Flávia Regina Almeida-Campos a,1, Carlos Eduardo Calzavara-Silva a,2,Rosiane Aparecida da Silva a,3, Frédéric Frézard b, Maria Fátima Horta a,*

a Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, 31270-901 Belo Horizonte, MG, Brazilb Departamento de Fisiologia e Biofísica, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, 31270-901 Belo Horizonte, MG, Brazil

a r t i c l e i n f o

Article history:Received 23 July 2009Revised 2 September 2009Accepted 3 September 2009Available online 6 September 2009

Edited by Michael Ibba

Keywords:LeishporinCytolysinPore-forming proteinLipid-binding proteinCholesterolLiposomeLeishmania

0014-5793/$36.00 � 2009 Federation of European Biodoi:10.1016/j.febslet.2009.09.005

Abbreviations: CHAPS, 3[(3-cholamidoprohydroxy-propanesulfonate; DPPC, dipalmitoylphospeylphosphatidylcholine; FBS, fetal bovine serum; GPI,H50, inverse of the dilution that caused 50% of hemolysmExt, promastigotes membrane detergent-solubleprotein(s); PBS, phosphate-buffered saline

* Corresponding author. Address: Av. Antônio CaHorizonte, MG, Brazil. Fax: +55 31 3409 2614.

E-mail address: phorta@icb.ufmg.br (M.F. Horta).1 Present address: Depto. Ciências Fisiológicas, Ins

Universidade Federal do Amazonas, Av. Gal. RodrigoBairro Coroado I, Manaus, AM 69077-000, Brazil.

2 Present address: Departamento de Imunologia, Laboe Molecular, Centro de Pesquisas René Rachou, Fundaçde Lima, 1715 – Barro Preto, Belo Horizonte 30190-00

3 Present address: Laboratório de Parasitologia CePesquisas René Rachou, Fundação Oswaldo Cruz, Av. APreto, Belo Horizonte 30190-002, MG, Brazil.

a b s t r a c t

To lyse cells, some pore-forming proteins need to bind to receptors on their targets. Studying thebinding requirements of Leishmania amazonensis leishporin, we have shown that protease-treatederythrocytes are as sensitive to leishporin-mediated lysis as untreated cells, indicating that proteinreceptors are dispensable. Similarly, carbohydrate receptors do not seem to be needed, since severalsugars do not inhibit leishporin-mediated hemolysis. Conversely, dipalmitoylphosphatidylcholine(DPPC), but not cholesterol, completely inhibits leishporin-mediated lysis. DPPC liposomes, withor without cholesterol, are lysed by leishporin and remove its lytic activity. Our results demonstratethat leishporin is a cholesterol-independent cytolysin that binds directly to phospholipids.� 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction phagolysosomes, they change into amastigotes, proliferate and,

Protozoa of the genus Leishmania comprise many species thatcause leishmaniasis, an endemic disease in several continents.The parasite is spread to mammalian hosts through the bite of aPhlebotomus or Lutzomia sandfly containing the infective metacy-clic promastigotes, which end up infecting macrophages. Inside

chemical Societies. Published by E

pyl)dimethyl-ammonium]-2-hatidylcholine; DOPC, diol-glycosylphosphatidylinositol;is; HuE, human erythrocytes;extract; PFP, pore-forming

rlos, 6627, 31270-901 Belo

tituto de Ciências Biológicas,Octávio Jordão Ramos, 3000,

ratório de Imunologia Celularão Oswaldo Cruz, Av. Augusto2, MG, Brazil.lular e Molecular, Centro deugusto de Lima, 1715 – Barro

ultimately, exit the host cell, to infect healthy ones. The parasite’slife cycle is completed when, during a blood meal, the sandfly in-gests amastigotes, which evolve again to metacyclic promastigotes.

A crucial but poorly understood step in the pathogenesis ofintracellular microorganisms is their exit from host cells. Its conse-quences are patent both in the amplification of the infection, thusexacerbating the disease and the rate of transmission, and in theoutcome of the immune response. Over the last years, we have de-scribed and studied a cytolytic activity in Leishmania amazonensisthat functions optimally at 37 �C and pH 5.5 (conditions found in-side phagolysosome), but also at lower temperatures and/or neu-tral pH [1,2]. This cytolytic activity is due to pore-formation ontarget membranes by the action or on the dependence of a protein(or proteins) [2,3] and results from either proteolysis [4] or disso-ciation of an oligopeptide inhibitor (unpublished result). We havebeen referring to the component(s) responsible for the pore-forma-tion as leishporin [3–5], although the putative protein(s) remain(s)unidentified. The above features are compatible with a function oflysing membranes from both inside the phagolysosome and, lateron, the cytosol. We have then postulated that the exit of Leishmaniafrom macrophages is not simply a consequence of pathogenburden or cell stress, as usually assumed, but could be causedby the action of leishporin [1–6]. In fact, the active egress of

lsevier B.V. All rights reserved.

3210 T. Castro-Gomes et al. / FEBS Letters 583 (2009) 3209–3214

intracellular pathogens from within the host cell can be orches-trated by the pathogens themselves through a variety of strategies,including pore-formation [7,8].

Pore-forming proteins (PFPs) require binding to specific mole-cules on their target membranes, which can be lipids, carbohy-drates or proteins [9–11]. Here, we have studied therequirements of leishporin to bind to and lyse cells. Using erythro-cytes and liposomes as targets, we showed that, to lyse mem-branes, leishporin bind directly to phospholipids not requiringcholesterol, proteins or carbohydrates.

2. Materials and methods

2.1. Parasites

The PH8 (IFLA:PA:67:PH8) strain of Leishmania (Leishmania)amazonensis was used. Promastigotes were grown in Schneidermedium (Sigma) with 10% heat-inactivated fetal bovine serum(FBS) (CULTILAB, Campinas, Brazil) and 50 lg/ml gentamycin(Sigma). Four-day cultured parasites were washed with phos-phate-buffered saline (PBS) at 1000�g, and pellets were kept at�80 �C until required.

2.2. Parasite membrane extract

Promastigotes were resuspended in 50 mM borate buffer, pH7.0, to a density of 2 � 109 cells/ml and subjected to five cycles offreeze-and-thaw [2]. The extract was centrifuged at 1000�g for10 min to sediment intact cells and nuclei and the supernatantcentrifuged for 1 h at 16 000�g. The membrane-rich pellet wasresuspended in the same buffer containing 0.4% (sub-lytical)CHAPS (3[(3-cholamidopropyl) dimethyl-ammonium]-2-hydroxy-propanesulfonate) and kept on ice for 1 h with occasional agitation.The suspension was centrifuged at 100 000�g for 1 h, and thesupernatant, corresponding to the solubilized membrane mole-cules, was referred to as promastigotes membrane detergent-solu-ble extract (mExt).

2.3. Liposomes

Liposomes were made of DPPC (La-dipalmitoylphosphatidyl-choline) or DOPC (dioleylphosphatidylcholine), with or withoutcholesterol (10, 15, 20 or 30% w/w). To prepare large unilamellarvesicles, a lipid film was hydrated with PBS containing 30 mMphospholipids. The suspension was subjected to 10 cycles offreeze-and-thaw and extruded through 200-nm pore size polycar-bonate membranes. To obtain calcein-containing small unilamellarliposomes, the lipid film was hydrated with a 75 mM calceinsolution, pH 7.4, and the suspension was ultrasonicated (SonicsVibra-Cell, USA). Calcein-loaded liposomes were separated fromfree calcein by Sephadex G50.

2.4. Hemolytic assay

Hemolysis was assessed in human erythrocytes (HuE) [2].Briefly, 5 � 106 cells in 200 ll 20 mM acetate buffer, pH 5.5, con-taining 150 mM NaCl, were incubated in 96 round-bottomed wellmicroplates with 10 ll of 1:2 serially diluted mExt treated or notas described below. After 30 min at 37 �C, microplates were centri-fuged for 10 min at 500 � g and hemolysis was quantified throughthe absorbance of the supernatant at 414 nm. The percentage of ly-sis was in relation to total lysis, obtained by addition of 10 ll of0.25% Triton X-100 to the same number of HuE. Hemolytic activitywas reported as percentage of lysis versus dilution factor or as H50,the inverse of the dilution that caused 50% of hemolysis.

Alternatively, HuE were previously incubated for 1 h at 37 �C withtrypsin, Pronase� or proteinase K (12.5, 25, 50 or 100 lg/ml) in10 mM Tris–HCl buffer, pH 7.4, containing 150 mM NaCl and10 mM CaCl2 and washed three times with PBS. For the competitionexperiments, mExt was previously incubated for 30 min at 37 �C withfructose, galactose, glucose, lactose, maltose or mannose (12.5, 25, 50or 100 lM), DPPC or cholesterol (12.5, 25, 50 or 100 mg/ll).

2.5. Removal of the lytic activity from mExt

Ten microliters of mExt in a final volume of 200 l were incu-bated on ice with HuE, Lactobacillus acidophilus or liposomes as fol-lows: (1) HuE: 0.2 � 107, 1 � 107 or 2 � 107, acetate buffer, pH 5.5,30 min; (2) bacteria: 1 � 1010/ml (heated at 100 �C, 10 min or trea-ted with 1 mg/ml lysozyme at 37 �C, 30 min), Tris–HCl buffer, pH8.0, 15 min; (3) liposomes: 5, 15 or 20 ll of suspension, acetatebuffer, pH 5.5, 30 s. Cells and liposomes were removed by centrifu-gation and mExt was assayed for hemolytic activity. All centrifuga-tions in this work were carried out at 4 �C.

2.6. Liposome lysis assay

Ten microliters of calcein-loaded liposomes were diluted in90 ll of acetate buffer, pH 5.5, and incubated at 0 �C or at 37 �Cwith (1) mExt heated or not at 100 �C or previously incubated withcalcein-free liposomes or (2) liposomes previously incubated withmExt. Twenty-microliters aliquots were collected at 5- or 10-mininterval and diluted in 2 ml acetate buffer in a quartz cuvette be-fore reading the fluorescence (wavelengths: exciting – 490 nm;emission: 515 nm) (Varian Cary Eclipse Fluorimeter). Lysis was re-ported as the percentage of total lysis, obtained after addition of5 ll of 4% CHAPS. All experiments in this work were repeated atleast three times and all data correspond to a typical result.

3. Results

3.1. Binding of the leishporin to cell membranes

At least two steps are required for pore-formation by mExt onerythrocytes: (1) binding of the cytolysin to the target membrane,which occurs even at 0 �C with no hemolysis and (2) the pore-for-mation itself that probably consists of oligomerization and inser-tion of subunits, occurring only at higher temperatures [2,3]. Toverify whether HuE remove hemolytic activity from mExt, we incu-bated both components on ice. As expected, HuE were sedimentedwithout hemolysis and the hemolytic activity of the supernatantwas determined. We verified that HuE removes the hemolyticactivity of mExt in a dose-dependent manner (Fig. 1A). Similar re-sults were observed when erythrocytes were replaced by prokary-otic cells. Fig. 1B shows that the Gram-positive L. acidophiluspartially or completely removes hemolytic activity of mExt if pre-viously heated at 100 �C or treated with lysozyme, respectively.The Gram-negative bacterium Escherichia coli also partially re-moves hemolytic activity if heated at 100 �C (not shown). This indi-cates that the elimination or disruption of bacterial cell wallexposes cytolysin-binding sites on the cell, showing that leishporinis able to bind to membranes other than eukaryotic ones. We nextsought whether the cytolysin had a particular receptor, investigat-ing the participation of carbohydrates, proteins and lipids.

3.2. Involvement of carbohydrates, proteins and lipids in the binding ofleishporin to target membranes

To investigate whether hemolysis was dependent on the bindingof the cytolysin to proteins, we treated HuE with trypsin, Pronase�

Trypsin Pronase Proteinase K0

20

40

60

80

100

120

Erythrocytes treatment

0 0.125 0.25 0.5 (µg/ml)

(µg/ml)

(µm)

A

B

Fruct Gluc Galact Lact Malt Man0

20

40

60

80

100

120

mExt treatment

0 12.5 25 50 100

Hem

olyt

ic A

ctiv

ity (H

50)

C

Cholesterol DPPC0

20

40

60

80

100

120 0 0.25 1 5 10 50

mExt Treatment

1

Fig. 2. Involvement of erythrocytes surface proteins, carbohydrates or lipids inleishporin-mediated lysis. Hemolytic activity of mExt was evaluated in HuEpreviously treated with trypsin, Pronase� or proteinase K (A) or in untreated cellsafter being previously incubated with the indicated sugars (B) or lipids (C). Eachpoint represents the mean of H50 from duplicate wells ±S.D.

1 2 4 8 16

0

20

40

60

80

100Erytrhocytes

- 0.2 x 107

1 x 107

2 x 107

A

B

Hem

olys

is(%

)

1 2 4 8 16

0

20

40

60

80

100Bacteria

- untreated heat-killed lisozyme-treated

Fig. 1. Removal of mExt hemolytic activity by erythrocytes or bacteria. Hemolyt-ically active mExt were incubated with HuE (A) or untreated, heat-killed orlisozyme-treated bacteria (B). After sedimentation of cells, the hemolytic activity ofmExt was evaluated. Each point represents the mean of percentage of hemolysisfrom duplicate wells.

T. Castro-Gomes et al. / FEBS Letters 583 (2009) 3209–3214 3211

or proteinase K before the hemolytic assay. We verified that thetreatment did not reduce hemolysis, indicating that target cell pro-teins are not essential for the binding of and lysis mediated by lei-shporin (Fig. 2A). Trypsin and proteinase K even caused an increasein HuE susceptibility to lysis at the highest concentrations used.

The requirement for carbohydrate in leishporin binding andhemolysis was also investigated, using a competition assay. Weincubated mExt with fructose, glucose, galactose, lactose, maltoseor mannose, at a wide range of concentrations, and evaluated itshemolytic activity. None of the sugars was able to reduce mExthemolytic activity, indicating that carbohydrates might also not beimportant for the cytolysin-binding to HuE and hemolysis (Fig. 2B).

Since phospholipids and sterols are major structural elementsof biological membranes, we used the phospholipid DPPC and thenon-phosphorilated lipid, cholesterol, to investigate whether lei-shporin was binding directly to these molecules. Fig. 2C shows thatwhereas cholesterol, in concentrations of 0.25–250 lg (higher con-centrations not shown), is not able to inhibit leishporin activity,DPPC can totally inhibit hemolysis mediated by mExt in a dose-dependent fashion. This result suggested that cholesterol doesnot participate in the binding of leishporin to HuE and that themost probable binding sites for the cytolysin are the membranephospholipids. To confirm this assumption, we used liposomeswith different compositions in various assays.

3.3. Direct binding of leishporin to lipids

We first investigated whether liposomes made of DPPC andcholesterol, similar to HuE or bacteria, were able to pull leishporin

down. We showed that the supernatant of mExt incubated withliposomes is totally devoid of its original hemolytic activity andthe removal of this activity occurs in a dose-dependent manner(Fig. 3). It occurs immediately after the addition of the vesiclessince they stay in contact with mExt only the time required forhomogenization and centrifugation (around 30 s). Longer incuba-tion periods (up to 5 min) and the replacement of DPPC by DOPCdid not affect the binding of the cytolysin to liposomes (notshown). Cholesterol does not seem to have any role in the removal

A

B

Cal

cein

rele

ase

(%)

C

0 5 10 15 20 25 30

0

20

40

60

80

100Cholesterol (%)

- 10 15 20 30

0 10 20 30 40 50 60

0

10

20

30

40

50Contact with liposomes:

previously incubated with Ext-ms not incubated with Ext-ms

Time (Minutes)

0 10 20 30 40 50 60

0

20

40

60

80

100

mExt (37ºC) untreated (37ºC) heated at 100ºC (37ºC) liposome-incubated (0ºC) untreated

Fig. 4. Liposome lysis by leishporin. Calcein-loaded DPPC liposomes without (A andC) or with cholesterol (B) were incubated with native (A and B), heat-inactivated (A)or liposome-incubated mExt (A) or with DPPC liposomes that had been extensivelywashed after previous incubation with mExt (C) at 0 �C (A) or 37 �C (A, B and C).Lysis was evaluated by the percentage of calcein released at the indicated time.

1 2 4 8 16

0

20

40

60

80

100

Dilution Factor

Liposomessuspension

- 5 µl 15 µl 20 µl

Hem

olys

is(%

)

Fig. 3. Removal of mExt hemolytic activity by liposomes. Hemolytically active mExtwere incubated with liposomes made of DPPC and cholesterol. After sedimentationof vesicles, the hemolytic activity of mExt was evaluated. Each point represents themean of percentage of hemolysis from duplicate wells.

3212 T. Castro-Gomes et al. / FEBS Letters 583 (2009) 3209–3214

of the lytic activity, since cholesterol-free DPPC vesicles were ableto remove the hemolytic activity to the same extent as the choles-terol-containing liposomes (not shown). This is in accordance withthe fact that mExt-mediated hemolysis is inhibited by DPPC butnot by cholesterol (Fig. 2C).

3.4. Lysis of liposomes

To confirm that the only requirement for leishporin-mediatedlysis is the presence of phospholipids, we used calcein-loadedDPPC liposomes and evaluated their susceptibility to lysis. We firstverified that calcein trapped in DPPC liposomes is released whenthey are incubated with mExt, demonstrating that vesicles madeof a single phospholipid can be lysed by leishporin (Fig. 4A). Lysisdoes not occur (1) if mExt is heated at 100 �C, (2) if mExt is previ-ously incubated with calcein-free DPPC liposomes, supporting ourresults that leishporin is removed from mExt by interaction withphospholipids (Fig. 3), (3) at 0 �C, or (4) when liposomes have high-er cholesterol content (Fig. 4B).

The next experiment was carried out to ensure that liposomeswere removing from mExt leishporin itself, and not other compo-nent essential for the lytic activity. Liposomes that had been previ-ously incubated with mExt at 0 �C (at which lysis does not occur),extensively washed (still at 0 �C) and had removed the hemolyticactivity from mExt were incubated with calcein-loaded liposomesthat had no contact with mExt. We found that liposomes that hadcontact with mExt, in a suspension devoid of any soluble/unboundmExt molecule, produced lysis in the calcein-loaded liposomes(Fig. 4C). This shows that it is the cytolysin that binds to the lipo-somes and, possibly by liposomes fusion, it is transferred to thecalcein-containing vesicles.

4. Discussion

To cause cytolysis, PFPs require the binding to target cell recep-tors. Some PFPs recognize lipids, while other use membrane pro-teins or carbohydrates as receptors [9–11]. Here, we provideseveral evidences that the L. amazonensis cytolisin we call leishpo-rin binds directly to membrane phospholipids leading to cytolysis.

We had shown that, whereas HuE incubated with mExt at 0 �Care not lysed, hemolysis occurs if these cells are extensivelywashed (still at 0 �C), to remove unbound/soluble molecules, andbrought to 37 �C [2]. This demonstrated that leishporin must bindto the target membrane before making the lytic pore. Here, we

confirmed these findings by showing that, at 0 �C, HuE (Fig. 1A)or bacteria that had their cell wall eliminated or disrupted(Fig. 1B) are able to remove leishporin-mediated hemolytic activityfrom mExt. For the bacteria, we assumed that the cell wall blocksleishporin binding and the treatments make plasma membraneaccessible to the cytolysin. In fact, intact bacteria remove almostnone of the hemolytic activity, are not lysed by leishporin andgrow normally in the presence of L. amazonensis mExt (unpub-lished results).

Some cytolysins bind to cells through protein or carbohydratesreceptors [10,12–15]. Leishporin does not require surface proteinsas a binding site on the erythrocytes membrane, since the treat-ment of these cells with different proteases did not impair mExt-mediated hemolysis (Fig. 2A). On the contrary, the susceptibilityto lysis increased, probably by exposing binding sites on the cellsurface. HuE carbohydrates also did not seem to be used as recep-

T. Castro-Gomes et al. / FEBS Letters 583 (2009) 3209–3214 3213

tors, since several types of sugars did not compete with bindingsites on targets (Fig. 2B).

The above findings suggested that leishporin uses lipids asreceptor and that they are sufficient for the cytolysin’s binding tomembranes and lytic activity. This assumption was corroboratedby the findings that (1) DPPC completely inhibits mExt hemolyticactivity (Fig. 2C), (2) liposomes made of DPPC (Fig. 3) or DOPC(not shown), similar to HuE or bacteria (Fig. 1A and B), promptlyremove hemolytic activity from mExt (suggesting that leishporinis highly hydrophobic) and (3) calcein-loaded DPPC liposomesare lysed by mExt (Fig. 4A). The absence of liposomes lysis by mExtheated at 100 �C or when the assay is carried out at 0 �C confirmsthe thermolability of the cytolysin and the temperature-depen-dence of the pore-formation, respectively [1,2]. The fact that vesi-cles made of a single lipid can be lysed by leishporin, demonstratesthat (1) it binds directly to the lipid portion of target membrane,(2) the binding occurs via phospholipids, (3) they are the only re-quired molecules and (4) they are sufficient for the cytolysin’smembrane binding and lytic activity. Other protozoan PFPs, suchas amoebapores [16] and naegleriapores [17], also insert to celllipids without the mediation of classic receptors. Likewise, somebacterial PFPs bind directly to phosphatidylcholine, phosphatidyl-serine, phosphatidylethanolamine, cholesterol or sphingomyelin[18–21].

Binding to cholesterol is a feature currently used to group bac-terial PFPs as a family of proteins, the cholesterol-binding cytoly-sins [22,23]. We showed that leishporin does not use cholesterolas binding site on target membranes. As opposed to DPPC, choles-terol is unable to inhibit mExt-mediated hemolytic activity(Fig. 2C). It is even dispensable for leishporin binding to thephospholipid and for cytolytic activity, since it does not improvethe ability of DPPC liposomes to remove leishporin from mExt(not shown) or the efficiency of the cytolysin to lyse DPPC lipo-somes (Fig. 4B). The cholesterol-independency is in accordancewith leishporin’s ability to bind to bacteria membrane (Fig. 1B).However, cholesterol may be a negative modulator of leishporin-mediated lytic activity since it can prevent liposome lysis (Fig. 4B).

Since previous data indicated that pore-formation by leishporinoccurs by polymerization of monomers [3], it is probable that thehigher content in cholesterol impairs either the insertion or theassembly of single subunits into cell membrane. However, ineukaryotic cells, cholesterol, which corresponds to about 25% oftheir total membrane lipids and is mainly concentrated in lipidrafts, is not an obstacle for leishporin activity. Accordingly, in ourexperiments, lysis, although reduced, still occurs in liposomes with20% cholesterol. Because leishporin requires only phospholipids asligands to cause cytolysis, it can destroy mammalian cell mem-branes easily and non-specifically, even in the presence of choles-terol. Therefore, susceptibility to lysis by leishporin may be shapedby the lipid composition of the membrane, reflected in the mem-brane fluidity.

Regarding the parasite, its surface lipid composition mayprotect it from autolysis since, although leishporin can bind toL. amazonensis promastigotes, parasites are resistant to mExt-mediated lysis and do not undergo autolysis (unpublished results).In Entamoeba histolytica, a high content of membrane cholesterol(about 40%) protects the protozoan from self-destruction by amoe-bapores [16]. Likewise, granulysin, a PFP from mammalian cyto-toxic T lymphocytes, similarly to what we observed here forleishporin (Figs. 3 and 4B), can bind to cholesterol-containing aswell as to cholesterol-free membranes, although, unlike leishporin(1–4 and Fig. 4B), lysis occurs only in the absence of cholesterol[24]. For staphylococcal alpha-toxin, clustering of phosphocholinehead groups of sphingomyelin in microdomains containing choles-terol is required, so that oligomerization, a prerequisite for stableattachment of the toxin to the membrane, efficiently occurs.

Outside these clusters, binding to phosphocholine is too transientfor monomers to find each other. The principle of membrane tar-geting in the absence of any genuine, high affinity receptor mayalso underlie the assembly of other lipid-inserted oligomers [20],and may include leishporin.

Regarding the host cell, it is worth mentioning that the parasi-tophorous vacuoles from macrophages infected with L. amazonensisdisplay high levels of phosphatidylcholine [25]. They must thenbind leishporin and be susceptible to permeabilization/lysis, as oc-curs with listeriolysin O [26], the Legionella pneumophila PFP [27],and the recently described TgPLP1 from Toxoplasma gondii [28].Another regulation mechanism that may make leisporin-mediatedlyses a vectorial event may rely on the leishporin’s need to be pro-teolitically activated to cause lysis [4], which could be easily ful-filled inside the parasitophorous vacuole. Therefore, ourhypothesis that leishporin can disrupt the parasitophorous vacuolemembrane and, later on, the plasma membrane [2,4,5], withoutparasite autolysis, is plausible and already currently accepted [5–8].

It is interesting that DPPC liposomes containing only boundmExt molecules cause the lysis of calcein-loaded liposomes thathad no previous contact with the parasite extract (Fig. 4C). SincemExt-free liposomes do not lyse calcein-loaded liposomes, wecan infer that leishporin is transferred from the calcein-free tothe calcein-loaded liposomes, presumably by fusion of the vesicles.This confirms that the L. amazonensis cytolysin lyse cells using asbinding sites only phospholipids, the only requirement on targetmembranes to form stable pores. This also provides a final demon-stration that it is the cytolysin that binds to the phospholipid, rul-ing out the possibility that liposomes (Fig. 3), HuE or bacteria(Fig. 1) were merely removing from mExt any other componentwithout which the hemolytic activity could not occur. This findingis in agreement with previous results showing that, although HuEincubated with mExt are not lysed at 0 �C, they are indeed lysed at37 �C after being extensively washed to remove all unbound/solu-ble molecules [2].

The binding of leishporin to liposomes lead us to the obvioussubsequent step, the identification of the protein(s) bound to thevesicles. Preliminary data show that various known and yetunidentified proteins are associated with mExt-treated DPPCliposomes, which we are currently identifying to further determinethe identity of the cytolytic pore-forming molecule we callleishporin.

Acknowledgments

We thank Elimar Faria for technical assistance and the financialsupport from: UNICEF/UNDP/World Bank/WHO Special Pro-gramme for Research and Training in Tropical Diseases (TDR), Fun-dação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG),Programa de Apoio a Núcleos de Excelência (PRONEX). T.C.G. andF.R.A.C. were supported by Coordenadoria de Aperfeiçoamento dePessoal do Ensino Superior (CAPES). C.E.C.S. was supported by Con-selho Nacional de Desenvolvimento Científico e Tecnológico(CNPq). R.A.S. was a WHO post-doctoral fellow. M.F.H. and F.F.are CNPq research fellows.

References

[1] Noronha, F.S., Ramalho-Pinto, F.J. and Horta, M.F. (1994) Identification of aputative pore-forming hemolysin active at acid pH in Leishmania amazonensis.Braz. J. Med. Biol. Res. 27, 477–482.

[2] Noronha, F.S., Ramalho-Pinto, F.J. and Horta, M.F. (1996) Cytolytic activity inthe genus Leishmania: involvement of a putative pore-forming protein. Infect.Immun. 64, 3975–3982.

[3] Noronha, F.S., Cruz, J.S., Beirão, P.S. and Horta, M.F. (2000) Macrophage damageby Leishmania amazonensis cytolysin: evidence of pore formation on cellmembrane. Infect. Immun. 68, 4578–4584.

3214 T. Castro-Gomes et al. / FEBS Letters 583 (2009) 3209–3214

[4] Almeida-Campos, F.R. and Horta, M.F. (2000) Proteolytic activation ofleishporin: evidence that Leishmania amazonensis and Leishmania guyanensishave distinct inactive forms. Mol. Biochem. Parasitol. 111, 363–375.

[5] Almeida-Campos, F.R., Noronha, F.S. and Horta, M.F. (2002) The multitalentedpore-forming proteins of intracellular pathogens. Microb. Infect. 4, 741–750.

[6] Horta, M.F. (1997) Pore-forming proteins in pathogenic protozoan parasites.Trends Microbiol. 5, 363–366.

[7] Hybiske, K. and Stephens, R.S. (2008) Exit strategies of intracellular pathogens.Nat. Rev. Microbiol. 6, 99–110.

[8] Roiko, M.S. and Carruthers, V.B. (2009) New roles for perforins and proteasesin apicomplexan egress. Cell. Microbiol. 11, 1444–1452.

[9] Geny, B. and Popoff, M.R. (2006) Bacterial protein toxins and lipids: poreformation or toxin entry into cells. Biol. Cell 98, 667–678.

[10] Bravo, A., Gill, S.S. and Soberón, M. (2007) Mode of action of Bacillusthuringiensis Cry and Cyt toxins and their potential for insect control.Toxicon 49, 423–435.

[11] Young, J.A. and Collier, R.J. (2007) Anthrax toxin: receptor binding,internalization, pore formation, and translocation. Annu. Rev. Biochem. 76,243–265.

[12] Cortajarena, A.L., Goñi, F.M. and Ostolaza, H. (2001) Glycophorin as a receptorfor Escherichia coli alpha-hemolysin in erythrocytes. J. Biol. Chem. 276, 12513–12519.

[13] Gordon, V.M., Nelson, K.L., Buckley, J.T., Stevens, V.L., Tweten, R.K., Elwood, P.C.and Leppla, S.H. (1999) Clostridium septicum alpha toxin usesglycosylphosphatidylinositol-anchored protein receptors. J. Biol. Chem. 274,27274–27280.

[14] Lang, S., Xue, J., Guo, Z. and Palmer, M. (2007) Streptococcus agalactiae CAMPfactor binds to GPI-anchored proteins. Med. Microbiol. Immunol. 196, 1–10.

[15] Farrand, S., Hotze, E., Friese, P., Hollingshead, S.K., Smith, D.F., Cummings, R.D.,Dale, G.L. and Tweten, R.K. (2008) Characterization of a streptococcalcholesterol-dependent cytolysin with a Lewis y and b specific lectin domain.Biochemistry 47, 7097–7107.

[16] Andrä, J., Berninghausen, O. and Leippe, M. (2004) Membrane lipidcomposition protects Entamoeba histolytica from self-destruction by its pore-forming toxins. FEBS Lett. 564, 109–115.

[17] Young, J.D. and Lowrey, D.M. (1989) Biochemical and functionalcharacterization of a membrane-associated pore-forming protein from thepathogenic ameboflagellate Naegleria fowleri. J. Biol. Chem. 264, 1077–1083.

[18] Potrich, C., Bastiani, H., Colin, D.A., Huck, S., Prévost, G. and Dalla Serra, M.(2009) The influence of membrane lipids in Staphylococcus aureus gammahemolysins pore formation. J. Membr. Biol. 227, 13–24.

[19] Bakrac, B., Gutiérrez-Aguirre, I., Podlesek, Z., Sonnen, A.F., Gilbert, R.J., Macek,P., Lakey, J.H. and Anderluh, G. (2008) Molecular determinants ofsphingomyelin specificity of a eukaryotic pore-forming toxin. J. Biol. Chem.283, 18665–18677.

[20] Valeva, A., Hellmann, N., Walev, I., Strand, D., Plate, M., Boukhallouk, F., Brack,A., Hanada, K., Decker, H. and Bhakdi, S. (2006) Evidence that clusteredphosphocholine head groups serve as sites for binding and assembly of anoligomeric protein pore. J. Biol. Chem. 281, 26014–26021.

[21] Flanagan, J., Tweten, R., Johnson, A. and Heuck, A. (2009) Cholesterol exposureat the membrane surface is necessary and sufficient to trigger perfringolysin Obinding. Biochemistry 48, 3977–3987.

[22] Alouf, J.E. (2000) Cholesterol-binding cytolytic protein toxins. Int. J. Med.Microbiol. 290, 351–356.

[23] Tweten, R.K. (2005) Cholesterol-dependent cytolysins, a family ofversatile pore-forming toxins. Infect. Immun. Infect. Immun. 73, 6199–6209.

[24] Barman, H., Walch, M., Latinovic-Golic, S., Dumrese, C., Dolder, M., Groscurth,P. and Ziegler, U. (2006) Cholesterol in negatively charged lipid bilayersmodulates the effect of the antimicrobial protein granulysin. J. Membr. Biol.212, 29–39.

[25] Henriques, C., Atella, G.C., Bonilha, V.L. and de Souza, W. (2003) Biochemicalanalysis of proteins and lipids found in parasitophorous vacuoles containingLeishmania amazonensis. Parasitol. Res. 89, 123–133.

[26] Portnoy, D.A., Auerbuch, V. and Glomski, I.J. (2002) The cell biology of Listeriamonocytogenes infection: the intersection of bacterial pahogenesis and cell-mediated immunity. J. Cell. Biol. 158, 409–414.

[27] Alli, A.O., Gao, L.Y., Pedersen, L.L., Zink, S., Radulic, M., Doric, M. and Abu Kwaik,Y. (2000) Temporal pore-formation-mediated egress from macrophages andalveolar epithelial cells by Legionella pneumophila. Infect. Immun. 68, 6431–6440.

[28] Kafsack, B.F., Pena, J.D., Coppens, I., Ravindran, S., Boothroyd, J.C. andCarruthers, V.B. (2009) Rapid membrane disruption by a perforin-like protein facilitates parasite exit from host cells. Science 323, 530–533.