Parasitol Res (2000) 86: 96±100
Ó Springer-Verlag 2000
ORIGINAL PAPER
M.N. Garrido á G. Racagni á B.M.I. Pereira M.A. RodrõÂ guez á H.D. LujaÂn á D.H. Bronia E.E. Machado-Domenech
Changes in Trypanosoma cruzi phospholipid turnover induced by parasite contact with cell membranes Received: 20 July 1999 / Accepted: 20 August 1999
Abstract To investigate the possibility that cell contact could initiate a series of signals in both the host cell and the ¯agellate protozoan Trypanosoma cruzi, we studied [32P]-phospholipid turnover during parasite interaction with cellular membranes in vitro. Lipid alterations were produced in the parasite during the initial period of contact with the plasma membranes of human erythrocytes. In the presence of calcium an increment in phosphatidylethanolamine was observed with a concomitant decrease in phosphatidic acid fractions, whereas these modi®cations were not observed in the absence of calcium. There was an evident decrease in phosphatidylcholine and a shift in the phosphatidylinositol/lysophosphatidylethanolamine fraction among the phospholipids of major turnover in the absence or presence of calcium. Among the minor labeled species, lysophosphatidylcholine reached levels that duplicated control values, whereas the amounts of lysophosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate diminished by over 50%. All of these variations indicate that the parasite's contact with plasma membranes induces changes involving T. cruzi phospholipids and suggest the participation of these compounds in the activation of intracellular mechanisms that might be important during the life cycle of this parasite. Key words Trypanosoma cruzi á Phospholipids á Adhesion á Invasion á Cellular signaling
M.N. Garrido á G. Racagni á E.E. Machado-Domenech (&) Departamento de BiologõÂ a Molecular, Facultad de Ciencias Exactas, FõÂ sico-QuõÂ micas y Naturales, Universidad Nacional de RõÂ o Cuarto, 5800 RõÂ o Cuarto, CoÂrdoba, Argentina e-mail:
[email protected]; Fax: +54-358-4676232 B.M.I Pereira á M.A. RodrõÂ guez á H.D. LujaÂn á D.H. Bronia CaÂtedra de QuõÂ mica BioloÂgica, Facultad de Ciencias MeÂdicas, Universidad Nacional de CoÂrdoba, 5016 CoÂrdoba, Argentina
Introduction Trypanosoma cruzi, the etiologic agent of American trypanosomiasis, or Chagas' disease, is a unicellular organism whose life cycle involves a series of intimate interactions with host and vector structures. The maintenance of its biologic cycle involves cell penetration, signal recognition for dierentiation, and evasion of the host immune system, phenomena reasonably presumed to be dependent on plasma membrane mediation. Lipids and calcium are relevant in many membranerelated events occurring in eukaryotic cells, including T. cruzi. Moreno et al. (1994) have indicated that in addition to an increase in intracellular Ca2+ in the host cells, an increase in cytosolic Ca2+ also occurs in T. cruzi trypomastigotes during invasion of the host cell. Therefore, it appears that Ca2+ is an important signal for both parasite and host cells during their interaction. In this regard, the formation of inositol triphosphate (IP3) and inositol bisphosphate (IP2) in the presence of CaCl2 has been stimulated in digitonin-permeabilized epimastigotes (Docampo and Pignataro 1991). Machado de Domenech et al. (1992) have shown that carbachol is capable of modifying phosphatidylinositol (PI) metabolism in this parasite as demonstrated by a shift in the levels of phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP), and phosphatidic acid (PA). CalderoÂn et al. (1986, 1989) have reported that T. cruzi can destabilize the plasma membrane of red blood cells, inducing either fusion or hemolysis of human and chicken erythrocytes. Although these cells are not invaded by the parasite in infected animals, this cellular system represents a simple and quantitative means of detecting changes in the protein and lipid composition of a target membrane during interaction with the parasite and of subsequent screening for the presence of lytic or fusogenic molecules (Andrews et al. 1987; CalderoÂn et al. 1989; LujaÂn and Bronia 1994). In those studies, biochemical analysis of human erythrocyte membranes
97
showed that their protein and lipid composition diered markedly after their interaction with the parasite in vitro. Adhesion of parasites to human erythrocytes induced membrane destabilization, promoting lysis or fusion of these cells, depending upon the interaction conditions. Speci®cally, the red-cell fusion process was calcium-dependent and associated with an increase in 1,2diacylglycerides (DAG), whereas an increment in lysophospholipids and free fatty acids was calcium-independent and common to both the lysis and fusion processes. In addition, the use of radiolabeled phosphatidylcholine (PC) allowed the demonstration that free fatty acids and lysoderivatives were transferred from the T. cruzi membrane to that of the red cell (LujaÂn and Bronia 1994), which suggests the activation of parasite phospholipases during this process. However, the biochemical basis of the adhesion step has not been studied. The aim of the present work was to determine the in¯uence of cell contact on phospholipid turnover during early stages of the parasite-erythrocyte membrane interaction.
Materials and methods Growth conditions and organisms The Tulahuen strain (stock 0) of Trypanosoma cruzi was used in this study. Parasites were grown at 28 °C in culture medium supplemented with 10% fetal calf serum and harvested according to Racagni et al. (1992). Then, the cells were resuspended in KrebsRinger-Tris (KRT) buer at a concentration of 2±4 ´ 108 cells/ml. Freshly drawn human blood was mixed 4:1 (v/v) with 3.8% sodium citrate. Erythrocytes were washed three times in HEPES buer (pH 7.55) containing 188 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid/NaOH, 5.5 mM glucose, and 100 mM sodium dimethylarsenate. Thereafter they were resuspended in identical buer at a concentration of 2±4 ´ 108 cells/ml. Radioisotope incorporation Parasites were incubated with [32P]-orthophosphate ([32P]-Pi) (100 lCi/250 mg of cells) in KRT buer at 28 °C for 3 h in a water bath. Erythrocytes were incubated with [32Pi]-Pi (15 lCi/1.5 ´ 108 cells) in HEPES buer at 37 °C for 3 h. These conditions resulted in a steady-state labeling of the lipids (Racagni et al. 1992). The cells were washed twice to eliminate the unincorporated radioisotope. Parasite-erythrocyte interaction and isolation Cell contact was studied by incubation of each cell type at a cell density of 2±4 ´ 108 cells/ml in KRT buer (pH 7.2) for 1 h at 37 °C in the presence of 2 mM Ca2+ or 5 mM ethylene glycol tetraacetic acid (EGTA). In these experiments, labeled cells interacted with unlabeled ones. 32P-labeled erythrocytes or 32P-labeled T. cruzi epimastigotes incubated in the presence or absence of Ca2+ were used as controls. After cell-adhesion reaction, parasites were separated from erythrocytes by a gradient of Ficoll-Triyosom (Boyum 1968). Ficoll and Triyosom were diluted to a ®nal density of 1.080 0.2. After centrifugation, the sediment containing isolated erythrocytes was resuspended in 0.015 M NaCl to yield erythrocyte ghosts. The milky interface present in the supernatant containing motile parasites was collected, and cells were washed with KRT buer. Recovery was 95% for erythrocytes and 90% for parasites.
Lipid extraction and phospholipid separation and quanti®cation Total lipids were extracted from the parasites and ghosts according to Schacht (1981). The resulting lower phase was removed, evaporated to dryness under a stream of nitrogen, and redissolved in a suitable volume of chloroform/methanol (9:1, v/v). Phospholipids were separated by thin-layer chromatography (TLC) on silica-gel plates (Merck, 200 lm thick) that had been pretreated with 1% potassium oxalate/2 mM EDTA in methanol/water (2:3, v/v) as described by Jolles et al. (1981). Prior to use the plates were activated at 110 °C for 60 min. An aliquot of the total lipid extract was applied onto the chromatography plate and developed with chloroform/methanol/acetone/acetic acid/water (40:14:15:12:7, by vol.). The developed chromatograms were exposed to iodine vapors. The position of radiolabeled lipids was determined by autoradiography on Agfa-Gevaert ®lm. Spots were scrapped o the plates, and fractions were counted in a liquid scintillation counter. The amount of protein was determined according to Bradford (1976) using bovine serum albumin (Sigma Chemical Co.) as the standard. Materials All solvents (Merck) were of analytical grade. Phospholipid standards were obtained from Sigma Chemical Co. [32P]-Orthophosphoric acid (carrier-free) was obtained from CNEA (ComisioÂn Nacional de EnergõÂ a AtoÂmica, Buenos Aires, Argentina). All other chemicals were of the highest purity available from commercial sources. Polyphosphoinositides were prepared from ox brain as described by Lees (1957).
Results There were signi®cant dierences between the labeled phospholipid patterns observed in human erythrocytes and Trypanosoma cruzi incubated with [32Pi]-Pi (Table 1). PIP and PA were the erythrocyte species of major turnover, as would be expected in an incubation medium lacking competing anions (King et al. 1987) and given that mature erythrocytes show only a very low turnover of membrane lipids and are essentially devoid of any newly biosynthesized lipid (Allan 1982). In the
Table 1 Phospholipid turnover as determined in Trypanosoma cruzi and human erythrocytes. [32Pi]-Pi (100 lCi/250 mg of cells) was incubated with parasites for 3 h at 28 °C in KRT medium and with human erythrocytes at 37 °C in HEPES medium. Phospholipids were resolved by TLC, and levels of radioactivity were determined as described in Materials and methods. Results are expressed as mean values SE (n = 5) (LPC Lysophosphatidylcholine) Phospholipids
Percentage of [32P] incorporation T. cruzi
PC PI/LPE PE LPI PIP PA LPC PIP2 Others
34.18 23.40 20.52 5.20 4.40 4.07 1.66 0.41 6.02
Red cells 3.8 3.0 2.7 1.4 1.3 0.9 0.4 0.1 2.4
1.52 1.61 1.24 2.16 42.90 43.83 0.36 1.70 6.39
0.3 0.8 0.3 0.6 6.6 5.5 0.1 0.7 3.0
98
present study the principal labeled fractions in parasite membranes corresponded to PC, the PI/lysophosphatidylethanolamine fraction (PI/LPE), and phosphatidylethanolamine (PE). Cellular interaction was induced by confrontation of one of the prelabeled cell types with the unlabeled one. During the interaction period (1 h), epimastigotes contacted and adhered to the erythrocyte plasma membrane (even in the presence or absence of calcium), but this interval was not sucient to allow red-cell lysis or fusion (LujaÂn and Bronia 1994). Possible variations in phospholipid patterns during the early stages of interaction were studied. When the ratio of the phospholipidic-phase label (counts per minute) to the amount of protein (micrograms) from membranes of noninteracting cells (controls) was analyzed, a remarkable decrease (50%) was found in membranes of T. cruzi, which occurred independently of the presence of calcium. No signi®cant variation was detected among erythrocyte membranes (data not shown). This ®nding was consistent with changes in the protein and/ or phospholipidic fraction that might have been triggered by cellular contact. Figure 1 shows the in¯uence of cellular interaction on T. cruzi phospholipid turnover. Among the phospholipids of major turnover there was an evident decrease in PC (30±40%) and a shift in the PI/LPE fraction (45±60%). Among the minor labeled species, lysophosphatidylcholine (LPC) reached levels that duplicated the control values, whereas the amounts of lysophosphatidylinositol (LPI), PIP, and PIP2 diminished by over 50%. All of these variations occurred independently of the presence of calcium, suggesting that this might be a direct consequence of cellular contact. In the presence of calcium (Fig. 1A) an increment in PE and a concomitant decrease in PA fractions were observed, whereas these modi®cations were not observed in the presence of EGTA (Fig. 1B). Among the major labeled human-erythrocyte phospholipids, no change was detected in the turnover rate under the conditions tested (data not shown).
investigation may involve a sequence of molecular changes in the interacting membranes, given that close contact between two lipid bilayers does not induce membrane destabilization per se but that they actually repel each other (Helm et al. 1992). This suggests that some extracellular factor(s) belonging to the parasite may be required for destabilization of the target membrane. That the changes in PC and LPC reported herein are calcium-independent may be an indication of their role in the adhesion process accomplished either between T. cruzi and red cells or between erythrocytes and erythrocyte membranes. Fang et al. (1997) and Zhu et al. (1997) have reported that apart from its well-established role as a fusogenic phospholipid, LPC is related to events such as monocyte adhesiveness to endothelial cells in the in¯ammatory process, probably acting as an inducer of intercellular adhesion molecules in a protein tyrosine kinase-dependent way. Other changes that were calcium-independent ± and therefore possibly related to the adhesion process ± involved the polyphosphoinositide and PI fractions, a ®nding that may depend upon modi®ed phosphoinosi-
Discussion Our ®ndings of a PC decrease and a concomitant increment in LPC turnover for [32P]-prelabeled Trypanosoma cruzi membranes as calcium-independent events support previous suggestions of a T. cruzi PLA activation triggered by cellular adhesion (LujaÂn and Bronia 1994; LujaÂn et al. 1997). LujaÂn and Bronia (1994) have demonstrated that epimastigote and trypomastigote forms of T. cruzi can cause red-cell membrane destabilization in vitro and have shown that the parasites ®rst attach to the erythrocytes, independently of the presence of calcium in the medium. They also demonstrated an increment in lysoderivatives and free fatty acids in human erythrocyte membranes after 120 min of cellular interaction. The adhesion step studied in the present
Fig. 1A, B Variations in Trypanosoma cruzi [32P]-phospholipid turnover after cellular interaction. [32P]-Pi-prelabeled epimastigote forms were allowed to interact with human erythrocytes for 1 h. Cells were separated, lipids were extracted and analyzed by TLC, and levels of individual phospholipid radioactivity were determined. A Interaction performed in the presence of 2 mM calcium. B Interaction performed in the presence of 5 mM EGTA. Controls constituted [32P]-Piprelabeled T. cruzi incubated under both conditions in the absence of interaction with erythrocytes (100%). Data represent median values for 5 experiments (SE)
99
tide kinase activities. In this regard, polyphosphoinositides are known to be involved in the regulation of several actin-binding proteins that in¯uence the organization of the actin cytoskeleton associated with vesicle tracking, agonist stimulation, and cellular adhesion (Fukami et al. 1992; De Camilli et al. 1996; Sun et al. 1997). In relation to this, we have demonstrated the activation of the inositol phosphate/DAG pathway in the parasite as a response to external signals such as carbachol and a synthetic peptide carrying a chicken a-D-globin fragment (Garrido et al. 1996a, b). The decrease found in the turnover of T. cruzi PA is another suggestive observation and could be related to the increment in the erythrocyte DAG level previously demonstrated by LujaÂn and Bronia (1994). Our results suggest that PA, directly or as a phospholipase D product, is hydrolyzed by a PA phosphatase in association with a subsequent transfer of the product from the parasite to the red-cell membrane. Also, the possibility of the production of LPA, a second messenger involved in events such as cell growth, motility, and focal adhesion, cannot be discarded (Durieux and Lynch 1993; Moolenar 1995). The results reported herein also show that phosphatidylethanolamine is another T. cruzi phospholipid that changed after cellular interaction by increasing its turnover only in the presence of calcium, which indicates that this compound may be related more to fusion than to the adhesion step. This phospholipid has been reported to be one of the lipid receptors in the adhesion of Helicobacter pylori and H. mustelae to mucosal surfaces (Gold et al. 1993). Bonaldo et al. (1988) have reported that T. cruzi adheres to culture ¯asks prior to their dierentiation to metacyclic trypomastigotes, a phenomenon similar to that observed inside the invertebrate host, whereby parasites are attached to the epithelium of the insect rectal gland but are released after they have transformed into metacyclic trypomastigotes. Adherent parasites are characterized by the expression of various polypeptides that are probably involved in the adhesion process. Our ®ndings of a decrease in the ratio of the phospholipidicphase label and the protein quantity after cellular interaction not only support the changes found in T. cruzi phospholipid turnover but also suggest that alterations in proteins or phospholipids are triggered by cellular contact. In conclusion, the results described herein provide evidence for the ®rst time that T. cruzi attachment to a target cell membrane produces changes involving parasite phospholipids, suggesting the possible participation of these compounds in either the sensing of cell contact by the parasite or the triggering of signal-transduction mechanisms for the activation of speci®c genes required for cell invasion and/or dierentiation. Acknowledgements We are grateful to language consultant Prof. Iliana A. Martinez. This work was supported by a grant from CONICOR (CoÂrdoba, Argentina), CONICET (Buenos Aires, Argentina), and SECYT (UNRC, RõÂ o Cuarto, Argentina).
References Allan D (1982) Inositol lipids and membrane function in erythrocytes. Cell Calcium 3: 451±465 Andrews NW, Hong LS, Robbins ES, Nussenzweig V (1987) Stagespeci®c surface antigens expressed during the morphogenesis of vertebrate forms of Trypanosoma cruzi. Exp Parasitol 64: 474± 484 Bonaldo MC, Souto-PadroÂn T, Souza W de, Goldenberg S (1988) Cell-substrate adhesion during Trypanosoma cruzi dierentiation. J Cell Biol 106: 1349±1358 Boyum A (1968) Isolation of mononuclear cells and granulocytes from human blood. Scand J Clin Lab Invest 97: 77±89 Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248±254 CalderoÂn RO, Aguerri AM, Bronia DH (1986) Trypanosoma cruzi: variable fusogenic ability by dierent growth phases of the epimastigote form. Exp Parasitol 62: 453±455 CalderoÂn RO, LujaÂn HD, Aguerri AM, Bronia DH (1989) Trypanosoma cruzi: involvement of proteolytic activity during cell fusion induced by the epimastigote form. Mol Cell Biochem 86: 189±200 De Camilli P, Emr SD, McPherson PS, Novick P (1996) Phosphoinositides as regulators in membrane trac. Science 271: 1533±1539 Docampo R, Pignataro OP (1991) The inositol phosphate/diacylglycerol signalling pathway in Trypanosoma cruzi. Biochem J 274: 407±410 Durieux ME, Lynch KR (1993) Signalling properties of lysophosphatidic acid. Trends Pharmacol Sci 14: 249±254 Fang XJ, Gibson S, Flowers M, Furui T, Bast RC, Mills GB (1997) Lysophosphatidylcholine stimulates activator protein-1 and the C-Jun N-terminal kinase-activity. J Biol Chem 272: 13683± 13689 Fukami K, Fuhurashi K, Inagaki M, Endo T, Hatano S, Takenama T (1992) Requirement of phosphatidylinositol 4,5-bisphosphate for b-actinin function. Nature 359: 150±152 Garrido MN, Bollo M, Machado-Domenech EE (1996a) Carbachol stimulates inositol phosphate formation transiently in Trypanosoma cruzi. Cell Mol Biol 42: 221±225 Garrido MN, Bollo M, Machado-Domenech EE (1996b) Biphasic and dose-dependent accumulation of InsP3 in Trypanosoma cruzi stimulated by a synthetic peptide carrying a chicken D-globin fragment. Cell Mol Biol 42: 859±864 Gold BD, Huesca M, Sherman PM, Lingwood CA (1993) Helicobacter mustelae and Helicobacter pylori bind to common lipid receptors in vitro. Infect Immun 61: 2632±2638 Helm CA, Israelachvili JN, Muguiggan PM (1992) Role of hydrophobic forces in bilayer adhesion and fusion. Biochemistry 31: 1794±1805 Jolles J, Zwiers H, Dekker A, Wirtz KWA, Gipsen WH (1981) Corticotrophin-(1±24)-tetracosapeptide aects protein phosphorylation and polyphosphoinositide metabolism in rat brain. Biochem J 194: 283±292 King CE, Stephens LR, Hawkings PT, Guy GR, Mitchell RH (1987) Multiple metabolic pools of phosphoinositides and phosphatidate in human erythrocytes incubated in a medium that permits rapid transmembrane exchange of phosphate. Biochem J 244: 209±217 Lees MB (1957) Preparation and analysis of phosphatides. In: Colowick S, Kaplan N (eds) Methods in enzymology, vol 3. Academic Press, New York, pp 328±345 LujaÂn HD, Bronia DH (1994) Intermembrane lipid transfer during Trypanosoma cruzi-induced erythrocyte membrane destabilization. Parasitology 108: 323±334 LujaÂn HD, Racagni G, Garrido M, Pereira BMI, Rodriguez M, Machado-Domenech EE, Bronia DH (1997) Compromiso de fosfolõÂ pidos y fosfolipasas durante la interaccioÂn del Trypanosoma cruzi y membranas bioloÂgicas (meeting abstract). Medicina (B Aires) 57: 11
100 Machado de Domenech EE, Garrido MN, GarcõÂ a M, Racagni G (1992) Phospholipids of Trypanosoma cruzi: increase of polyphosphoinositides and phosphatidic acid after cholinergic stimulation. FEMS Microbiol Lett 95: 267±270 Moolenar WH (1995) Lysophosphatidic acid, a multifunctional phospholipid messenger. J Biol Chem 270: 12949± 12952 Moreno SNJ, Silva J, Vercesi AE, Docampo R (1994) Cytosolicfree calcium elevation in Trypanosoma cruzi is required for cell invasion. J Exp Med 180: 1535±1540 Racagni G, GarcõÂ a de Lema M, Domenech CE, Machado de Domenech EE (1992) Phospholipids in Trypanosoma cruzi:
phosphoinositide composition and turnover. Lipids 27: 275± 278 Schacht J (1981) Extraction and puri®cation of polyphosphoinositides. In: Lowenstein JM (ed) Methods in enzymology, vol 72. Academic Press, New York, pp 626±631 Sun HQ, Lin KM, Yin HL (1997) Gelsolin modulates phospholipase-C activity in-vivo through phospholipid-binding. J Cell Biol 138: 811±820 Zhu Y, Lin JHC, Verna L, Stemerman MB (1997) Activation of ICAM-1 promoter by lysophosphatidylcholine. Possible involvement of protein-tyrosine kinases. Biochim Biophys Acta 1345: 93±98