Entamoeba histolytica: Signaling through G Proteins

Experimental Parasitology 91, 170–175 (1999) Article ID expr.1998.4361, available online at http://www.idealibrary.com on

Entamoeba histolytica: Signaling through G Proteins

´ ´ Cristina Paveto,* Hector N. Torres,* Mirtha M. Flawia,* Matilde Garcıa-Espitia,† , , Arturo Ortega,‡ § and Esther Orozco† § *Instituto de Investigaciones en Ingenierı´a Gene´tica y Biologı´a Molecular and Facultad de Ciencias Exactas y Naturales, Obligado 2490, 1428, Buenos Aires, Argentina; †Departamento de Patologı´a Experimental ´ ´ ´ and ‡Departamento de Genetica y Biologıa Molecular, CINVESTAV-IPN, Apartado Postal 14-740 Mexico DF 07000 Me´xico; and §Posgrado en Biomedicina Molecular, CICATA-IPN, Me´xico DF, Me´xico

´ Paveto, C., Torres, H. N., Flavia, M. M., Garcıa-Espitia, M., Ortega, A., and Orozco, E. 1999. Entamoeba histolytica: Signaling through G proteins. Experimental Parasitology 91, 170–175. The intracellular signaling pathways of Entamoeba histolytica are largely unknown. Although the expression of guanine nucleotide binding proteins (G proteins) is expected from functional studies, their biochemical characterization remains elusive in this protozoan. Using a combination of biochemical and immunological studies, we provide strong evidence for the presence of a Gs protein in amoeba. Our results streghthen our understanding of the signal transduction mechanisms in E. histolytica as potential sites of a new therapeutic strategy. 䉷 1999 Academic Press Index Descriptors and Abbreviations: Entamoeba histolytica; guanine nucleotide binding proteins; signal transduction; ECM, extracellular matrix; EDGs, electron-dense granules; pp125FAK, focal adhesion kinase; MAPK, mitogen-activated protein kinase; FN, fibronectin; PKC, Ca2⫹/diacylglycerol-dependent protein kinase; G proteins, guanine nucleotide binding proteins; PHBM, p-hydroximercuribenzoic acid; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; CTX, cholera toxin; DTT, dithiothreitol; BSA, bovine serum albumin.

capability of host connective tissues during amoebic inva˜ sion (Munoz et al., 1982). The attachment of trophozoites to ECM induces focal contact formation and release of electron-dense granules (EDGs) containing collagenase (Gadassi and Kesseler, 1983). The signaling pathways activated in the amoeba have begun to be elucidated. Among these, a collagen-dependent tyrosine kinase cascade that includes pp125FAK and p42MAPK ´ homologues takes place (Perez et al. 1996). Fibronectin induces a sustained rise in intracellular Ca2⫹ concentrations, with a subsequent activation of the Ca2⫹/diacylglycerol-dependent protein kinase (PKC) and the Ca2⫹/calmodulin-dependent protein kinase (Carbajal et al. 1996). Inositol phosphates apparently play a major role as second messengers that control the parasite’s Ca2⫹ homeostasis (Giri et al. 1996). Cell–cell and cell–matrix adhesion molecules participate in processes which are in turn controlled by extracellular factors that act through G-protein-coupled receptors and receptor tyrosine kinases, as well as bioactive lipids such as lysophosphatidic acid, sphingosine, and shingosylphosphorylcholine. In principle, transmembrane signaling processes are governed by two fundamentally different and seemingly unrelated mechanisms. Some membrane receptors intramolecularly combine an extracellular ligand binding domain and an intracellular or transmembrane effector domain. This transduction principle is characterized by a rigidly coupled

INTRODUCTION The enteric protozoan parasite Entamoeba histolytica causes amoebic dysentery, a disease that is prevalent in tropical countries (Walsh, 1986). Binding of trophozoites to target cells is mediated by extracellular matrix (ECM) components. Therefore, it has been suggested that the secretion of collagenase activity is associated to the penetration

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receptor–effector system (Ismaa et al. 1995). Most intracellular signaling molecules, however, bind to membranous receptors that represent one element of a three-component transmembrane signaling system whose individual elements interact sequentially and reversably. Agonist binding to a specific receptor results in the activation of heteromeric guanine nucleotide binding proteins (G proteins), which act as transducers and signal amplifiers. G proteins subsequently modulate the activity of effectors, resulting in rapid alterations of second messenger concentration (Neer 1995). The molecular identity of the amoeba ECM receptors is unknown. To date, no G protein has been unequivocally demonstrated to participate in the signal transduction cascades in E. histolytica. The common evolutionary origin of these proteins and the presence of some of its effectors favor the expression of these important components of the signaling machinery in the parasite. In this context, the present studies provide evidence for the presence and functionality of G proteins in the parasitic protozoan E. histolytica.

MATERIALS AND METHODS

Chemicals. All reagents were of analytical grade. Radiochemicals and antiserum were purchased from New England Nuclear and cholera toxin (CTX) from Sigma. The cAMP assay kit was obtained from Amersham. Cell cultures. E. histolytica strain HM-1:IMSS trophozoites were cultured in TYI-S-33 medium (Diamond et al. 1978). All experiments were carried out with trophozoites harvested after 48 h of culture. cAMP accumulation. Approximately 500,000 amoebas plated on 24-well culture dishes were stimulated for the indicated time periods with 5 million freshly isolated human red blood cells. The stimulation was finished by rapid aspiration of the media followed by three washes with phosphate-buffered saline (PBS). The cyclic nucleotides were extracted with perchloric acid. The samples were neutralized with KOH and the cAMP levels were determined by means of a protein binding assay. Preparation of crude membranes. Cells were collected by centrifugation, washed twice with PBS, and resuspended in 20 mM Tris–HCl buffer, pH 7.5, 0.5 mM PMSF, 1 mM PHBM, 10 ␮g/ml pepstatin, 10 ␮g/ml leupeptin, and 1 mM benzamidine. Cells in suspension were broken by freezing and thawing three times with liquid N2. The homogenate was centrifuged in a microcentrifuge for 10 min at 4000 rpm, the pelleted unbroken cells were discarded, and the supernatant was recentrifuged at 14,000 rpm for 30 min at 4⬚C. The resultant pellet, resuspended in the appropriate buffer, was considered the crude membrane fraction. Protein concentration was determined by the Bradford (1976) method. Adenylyl cyclase assay. Adenylyl cyclase activity was assayed as already described (Farber et al. 1995) in a reaction mixture containing 50 mM Tris–HCl buffer, pH 7.4, 1 mg/ml BSA, 0.2 mM 3-isobutyl1-methylxantine, 2.5 mM MnCl2 or MgCl2, 0.5 mM [␥-32P] ATP (sp

act 200 cpm/pmol), 2 mM phosphocreatine, 0.2 mg creatine kinase, and 20–100 ␮g membrane protein, as well as the indicated additions, in a final assay volume of 0.1 ml. Incubation was performed at 30⬚C for 15 min and the reaction was stopped by the addition of 0.12 ml of 0.5 N HCl with 0.005 ␮Ci of [3H]cAMP. The [32P]cAMP produced was determined according to Alvarez and Daniels (1990). Toxin treatment. ADP ribosylation of crude membranes by CTX was performed as described previously (Paveto et al., 1992) with minor modifications. CTX (1 mg/ml) was activated for 30 min at 37⬚C in 20 mM DTT and 1 mg/ml BSA. Crude membrane preparation (1 mg/ml) and preactivated toxin (100 ␮g/ml) were mixed vol/vol and incubated for 1 h at 37⬚C in the presence of 20 mM Tris–HCl buffer, pH 7.5, 1 mM ATP, 0.1 mM GTP, and either 100 ␮M unlabeled NAD or 1 ␮M [32P]NAD (sp act 5 Ci/mmol). The reaction mixture was then centrifuged at 14,000 rpm for 15 min and the resulting pellet assayed for adenylyl cyclase activity or subjected to 10% SDS–PAGE, stained with Coomassie blue, destained, dried, and autoradiographed. GTP binding assay. Binding of [35S]GTP-␥-S to amoeba membrane preparations was determined by the method of Northup (Northup et al., 1982). Approximately 20 ␮g of crude membrane preparation was diluted with 80 ␮l of 20 mM Tris–HCl, pH 8, 1 mM EDTA, 1 mM DTT, the previously mentioned protease inhibitors, and 0.1% Lubrol PX (50–100 ␮g protein). The binding reaction was initiated by the addition of 100 ␮l of a solution containing 50 mM Tris–HCl, pH 8, 1 mM EDTA, 20 mM MgCl2, 100 mM NaCl, 100 nM GTP-␥-S, and 5 ⫻ 105 cpm [35S]GTP-␥-S. The mixture was incubated for 60 min at 28⬚C with shaking and stopped by dilution with 2 ml of ice-cold washing buffer containing 20 mM Tris–HCl, pH 8, 25 mM MgCl2, and 100 mM NaCl. It was immediately filtered through BA 85 nitrocellulose filters, washed five times with 2 ml of cold washing buffer, dried, and assayed for radioactivity in a liquid scintillation counter. Saturation experiments were performed within a concentration range of 1–1000 nm GTP-␥-S with a fixed amount of [35S]GTP-␥-S. Nonspecific binding was calculated in the presence of 100 ␮M GTP-␥-S and it represented less than 10% of the total binding. Immunological detection of G protein subunits. Samples of the membrane preparations (20–40 ␮g protein) were subjected to 10% SDS–PAGE, transferred to nitrocellulose membranes, and analyzed by Western blotting with the following specific antisera (from New England Nuclear): GA/1 (anti-␣ common), MS/1(anti-␤ common), and AS/7 (anti-␣t, anti-␣I1, anti-␣i2). For immunocytochemical studies, trophozoites were plated on gelatin-coated coverslips. Cells were fixed and permeabilized with methanol at room temperature for 30 min and washed with 0.2% Triton X-100 in PBS (PBS–Triton). Trophozoites were then incubated with 1% bovine serum albumin (BSA) for 20 min at room temperature, washed twice with PBS–Triton, and incubated overnight at 4⬚C with the primary antibody. The cells were washed twice with PBS–Triton, incubated with fluorescein-labeled goat antirabbit antisera for 90 min at 37⬚C, and washed with PBS–Triton. Trophozoites were examined with a Zeiss Axiovert 35 M microscope attached to a laser confocal scanning system MRC 600 (Bio–Rad).

RESULTS Characterization of E. histolytica adenylyl cyclase activity. When we decided to investigate the expression of

172 functional trimeric G proteins in amoeba, one of the first criteria that we had to investigate was the presence of a typical effector. In this context, we explored adenylyl cyclase activity. The stimulation of E. histolytica trophozoites with freshly isolated human red blood cells (RBC) elicits a timedependent accumulation of cAMP, which reaches its maximum value after 5 min of stimulation and returns to basal levels after 30 min (Fig. 1). As expected a 15-min exposure of the trophozoites to 100 ␮M Forskolin results in a threefold stimulation of cAMP levels. The adenylyl cyclase activity associated to E. histolytica membranes is shown in Table 1. Pretreatment of membranes with CTX increased threefold the Mg2⫹-stimulated activity. This fact is functional evidence for a G␣s protein involved in the regulation of E. histolytica adenylyl cyclase. [32P]NAD ribosylation of E. histolytica membranes. The above-mentioned CTX-induced changes in adenylyl cyclase enzymatic activity were correlated to the analysis of toxin-treated membranes by SDS–PAGE. As is depicted in Fig. 2, CTX in the presence of adenylate [32P]NAD produced the incorporation of radioactivity to a main protein with an apparent molecular weight of 40 kDa. When the membranes were incubated in the absence of toxin or with an excess of nonradioactive NAD no protein was detected, clearly suggesting the identity of the labeled polypeptide as a G protein ␣ subunit. GTP binding activity. To further confirm the expression of a trimeric G protein in amoeba, we performed [35S]GTP␥-S binding assays in E. histolytica membrane preparations.

FIG. 1. RBC-induced increase in cAMP levels in Entamoeba histolytica trophozoites. After several washes with PBS, the cells were exposed to 5 million red blood cells for the indicated times, after which the medium was rapidly aspireted, the cells were washed three times with PBS, and the cyclic nucleotides were extracted with perchloric acid. Forskolin (100 ␮M) was included as a positive control.

PAVETO ET AL.

TABLE I Adenylyl Cyclase Activity from Control and Toxin-Treated Membranes

Metal

Additions

Treatment

2⫹

None GTP-␥-S GTP-␥-S None GTP-␥-S GTP-␥-S GDP-␤-S

None None CTX None None CTX None

Mn Mn2⫹ Mn2⫹ Mg2⫹ Mg2⫹ Mg2⫹ Mg2⫹

Adenylyl cyclase (pmol/min/mg/ protein) 9.43 8.98 14.06 0.89 2.66 9.17

⫾ ⫾ ⫾ ⫾ ⫾ ⫾ 0

0.44 0.49 0.54 0.02 0.37 0.45

Note. Aliquots of membrane preparations were preincubated for 15 min at 30⬚C with 100 mM NAD+ in the presence or the absence of preactivated CTX and assayed for adenylyl cyclase with the indicated additions. Data are the mean ⫾ standard deviation of triplicate experiments.

As depicted in Fig. 3, we were able to detect specific [35S]GTP-␥-S binding, which was effectively competed by increasing concentrations of GTP but insentitive to 100 ␮M ATP or GMP; these results are in line with the presence of G protein ␣ subunits in the membrane preparations. Immunological detection of G protein subunits. Finally, an immunological approach was undertaken to fully confirm the biochemical data detailed above concerning the presence of trimeric G proteins in amoeba. For this purpose, we electrophoretically separated proteins of E. histolytica membranes and analyzed them via Western blots with commercial antibodies directed against G protein common ␣ subunit (GA/1), G␣i subunit (AS/7), and G protein ␤ subunit (MS/

FIG. 2. CTX-induced [32P]NAD ribosylation of E. histolytica membranes. Ribosylation reaction was carried out as described under Materials and Methods and analyzed in 10% SDS–PAGE slab gels. Lane 1; heart membranes (15 ␮g) plus CTX; lane 2, E. histolytica membranes (7 ␮g); lane 3; E. histolytica membranes (7 ␮g) plus CTX; lane 4; 15 ␮g E. histolytica membranes plus CTX; lane 5, 15 ␮g E. histolytica membranes plus CTX and 1 mM NAD. After drying, the gels were exposed to X-ray films for 12 h.

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DISCUSSION

FIG. 3. Displacement by increasing concentrations of GTP of the binding of 100 nM [35S]GTP-␥-S to E. histolytica membranes. Each point is the mean of at least three determinations.

1). The results are shown in Fig. 4; both anti-␣ common and anti-␣i antisera strongly detected a single band, with an apparent molecular weight of 40 kDa. When the blots were revealed with anti-␤ antiserum a main band of about 30 kDa was observed. As expected, no immunoreactive bands were detected in cytosolic preparations. When these same antibodies were used in immunocytochemical studies, Fig. 5, all of them were able to decorate fixed trophozoites. It is important to note that immunoreactivity is not restricted to the plasma membrane, suggesting the presence of G proteins associated to intracellular membranes and/or to the cytoskeleton.

FIG. 4. Western blots of proteins from E. histolytica and bovine tissue after incubation with GA/1, AS67, and MS61 antisera and peroxidase-linked goat anti-rabbit IgGs. In the three panels: lane 1, E. histolytica membranes; lane 2, bovine heart membranes; lane 3, bovine brain membranes. Immunopositive polypeptides were revealed with the ECL kit (Amersham).

The signal transduction components in E. histolytica are ´ far from being completely understood (Perez et al. 1996). Since the functioning of cells relies in the fidelity of signaling pathways, the manipulation of them offers an alternative to anti-parasitic drug development. In this context, the results presented in this study provide compelling evidence for the expression of functional trimeric G proteins in E. histolytica trophozoites. Although it has been suggested that fibronectin (FN) stimulates the accumulation of inositol phosphates, diacilglycerol, and even cAMP in a G-protein-dependent pathway (Raggi and Meza 1997), no evidence for the expression of bona fide trimeric G proteins is available. We thus decided to investigate the issue further. First we decided to explore the biochemical characteristics of a typical G protein effector: adenylyl cyclase. When E. histolytica trophozoites are stimulated with human RBC a time-dependent increase of cAMP levels is observed (Fig. 1). This increase is also detected when the cells are exposed to 100 ␮M Forskolin, a direct activator of adenylyl cyclase. If a G protein participates in the modulation of cAMP levels in amoeba, then adenylyl cyclase activity has to be modified in the presence of nonhydrolyzable analogues of GTP. This is indeed the case. The values of specific activity detected in the presence of Mn2⫹ were higher than those with Mg2⫹ and GTP-␥-S, resembling the classical model of the vertebrate system in which Mn2⫹ uncouples G protein regulation from adenylyl cyclase (Paveto et al., 1990). Moreover, Mg2⫹-dependent activity could scarcely be demonstrated when GTP or GTP-␥-S was absent in the reaction mixture (Table I). In line with these observations, treatment of the membranes with CTX resulted in a significant increase in activity that was perfectly correlated with the appearance of a [32P]NAD-labeled polypeptide with an apparent molecular weight of 40 kDa in membrane preparations treated with the toxin. It can be argued that the labeled polypeptide does not migrate as a defined sharp band. This most probably refects the fact that these experiments were done in the absence of protease inhibitors. Nevertheless, when an excess of NAD is included in the assay, the radiolabeled material indicated by the arrow disapears, establishing its specificity. These results together with the binding activity depicted in Fig. 3 were all suggestive of the expression of G proteins in amoeba. Taking advantage of the reported cross-reactivity of antiG protein subunit (MS/1, GA/1, and AS/7) antibodies to Phytomonas proteins (Farber et al. 1995), we decided to use

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FIG. 5. Immunohistochemical detection of G protein subunits in E. histolytica trophozoites. (A) Flourescence background detected in cells exposed to fluorescein-labeled goat anti-rabbit Abs. (B) Staining with GA/1 antiserum (␣ common). (C) Staining with anti-G␣i antibodies (AS/7). (D) Staining with anti-␤ antiserum.

these antisera in order to identify E. histolytica G proteins. As shown in Fig. 4 we could detect the ␣i and the ␤ subunits. The specificity of the immune reaction was controlled using preimmune rabbit serum. An unexpected finding was observed when these antibodies were used in immunocytochemical studies (Fig. 5). Cytoplasmatic labeling was detected rather than a plasma membrane preferential decorating, as one would have expected. A plausible explanation for this labeling pattern could be the reported association of the ␣ and ␤ subunits to putative plasmalemmal caveolae (Chang et al. 1994; Sergiacomo et al. 1993). Another possibility is that association is taking place with the cytoskeletal network (Clapham and Neer 1997). In summary, the present findings demonstrate the expression of functional heteromeric G proteins in amoeba. Their participation in the signal transduction mechanisms in E. histolytica is currently being investigated in our laboratories.

ACKNOWLEDGMENTS

E.O. is an International Fellow at the Howard Hughes Medical ´ Institute. This work was supported by grants from CONACYT-Mexico ´ to E.O. and A.O. and from Fundacion Antorchas to C.P.

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