Giardia lamblia: Biochemical characterization of an ecto-ATPase activity

Experimental Parasitology 119 (2008) 279–284

Contents lists available at ScienceDirect

Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr

Giardia lamblia: Biochemical characterization of an ecto-ATPase activity Ana Acacia de Sá Pinheiro a, Daniela Cosentino-Gomes a, Adriana Lanfredi-Rangel b, Rodrigo Barbosa Ferraro a, Wanderley De Souza c, José Roberto Meyer-Fernandes a,* a

Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, CCS, Bloco H, 2°. andar, sala 013, Cidade Universitária, Ilha do Fundão, 21941-590, Rio de Janeiro, RJ, Brazil Centro de Pesquisas Goncßalo Muniz, Fundacßão Oswaldo Cruz (FIOCRUZ), Salvador, Bahia, BA, Brazil c Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, CCS, Bloco C, Cidade Universitária, Ilha do Fundão, 21941-590, Rio de Janeiro, RJ, Brazil b

a r t i c l e

i n f o

Article history: Received 2 October 2007 Received in revised form 19 February 2008 Accepted 27 February 2008 Available online 7 March 2008 This work is dedicated to Leopoldo De Meis on his 70th birthday. Index Descriptors and Abbreviations: Giardia lamblia Ecto-ATPase ENTPase

a b s t r a c t In this work, we describe the ability of living trophozoites of Giardia lamblia to hydrolyze extracellular ATP. In the absence of any divalent cations, a low level of ATP hydrolysis was observed (0.78 ± 0.08 nmol Pi  h1  106 cells). The ATP hydrolysis was stimulated by MgCl2 in a dose-dependent manner. Half maximum stimulation of ATP hydrolysis was obtained with 0.53 ± 0.07 mM. ATP was the best substrate for this enzyme. The apparent Km for ATP was 0.21 ± 0.04 mM. In the pH range from 5.6 to 8.4, in which cells were viable, this activity was not modified. The Mg2+-stimulated ATPase activity was insensitive to inhibitors of intracellular ATPases such as vanadate (P-ATPases), bafilomycin A1 (V-ATPases), and oligomycin (F-ATPases). Inhibitors of acid phosphatases (molybdate, vanadate and fluoride) or alkaline phosphatases (levamizole) had no effect on the ecto-ATPase activity. The impermeant agent DIDS and suramin, an antagonist of P2 purinoreceptors and inhibitor of some ecto-ATPases, decreased the enzymatic activity in a dose-dependent manner, confirming the external localization of this enzyme. Besides ATP, trophozoites were also able to hydrolyse ADP and 5´ AMP, but the hydrolysis of these nucleotides was not stimulated by MgCl2. Our results are indicative of the occurrence of a G. lamblia ecto-ATPase activity that may have a role in parasite physiology. Ó 2008 Elsevier Inc. All rights reserved.

1. Introduction The protozoan Giardia lamblia is one of the most important human’s enteric parasite and it has been included in the WHO Neglected Diseases Initiative (Savioli et al., 2006). In developing countries, the prevalence of human giardiasis is 20% in comparison to 5% in developed countries, where it is associated mainly with traveling and waterborne outbreaks (Roxström-Lindquist et al., 2006). Water is increasingly recognized as an important vehicle for transmission, even a zoonotic and person-to-person transmission have been considered (Thompson, 2000). Giardiasis, along with cryptosporidiosis represent the major public health concerns of water utilities in developed nations (Thompson et al., 2000). Infection is initiated by the ingestion of the cyst form, followed by excystation and colonization of the gut mucosa by Giardia trophozoites. The parasite displays distinct tissue tropism and this infection is restricted to the small intestine where Giardia attaches to the mucosal surface, exerting pathological effects (Müller and von Allmen, 2005). Attachment of the parasite to the substratum is thought to be mediated by the ventral adhesive disk. However, this mechanism may not account for the selective colonization of the proximal small intestine (Müller and von All* Corresponding author. Fax: +55 21 270 8647. E-mail address: [email protected] (J.R. Meyer-Fernandes). 0014-4894/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2008.02.006

men, 2005). Therefore, recognition and adherence must be mediated by specific host and parasite membrane determinants. Membrane-bound lectins are believed to mediate several specific cell-cell interactions, including those between parasite and host cells. In Giardia, lectins have been proposed to mediate attachment of trophozoites (Lev et al., 1986) and for causing microvillus shortening (Farthing et al., 1986). Nevertheless, variant specific proteins (VSPs) have been described as a major surface component presenting an important role in antigenic variation of the trophozoites infection (Kulakeva et al., 2006). Cell–cell recognition and adherence are central processes to many fundamental areas of biology. Surface membrane interactions between parasites and their host cells are of critical importance for the survival of the parasite, from both the immunological and the physical viewpoints (Alexander and Russell, 1992; Martiny et al., 1999, 1996). Parasite membrane components may play a role in uptake of these organisms by mammalian macrophages (Alexander and Russell, 1992; Martiny et al., 1999). Plasma membrane contains enzymes whose active sites face the external medium rather than the cytoplasm. The activities of these enzymes, referred to as ectoenzymes, can be measured using intact cells (Meyer-Fernandes, 2002). Cell membrane ecto-ATPases are integral membrane glycoproteins that are millimolar divalent-cation dependent, low-specificity enzymes that hydrolyze all nucleoside triphosphates (Kirley,

280

A.A. de Sá Pinheiro et al. / Experimental Parasitology 119 (2008) 279–284

1997; Meyer-Fernandes, 2002; Plesner, 1995). The identity and functions of ecto-ATPases have been reviewed and the nomenclature of ‘‘E-type-ATPases” was proposed to describe these enzymes (Plesner, 1995). Several hypotheses have been suggested for the physiological role of these enzymes. They include: (i) protection from cytolytic effects of extracellular ATP (Filippini et al., 1990; Steinberg and Di Virgilio, 1991; Zanovello et al., 1990); (ii) regulation of ectokinase substrate concentration (Plesner, 1995); (iii) termination of purinergic signaling (Weisman et al., 1996; Westfall et al., 1997); (iv) involvement in signal transduction (Dubyak and El-Moatassim, 1993; Margolis et al., 1990) and (v) involvement in cellular adhesion (Bisagio et al., 2003; Kirley, 1997; Pinheiro et al., 2006). In this study, we show for the first time the presence of an Mg2+-dependent ecto-ATPase activity on the cell surface of living trophozoites of G. lamblia and characterize the biochemical properties of this enzyme. The potential relevance of this enzyme to the G. lamblia physiology is also discussed. 2. Materials and methods 2.1. Chemicals All reagents were purchased from E. Merck (D-6100 Darmstadt, Germany) or Sigma–Aldrich (Sigma Co., St. Louis, MO). [c-32Pi]ATP was prepared as described by Glynn & Chappell (Glynn and Chappell, 1964). Distilled water was deionized using a MilliQ system of resins (Millipore Corp., Bedford, MA) and was used in the preparation of all solutions. 2.2. Microorganisms Trophozoites of the Portland-1 strain of G. lamblia (Meyer, 1976) were cultivated in TYI-S-33 medium supplemented with 10% heat inactivated bovine serum and 0.1% bovine bile at 37 °C (Keister, 1983). Subcultures were made twice a week and vials containing cells that had grown for 72 h (late exponential phase) were incubated on ice at 4 °C for 15 min. The free parasites were harvested by centrifugation at 500g for 7 min, washed three times and kept in 116.0 mM NaCl, 5.4 mM KCl, 5.5 mM D-glucose, 50.0 mM Hepes–Tris buffer, pH 7.2. Cellular viability was assessed, before and after incubations, by motility and trypan blue dye exclusion (de Almeida-Amaral et al., 2006). The viability of cells was not affected under the conditions employed here.

were assessed. The calibration graphs were constructed by plotting the peak area ratios against amounts inject. The hydrolysis of ATP and generation of ADP, AMP and adenosine were determinate incubating 1  107 cells/mL in a mixture containing 116.0 mM NaCl, 5.4 mM KCl, 5.5 mM D-glucose, 5.0 mM MgCl2, 50.0 mM Hepes–Tris buffer, pH 7.2 and 100 lM ATP. After 5, 10, 15, 30, 45, 60 and 120 min, aliquots of 200 lL were taken and loaded in the system for the separation. The amount of nucleotides and adenosine were calculated using the peak ratio area in the calibration graph. 2.4. Ecto-ATPase activity measurements Intact cells were incubated for 1 h at 30 °C in 0.5 mL of a mixture containing, unless otherwise specified, 116.0 mM NaCl, 5.4 mM KCl, 5.5 mM D-glucose, 50.0 mM Hepes–Tris buffer, pH 7.2, 5.0 mM ATP, and 3.0  107 cells/mL, in the absence or in the presence of 5.0 mM MgCl2. The Mg2+-dependent ecto-ATPase activity was calculated from the total activity, measured in the presence of 5 mM MgCl2, minus the basal activity, measured in the absence of MgCl2. The ATPase activity was determined by measuring the hydrolysis of [c-32Pi]ATP (104 Bq/nmol ATP) (de Almeida-Amaral et al., 2007). The experiments were started by the addition of living cells and terminated by the addition of 1.0 mL of a mixture containing 0.2 g charcoal in 1.0 M HCl at 4 °C. The tubes were then centrifuged at 1500g for 10 min at 4 °C. Aliquot (0.5 mL) of the supernatants containing the released 32Pi were transferred to scintillation vials containing 9.0 mL of scintillation fluid (2.0 g PPO in 1 L of toluene). In all experiments the ATPase activity values were calculated by subtracting the nonspecific ATP hydrolysis measured in the absence of cells. The ATP hydrolysis was linear with time under the assay conditions used and was proportional to cell number. In the experiments where other nucleotides were used, the hydrolytic activities measured under the same conditions described above were assayed spectrophotometrically by measuring the release of Pi from the nucleotides (Lowry and Lopez, 1946). The values obtained for ATPase activities measured using both methods (spectrophotometric and radioactive) were exactly the same. In the experiments where high concentrations of Mn2+ and Ca2+ were tested, possible precipitates formed were checked as previously described (Meyer-Fernandes and Vieyra, 1988). Under the conditions employed, in the reaction medium containing 50.0 mM Hepes, pH 7.2, 116.0 mM NaCl, 5.4 mM KCl, 5.5 mM D-glucose, and 5 mM ATP, no phosphate precipitates where observed in the presence of these cations. 2.5. Statistical analysis

2.3. Reverse-phase HPLC analysis The HPLC system consisted of LC-10 At pump, FCV-10AL solvent mixer, DGU-14A degasser, SPD-M10A diodearray detector, and a CLASS-LC10A (version 1.41) computing integrator; Shimadzu (Kyoto, Japan). The flow rate was maintained at 2 mL/min. The separation of the nucleotides and adenosine was achieved by ion-pair reverse-phase chromatography on an analytical SupelcosilLC-18 (46  250 mm, 5 lm particle diameter; Supelco, St. Louis, USA) equipped with a guard column Supelguard (4  20 mm, 5 lm, Supelco). The eluents, 50 mM KH2PO4, 50 mM K2HPO4, 4 mM TBAR, and 10% methanol, adjusted to pH 6.0 with H3PO4, were prepared in the day of use filtered through a 0.22-lm filter (Millipore). The methodology used was modified from the original protocol described by Kawamoto et al., 1998), for the best separation and reproducibility under ours conditions. The nucleotides and adenosine were separated (retention times, min: adenosine, 4.52 ± 0.10; AMP, 3.61 ± 0.07; ADP, 5.30 ± 0.18; ATP, 7.47 ± 0.24) and detected by UV spectroscopy at 254 nm. For calibration graphs, five replicate determinations at each concentration of a standard mixture

All experiments were performed in triplicate, with similar results obtained in at least three separate cell suspensions. Apparent Km and Vmax values were calculated using a computerized nonlinear regression analysis of the data to the Michaelis–Menten equation (Meyer-Fernandes et al., 1997). Statistical significance was determined by Student’s t test. Significance was considered as P < 0.05.

3. Results and discussion This paper reports on the presence of an Mg2+-dependent ectoATPase activity in G. lamblia trophozoites and its biochemical characterization. In all experiments, cellular integrity and viability were assessed before and after the reactions by the motility and Trypan blue dye exclusion (de Almeida-Amaral et al., 2006). The integrity of the cells was not affected by any conditions used in the assays. At pH 7.2, in the absence of any divalent metal (1 mM EDTA), ATP hydrolysis was low (0.78 ± 0.08 nmol Pi  h1  106

A.A. de Sá Pinheiro et al. / Experimental Parasitology 119 (2008) 279–284

cells). However, the addition of 5 mM MgCl2 stimulated the ATP hydrolysis and the Mg2+-dependent ecto-ATPase activity [difference between total (measured in the presence of 5 mM MgCl2) minus basal ecto-ATPase activity (measured in the presence of 1 mM EDTA)] present in this parasite hydrolyzed ATP at 5.77 ± 0.40 nmol Pi  h1  106 cells. To check the possibility that the observed ATP hydrolysis was the result of secreted soluble enzymes, as seem in other parasites (Gottlieb and Dwyer, 1981; Smith et al., 1997), a reaction mixture with cells was prepared and incubated in the absence of the substrate ATP. Subsequently, the suspension was centrifuged to remove the cells and the supernatant was checked for ATPase activity. This supernatant failed to show ATP hydrolysis either in the absence or in the presence of MgCl2 (Fig. 1A, inset). These data also rules out the possibility that the ATPase activity here described could be from lysed trophozoites of G. lamblia. The time course of ATP hydrolysis by the G. lamblia Mg2+dependent ecto-ATPase activity was linear (r2 = 0.9812) for at least 60 min (Fig. 1A), suggesting that eventual cell disruption during the course of incubation was not adding appreciably to the total activity. Similarly, in assays to determine the influence of cell density, the Mg2+-dependent ecto-ATPase activity measured over 60 min was linear (r2 = 0.9840) over a nearly tenfold range of cell density (Fig. 1B). The optimum pH for ecto-ATPases described in other parasites such as Entamoeba histolytica (Barros et al., 2000), Trichomonas vaginalis (Jesus et al., 2002a), Leishmania amazonensis (Berredo-Pinho et al., 2001) and Trypanosoma rangeli (Fonseca et al., 2006) lies on the alkaline range. Differently, in G. lamblia trophozoites, using a pH range from 5.6 to 8.4, where cells were alive throughout the time course of reaction, the Mg2+-dependent ecto-ATPase activity was not modified (Fig. 1C), as also observed in Trypanosoma cruzi

281

(Meyer-Fernandes et al., 2004) and Trypanosoma brucei brucei (Leite et al., 2007) . It has been shown that Mg2+ is an important extracellular signal in the regulation of bacteria virulence (Vescovi et al., 1996). It has also been shown that Mg2+ increased the ecto-ATPase activity from Mycoplasma homínis in a dose-dependent manner (Hopfe and Henrich, 2004). We have shown that a pathogenic E. histolytica strain has much higher Mg2+-dependent ecto-ATPase activity than the noninvasive E. histolytica or the free-living E. moshkoviskii (Barros et al., 2000). The addition of MgCl2 to the extracellular medium increased the ecto-ATPase activity of G. lamblia trophozoites in a dose-dependent manner (Fig. 1D). At 5 mM ATP, half maximal stimulation of ATP hydrolysis was obtained with 0.53 ± 0.07 mM. The addition of MnCl2, CaCl2 or SrCl2 to the extracellular medium, increased this ecto-ATPase activity to a lesser extent when compared to the addition of MgCl2, (Fig. 1D). In order to exclude the possibility that the ATP hydrolysis was due to the action of phosphatases or other type of ATPases with internal ATP binding sites, different inhibitors for those enzymes were tested. We observed that 10.0 mM sodium fluoride (NaF) and 1.0 mM sodium molybdate, two potent inhibitors of acid phosphatases (de Almeida-Amaral et al., 2006; Furuya et al., 1998; Meyer-Fernandes et al., 1997) had no effect on the ecto-ATPase activity. Similarly, levamizole (1.0 mM), a specific inhibitor of alkaline phosphatases (Van Belle, 1976) and sodium tartrate (10.0 mM), an inhibitor of secreted phosphatases (Santos et al., 2002) also failed to inhibit the ATP hydrolysis catalyzed by intact trophozoites of G. lamblia. The lack of effect observed to sodium tartrate corroborates with the absence of detectable ATPase activity in the supernatants of cells as described above (Fig. 1A, inset). The Mg2+-dependent ATPase activity was insensitive to oligomycin (10.0 lg/mL) and sodium azide (10.0 mM), two mitochondrial

Fig. 1. Time course (A), dependence on cell density (B), dependence of pH (C) and influence of different divalent cation concentrations (D) on the ecto-ATPase activity of Giardia lamblia trophozoites. Intact cells were incubated for different periods of time (A) or for 1 h (B) at 30 °C, in a reaction medium described in the Section 2. In (A) inset: comparison between ATP hydrolysis measured in supernatant and intact cells. (C) Intact cells were incubated with 50.0 mM Mes–Hepes buffer adjusted to pH values between 5.6 and 8.4 with HCl and Tris, in the presence of all components of the reaction medium. In this pH range, cells were viable throughout the course of the reaction. It was not possible observe maximal cellular viability below or above of this pH range. (D) Intact cells were incubated with increasing concentrations of Mg2+ (j), Mn2+ (N), Ca2+ (d) or Sr2+ (.) The Data are means ± SE of three determinations with different cell suspensions.

282

A.A. de Sá Pinheiro et al. / Experimental Parasitology 119 (2008) 279–284

Mg-ATPase inhibitors (Meyer-Fernandes et al., 1997). The same lack of response was observed to bafilomycin A1 (10.0 lM), a VATPase inhibitor (Bowman et al., 1988), ouabain (1.0 mM), a Na+/ K+-ATPase inhibitor (Caruso-Neves et al., 1998a) and furosemide (1.0 mM), a Na+-ATPase inhibitor (Caruso-Neves et al., 1998b). Similarly, 5´AMP and p-NPP, substrates for 50 nucleotidase and phosphatase activities, respectively, also failed to inhibit the ecto-ATPase activity. Taken together, these results clearly eliminate the possible participation of any other type of ATPases and phosphatases in the ATP hydrolysis measured in the conditions described here. Since we used intact cells to measure the hydrolytic activity in all experiments performed in this work, it is likely to suggest that the described Mg2+-dependent ATPase activity is an ecto-enzyme. So, to confirm this hypothesis, we applied the criteria that an authentic ecto-enzyme should be inhibited by an extracellular impermeant inhibitor such as 4,40 -diisothiocyanostylbene 20 ,20 disulfonic acid (DIDS) (Barros et al., 2000; Hopfe and Henrich, 2004; Meyer-Fernandes et al., 1997) and possibly by an ecto-ATPase inhibitor, such as suramin, which is also an antagonist of P2purinergic receptors (Ziganshin et al., 1995). As shown in Fig. 2A, the Mg2+-dependent activity was inhibited by DIDS in a dosedependent manner with maximal inhibitory effect (50% inhibition) obtained at 500 lM. Similar to DIDS, suramin exhibited the same

inhibition profile with maximal effect obtained with 100 lM (Fig. 2B). The dependence on ATP concentration showed a normal Michaelis–Menten kinetic for this ecto-ATPase activity and the values of Vmax and apparent Km for ATP were 5.04 ± 0.57 nmol Pi  h1  106 cells and 0.21 ± 0.04 mM, respectively (Fig. 3A). Our data are consistent with observations showing the presence of ecto-ATPase in other protozoa, including Acathamoeba sp. (Sissons et al., 2004), Toxoplasma gondii (Asai et al., 1995; Nakaar et al., 1998), Entamoeba histolytica (Barros et al., 2000), Tetrahymena thermophila (Smith et al., 1997), Crithidia deanei (Lemos et al., 2002), Herpetomonas muscarum muscarum (Alves-Ferreira et al., 2003), Leishmania sp. (Berredo-Pinho et al., 2001; Meyer-Fernandes et al., 1997; Pinheiro et al., 2006), T. cruzi (Fietto et al., 2004; Meyer-Fernandes et al., 2004), Tritrichomonas foetus (Jesus et al., 2002) and T. vaginalis (de Jesus et al., 2002). However, in Entamoeba histolytica, it was shown that the Mg2+dependent ecto-ATPase activity present on its surface was able to hydrolyze ATP and also ADP, even in less extent. This characteristic makes evident the presence of an ecto-nucleotide diphosphohydrolase activity in this parasite (Barros et al., 2000). Although E. histolytica and G. lamblia are both considered cavitary parasites, in this work we show that in G. lamblia trophozoites surface there is an Mg2+-dependent ecto-ATPase activity different from that

Fig. 2. Effect of increasing concentrations of DIDS and suramin on the Mg2+-dependent ecto-ATPase activity of Giardia lamblia trophozoites. Intact cells were incubated for 1 h at 30 °C in the same reaction medium as that described in the legend of Fig. 1, in the absence or in the presence of 5.0 mM MgCl2 with increasing concentrations of DIDS (A) or suramin (B). The Mg2+-dependent ecto-ATPase activity was calculated from the total activity, measured in the presence of 5.0 mM MgCl2, minus the basal activity, measured in the absence of MgCl2. Data are means ± SE of three determinations with different cell suspensions.

Fig. 3. Dependence on ATP concentrations on the Mg2+-dependent ecto-ATPase activity of Giardia lamblia trophozoites (A). The ATPase activity was measured at different periods of time at 30 °C in the same reaction medium as that described in the legend of Fig. 1, in the presence of increasing concentrations of ATP as shown on the abscissa. For all ATP concentrations, ATP hydrolysis did not exceed 10%. Curve represents the fit of experimental data by nonlinear regression using the Michaelis–Menten equations as described under Section 2. (Inset) Lineweaver–Burk plot. Data are means ± SE of three determinations with different cell suspensions. (B) Influence of MgCl2 on the ectophosphohydrolase activities of G. lamblia trophozoites. Intact cells were incubated for 1 h at 30 °C in the same reaction medium as that described in the legend of Fig. 1, in the presence of 5.0 mM ATP, ADP or 50 AMP. Black bars represent total activity, measured in the presence of 5.0 mM MgCl2 and blank bars represent basal activity, measured in the absence of MgCl2. In these experiments ATP hydrolysis was measured using the same spectrophotometric assay (described under Section 2) for Pi release as that used for other nucleotides. Data are means ± SE of three determinations with different cell suspensions. Significant statistical difference after comparison with ecto-phosphohydrolase activities measured in the absence of MgCl2 (p < 0.05) is marked with an asterisk.

A.A. de Sá Pinheiro et al. / Experimental Parasitology 119 (2008) 279–284

characterized in E. histolytica because, in this case, only ATP hydrolysis was stimulated by Mg2+. We also analyzed the specificity of this ecto-ATPase activity for different nucleoside 50 -triphosphate. ATP was the best substrate for this enzyme although it also hydrolyzed UTP, GTP and ITP even at very low reaction rates (data not shown). Besides ATP, G. lamblia trophozoites are also able to hydrolyze different nucleotides other than ATP, such as ADP and 50 AMP in the extracellular medium (Fig. 3B). However, as depicted in Fig. 3B, the addition of MgCl2 to the extracellular medium uniquely increased the ecto-ATPase activity whereas no effect was observed on AMP and ADP hydrolysis. This result corroborates with the idea that the 50 -nucleotidase activity present on the surface of this parasite is not responsible for the ATP hydrolysis observed. In addition, the lack of response to sodium molybdate, a potent inhibitor of 50 -nucleotidases (Gottlieb and Dwyer, 1983), indicates that this enzyme did not contribute for the ATP hydrolysis. These data also confirm that the ATP hydrolysis stimulated by Mg2+ is catalyzed by an ecto-ATPase. Extracellular ATP and its degradation products ADP, AMP and adenosine are normal components of extracellular milieu. Extracellular nucleotides do not cross the cell membrane, but rather mediate their biological actions through specific receptors on the cell surface, where they are locally metabolized by ecto-nucleotidases (Dombrowski et al., 1998; El-Moatassim et al., 1992). The three different enzymatic activities (ecto-ATPase, ecto-ADPase and ecto-50 nucleotidase) present on the surface of G. lamblia trophozoites might sequentially dephosphorylate ATP to adenosine, ATP ? ADP ? AMP ? adenosine (Fig. 3B), making adenosine available to G. lamblia from nucleotides which, because of their charge, are not permeable to the plasma membrane. Therefore, using ion-pair reversed-phase HPLC, we identified and quantified the nucleotides sequentially generated from ATP degradation by the surface-located enzymes in G. lamblia (Fig. 4). The concentration of ATP decreased by approximately 70% in 120 min of reaction, generating 25.3 ± 3.7 lM of ADP. During the course of reaction, the highest AMP concentration (17.1 ± 1.9 lM) was accompanied by its hydrolysis yielding adenosine (Fig. 4). The decrease of Mg-dependente ecto-ATPase activity from G. lamblia grown in the presence of ATP or adenosine (Fig. 5), lead us to speculate that this enzyme could play a role in the

283

Fig. 5. Influence of adenosine and ATP during growth of Giardia lamblia on the Mg+2-dependent ecto-ATPase activity. Parasites were cultivated as described in Section 2 for 2 days, in the absence (black bar) or in the presence of 1 mM adenosine (blank bar) or in the presence of 1 mM ATP (gray bar). Intact cells were incubated for 1 h at 30 °C, in a reaction medium containing 50.0 mM Hepes–Tris buffer, pH 7.2, 116.0 mM NaCl, 5.4 mM KCl, 5.5 mM D-glucose, and 5.0 mM Tris–ATP (c-32Pi)ATP (sp act 104 Bq/nmol ATP) in the absence or in the presence of 5.0 mM MgCl2. The Mg2+-dependent ecto-ATPase activity was calculated from total activity, measured in the presence of 5.0 mM MgCl2 minus the basal activity, measured in the absence of MgCl2. Data are means ± SE of three determinations with different cell suspensions. Significant statistical difference after comparison with control (p < 0.05) is marked with an asterisk.

salvage of purines from extracellular medium. The physiological role of ecto-ATPases is still unknown, however many functions have been hypothesized, including role in termination of purinergic signaling, purine recycling, and cellular adhesion (Dubyak and El-Moatassim, 1993; Kirley, 1997; Plesner, 1995). Ecto-ATPases have been described in some protozoan parasites such as Acathamoeba sp. (Sissons et al., 2004), Toxoplasma gondii (Asai et al., 1995; Nakaar et al., 1998), E. histolytica (Barros et al., 2000), T. thermophila (Smith et al., 1997), Leishmania sp. (Berredo-Pinho et al., 2001; Meyer-Fernandes et al., 1997; Pinheiro et al., 2006), T. cruzi (Fietto et al., 2004; Meyer-Fernandes et al., 2004), T. rangeli (Fonseca et al., 2006), T. brucei brucei (Leite et al., 2007), T. foetus (Jesus et al., 2002) and T. vaginalis (de Jesus et al., 2002). The fact that virulent promastigotes of L. amazonensis has higher Mg2+-dependent ecto-ATPase activity then avirulent promastigotes (Berredo-Pinho et al., 2001), as well as the invasive form of E. histolytica presents much higher Mg2+dependent ecto-ATPase activity than noninvasive form (Barros et al., 2000), support the speculative idea that the presence of the ecto-ATPase activity in trophozoites of G. lamblia could also be considered a pathogenesis marker for this cell. Acknowledgments We acknowledge the excellent technical assistance of Fabiano Ferreira Esteves. This work was supported by grants from the Brazilian Agencies Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundacßão Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Fundacßão de Amparo á Pesquisa do Estado da Bahia (FAPESB). References

Fig. 4. Analysis of ATP hydrolysis by Giardia lamblia trophozoites. The parasites (1  107 cells/mL) were incubated for each indicated period of time at 30 °C in the presence of 116.0 mM NaCl, 5.4 mM KCl, 5.5 mM D-glucose, 5.0 mM MgCl2, 50.0 mM Hepes–Tris buffer, pH 7.2, and 100 lM ATP. The amounts of nucleotides (ATP, ADP, and AMP) and adenosine were determined by HPLC, as described in Section 2. Data are means ± SE of three determinations with different cell suspensions.

Alexander, J., Russell, D., 1992. The interaction of Leishmania species with macrophages. Advance Parasitology 31, 175–254. Alves-Ferreira, M., Dutra, P.M.L., Lopes, A.H.C.S., Ferreira-Pereira, A., Scofano, H.M., Meyer-Fernandes, J.R., 2003. Magnesium-dependent ecto-ATP diphosphohydrolase activity in Herptomons muscarum muscarum. Current Microbiology 47, 265–271.

284

A.A. de Sá Pinheiro et al. / Experimental Parasitology 119 (2008) 279–284

Asai, T., Miura, S., Sibley, L.D., Okabayashi, H., Takeuchi, T., 1995. Biochemical and molecular characterization of nucleoside triphosphate hydrolase isozymes from the parasitic protozoan Toxoplasma gondii. The Journal of Biological Chemistry 270, 11391–11397. Barros, F.S., De Menezes, L.F., Pinheiro, A.A.S., Silva, E.F., Lopes, A.H.C.S., De Souza, W., Meyer-Fernandes, J.R., 2000. Ectonucleotide diphosphohydrolase activities in Entamoeba histolytica. Archives of Biochemistry and Biophysics 375, 304–314. Berredo-Pinho, M., Peres-Sampaio, C.E., Chrispim, P.P., Belmont-Firpo, R., Lemos, A.P., Martiny, A., Vannier-Santos, M.A., Meyer-Fernandes, J.R., 2001. A Mgdependent ecto-ATPase in Leishmania amazonensis and its possible role in adenosine acquisition and virulence. Archives of Biochemistry and Biophysics 391, 16–24. Bisagio, D.F.R., Peres-Sampaio, C.E., Meyer-Fernandes, J.R., Souto-Padrón, T., 2003. Ecto-ATPase activity on the surface of Trypanosoma cruzi and its possible role in the parasite–host cell interaction. Parasitology Research 91, 273–282. Bowman, E.J., Siebers, A., Altendorf, K., 1988. Bafilomycins: a class of inhibitors of membrane ATPases from microorganisms, animal cells, and plant cells. Proceedings of the National Academy of Science of the United States of America 85, 7972–7976. Dombrowski, K.E., Ke, Y., Brewer, K.A., Kapp, J.A., 1998. Ecto-ATPase: an activation marker necessary for effector cell function. Immunological Reviews 161, 111–118. Dubyak, G., El-Moatassim, C., 1993. Signal transduction via P2-purinergic receptors for extracellular ATP and other nucleotides. The American Journal of Physiology 265, C577–C606. Caruso-neves, C., Meyer-Fernandes, J.R., Saad-Nehme, J., Lopes, A.G., 1998a. Osmotic modulation of the ouabain-sensitive (Na+ +K+) ATPase from Malpighian tubules of Rhodnius prolixus. Zeitschrift fùr Naturforschung. 53, 911–917. Caruso-neves, C., Meyer-Fernandes, J.R., Saad-Nehme, J., Proverbio, F., Marin, R., Lopes, A.G., 1998b. Ouabain insensitive Na+-ATPase activity of malpighian tubules from Rhodnius prolixus. Comparative Biochemistry and Physiology B 119, 807–811. de Almeida-Amaral, E.E., Belmont-Firpo, R., Vannier-Santos, M.A., Meyer-Fernandes, J.R., 2006. Leishmania amazonensis: Characterization of an ecto-phosphatase activity. Experimental Parasitology 114, 334–340. de Almeida-Amaral, E.E., Caruso-Neves, C., Lara, L.S., Pinheiro, C.M., MeyerFernandes, J.R., 2007. Leishmania amazonensis: PKC-like protein kinase modulates the (Na+ + K+)ATPase activity. Experimental Parasitology 116, 419– 426. de Jesus, J.B., Pinheiro, A.A.S., Lopes, A.H.C.S., Meyer-Fernandes, J.R., 2002b. An ectonucleotide ATP-diphosphohydrolase activity in Trichomonas vaginalis stimulated by galactose and its possible role in virulence. Zeitschrift fur Naturforschung 57c, 890–896. El-Moatassim, C., Dornand, J., Mani, J., 1992. Extracellular ATP and cell signalling. Biochimica et Biophysica Acta 1134, 31–45. Farthing, M.J., Pereira, M.E., Keusch, G.T., 1986. Description and characterization of a surface lectin from Giardia lamblia. Infection and Immunity 51, 661–667. Fietto, J.L., DeMarco, R., Nascimento, I.P., Castro, I.M., Carvalho, T.M., de Souza, W., Bahia, M.T., Alves, M.J., Verjovski-Almeida, S., 2004. Characterization and immunolocalization of an NTP diphosphohydrolase of Trypanosoma cruzi. Biochemical and Biophysical Research Communications 316, 454–460. Filippini, A., Taffs, R.E., Agui, T., Sitkovsky, M.V., 1990. Ecto-ATPase activity in cytolytic T-lymphocytes. Protection from the cytolytic effects of extracellular ATP. The Journal of Biological Chemistry 265, 334–340. Fonseca, F.V., de Souza, A.L.F., Mariano, A.C., Entringer, P.F., Gondim, K.C., MeyerFernandes, J.R., 2006. Trypanosoma rangeli: characterization of a Mgdependent ecto-ATP-diphosphohydrolase activity. Experimental Parasitology 112, 76–84. Furuya, T., Zhong, L., Meyer-Fernandes, J.R., Lu, H.G., Moreno, S.N., Docampo, R., 1998. Ecto-protein tyrosine phosphatase activity in Trypanosoma cruzi infective stages. Molecular and Biochemical Parasitology 92, 339–348. Glynn, I.M., Chappell, J.B., 1964. A simple method for the preparation of 32P-labelled adenosine triphosphate of high specific activity. Biochemical Journal 90, 147– 149. Gottlieb, M., Dwyer, D.M., 1981. Protozoan parasite of humans: surface membrane with externally disposed acid phosphatase. Science 212, 939–941. Gottlieb, M., Dwyer, D.M., 1983. Evidence for distinct 50 - and 30 -nucleotidase activities in the surface membrane fraction of Leishmania donovani promastigotes. Molecular and Biochemical Parasitology 7, 303–317. Hopfe, M., Henrich, B., 2004. OppA, the substrate-binding subunit of the oligopeptide permease, is the major ecto-ATPase of Mycoplasma hominis. Journal of Bacteriology 186, 1021–1028. Jesus, J.B., Lopes, A.H., Meyer-Fernandes, J.R., 2002. Characterization of an ectoATPase of Tritrichomonas foetus. Veterinary Parasitology 103, 29–42. Kawamoto, Y., Shinozuka, K., Kunitomo, M., Haginaka, J., 1998. Determination of ATP and its metabolites released from rat caudal artery by isocratic ion-pair reversed-phase high-performance liquid chromatography. Analytical Biochemistry 262, 33–38. Keister, D.B., 1983. Axenic culture of Giardia lamblia in TYI-S-33 medium supplemented with bile. Transactions of the Royal Society of Tropical Medicine and Hygiene 77, 487–488. Kirley, T.L., 1997. Complementary DNA cloning and sequencing of the chicken muscle ecto-ATPase. Homology with the lymphoid cell activation antigen CD39. The Journal of Biological Chemistry 272, 1076–1081. Kulakeva, L., Singer, S.M., Conrad, J., Nash, T.R., 2006. Epigenetic mechanisms are involved in the control of Giardia lamblia antigenic variation. Molecular Microbiology 61, 1533–1542.

Leite, M.D.S., Thomaz, R., Fonseca, F.V., Panizzutti, R., Vercesi, A.E., Meyer-Fernandes, J.R., 2007. Trypanosoma brucei brucei: biochemical characterization of ectonucleoside triphosphate diphosphohydrolase activities. Experimental Parasitology 115, 315–323. Lemos, A.P., Pinheiro, A.A.S., Berredo-Pinho, M., Souza, A.L.F., Motta, C.M., De Souza, W., Meyer-Fernandes, J.R., 2002. Ectonucleotide diphosphohydrolase activity in Crithidia deanei. Parasitology Research 88, 905–911. Lev, B., Ward, H., Keusch, G., Pereira, M., 1986. Lectin activation in Giardia lamblia by host protease: a novel host–parasite interaction. Science 232, 71–73. Lowry, H.O., Lopez, M., 1946. The determination of inorganic phosphate in the presence of labile phosphate esters. The Journal of Biological Chemistry 162, 421–428. Margolis, R.N., Schell, M.J., Taylor, S.I., Hubbard, A.L., 1990. Hepatocyte plasma membrane ECTO-ATPase (pp120/HA4) is a substrate for tyrosine kinase activity of the insulin receptor. Biochemical and Biophysical Research Communications 166, 562–566. Martiny, A., Meyer-Fernandes, J., De Souza, W., Vannier-Santos, M., 1999. Altered tyrosine phosphorylation of ERK1 MAP kinase and other macrophage molecules caused by Leishmania amastigotes. Molecular and Biochemical Parasitology 102, 1–12. Martiny, A., Vannier-Santos, M., Borges, V., Meyer-Fernandes, J., Assreuy, J., Cunha e Silva, N., De Souza, W., 1996. Leishmania-induced tyrosine phosphorylation in the host macrophage and its implication to infection. European Journal of Cell Biology 71, 206–215. Meyer-Fernandes, J., 2002. Ecto-ATPases in protozoa parasites: looking for a function. Parasitology International 51, 299–303. Meyer-Fernandes, J.R., Saad-Nehme, J., Peres-Sampaio, C.E., Belmont-Firpo, R., Bissagio, D.F.R., Couto, L.C., Fonseca de Souza, A.L., Lopes, A.H.S.C., SoutoPadrón, T., 2004. A Mg-dependent ecto-ATPase is increased in the infective stages of Trypanosoma cruzi. Parasitology Research 93, 567–576. Meyer-Fernandes, J.R., Dutra, P.M., Rodrigues, C.O., Saad-Nehme, J., Lopes, A.H.S.C., 1997. Mg-dependent ecto-ATPase activity in Leishmania tropica. Archives of Biochemistry and Biophysics 341, 40–46. Meyer-Fernandes, J.R., Vieyra, A., 1988. Pyrophosphate formation from acetyl phosphate and orthophosphate: evidence for heterogeneous catalysis. Archives of Biochemistry and Biophysics 266, 132–141. Meyer, E.A., 1976. Giardia lamblia: isolation and axenic cultivation. Experimental Parasitology 39, 101–105. Müller, N., von Allmen, N., 2005. Recent insights into the mucosal reactions associated with Giardia lamblia infections. International Journal for Parasitology 35, 1339–1347. Nakaar, V., Beckers, C., Polotsky, V., Joiner, K., 1998. Basis for substrate specificity of the Toxoplasma gondii nucleoside triphosphate hydrolase. Molecular and Biochemistry Parasitology 97, 209–220. Pinheiro, C.M., Martins-Duarte, E.S., Ferraro, R.B., Fonseca de Souza, A.L., Gomes, M.T., Lopes, A.H., Vannier-Santos, M.A., Santos, A.L., Meyer-Fernandes, J.R., 2006. Leishmania amazonensis: biological and biochemical characterization of ectonucleoside triphosphate diphosphohydrolase activities. Experimental Parasitology 114, 16–25. Plesner, L., 1995. Ecto-ATPases: identities and functions. International Review of Cytology 158, 141–214. Roxström-Lindquist, K., Palm, D., Reiner, D., Ringqvist, E., Svärd, S.G., 2006. Giardia immunity—an update. Trends in Parasitology 22, 26–31. Savioli, L., Smith, H., Thompson, A., 2006. Giardia and Cryptosporidium join the ‘Neglected Diseases Initiative’. Trends in Parasitology 22, 203–208. Santos, A.L.S., Souto-Padrón, T., Alviano, C.S., Lopes, A.H.C.S., Soares, R.M.A., MeyerFernandes, J.R., 2002. Secreted phosphatase activity induced by dimethyl sulfoxide in Herpetomonas samuelpessoai. Archives of Biochemistry and Biophysics 405, 191–198. Smith Jr., T.M., Kirley, T.L., Hennessey, T.M., 1997. A soluble ecto-ATPase from Tetrahymena thermophila: purification and similarity to the membrane-bound ecto-ATPase of smooth muscle. Archives of Biochemistry and Biophysics 337, 351–359. Sissons, J., Alsam, S., Jayasekera, S., Khan, N.A., 2004. Ecto-ATPases of clinical and non-clinical isolates of Acanthamoeba. Microbial Pathogenesis 37, 231–239. Steinberg, T., Di Virgilio, F., 1991. Cell-mediated cytotoxicity: ATP as an effector and the role of target cells. Current Opinion in Immunology 3, 71–75. Thompson, R.C., 2000. Giardiasis as a re-emerging infectious disease and its zoonotic potential. International Journal for Parasitology 30, 1259–1267. Thompson, R.C., Hopkins, R.M., Homan, W.L., 2000. Nomenclature and genetic groupings of Giardia infecting mammals. Parasitology Today 16, 210–213. Van Belle, H., 1976. Alkaline phosphatase. I. Kinetics and inhibition by levamisole of purified isoenzymes from humans. Clinical Chemistry 22, 972–976. Vescovi, E.G., Soncini, F.C., Groisman, E.A., 1996. Mg2+ as an extracellular signal: environmental regulation of Salmonella virulence. Cell 84, 165–174. Weisman, G., Turner, J., Fedan, J., 1996. Structure and function of P2 purinocepters. The Journal of Pharmacology and Experimental Therapeutics 277, 1–9. Westfall, T., Kennedy, C., Sneddon, P., 1997. The ecto-ATPase inhibitor ARL 67156 enhances parasympathetic neurotransmission in the guinea-pig urinary bladder. European Journal of Pharmacology 329, 169–173. Zanovello, P., Bronte, V., Rosato, A., Pizzo, P., Di Virgilio, F., 1990. Responses of mouse lymphocytes to extracellular ATP. II. Extracellular ATP causes cell typedependent lysis and DNA fragmentation. Journal of Immunology 145, 1545– 1550. Ziganshin, A., Ziganshina, L., King, B., Burnstock, G., 1995. Characteristics of ectoATPase of Xenopus oocytes and the inhibitory actions of suramin on ATP breakdown. Pflügers Archives 429, 412–418.