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Secreted phosphatase activity induced by dimethyl sulfoxide in Herpetomonas samuelpessoai
ABB Archives of Biochemistry and Biophysics 405 (2002) 191–198 www.academicpress.com
Secreted phosphatase activity induced by dimethyl sulfoxide in Herpetomonas samuelpessoai Andre L.S. Santos,a Thais Souto-Padr on,a Celuta S. Alviano,a Angela H.C.S. Lopes,a Rosangela M.A. Soares,a and Jose R. Meyer-Fernandesb,* a
Instituto de Microbiologia Prof. Paulo de G oes, CCS, Universidade Federal do Rio de Janeiro, Cidade Universit aria, Ilha do Fund~ ao, Rio de Janeiro, RJ 21941-590, Brazil b Departamento de Bioquımica M edica, ICB, Universidade Federal do Rio de Janeiro, CCS, Bloco H-2 andar – sala, 13 Cidade Universit aria, Ilha do Fund~ ao, Rio de Janeiro, RJ 21941-590, Brazil Received 26 March 2002, and in revised form 15 July 2002
Abstract A phosphatase activity of the trypanosomatid parasite Herpetomonas samuelpessoai was characterized using intact living cells. The effects of dimethyl sulfoxide (DMSO) on this activity were investigated. This phosphatase activity (2:53 0:01 nmol Pi /mg protein min) was linear with cell density and with time for at least 60 min. The optimum pH for the H. samuelpessoai phosphatase lies in the acid range. This phosphatase activity was inhibited by metal chelators and classical phosphatase inhibitors. A robust stimulation of the phosphatase activity was observed when the flagellates were grown in the presence of 4% DMSO, both when intact flagellates and when culture supernatant from those cells were assayed, as observed by biochemical and cytochemical analysis. We also demonstrate that DMSO induced the secretion and/or shedding of this phosphatase to the extracellular medium, with a possible involvement of protein kinase C in this process. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Herpetomonas samuelpessoai; Secreted phosphatase; Trypanosomatids; Dimethyl sulfoxide; Cell differentiation
The genus Herpetomonas, which has been extensively used as a model in physiological, biochemical, and ultrastructural studies, is composed of nonpathogenic trypanosomatids that display promastigote, paramastigote, and opisthomastigote developmental stages [1–3]. Herpetomonas samuelpessoai presents humoral and cellular cross-immunity against Trypanosoma cruzi and Leishmania sp. [4–7]. Intriguingly, a strain of Herpetomonas sp. was presumably the causative agent of a diffuse cutaneous ‘‘leishmaniasis-like’’ both in a patient infected with the human immunodeficiency virus [8] and in an immunocompetent patient [9].
*
Corresponding author. Fax: +55-21-2270-8647. E-mail address: [email protected] (J.R. Meyer-Fernandes).
Dimethyl sulfoxide (DMSO)1 is a widely used agent in cell biology. It is well known as a cryoprotectant, cell fusogen, and a permeability-enhancing agent. These applications depend, to a greater or lesser extent, on the effects of DMSO on the stability and dynamics of biomembranes [10]. Certain highly polar solvents, such as DMSO, can, at times, decrease the effects of carcinogens or some anesthetics by ‘‘cleansing’’ the membrane. On the other hand, they can interact directly with the membrane, producing effects similar to these surfactants [11]. DMSO triggers the process of cellular differentiation in H. samuelpessoai [12], which involves changes in the composition of membrane-associated polysaccharides 1 Abbreviations used: DMSO, dimethyl sulfoxide; p-NP, p-nitrophenol; p-NPP, p-nitrophenylphosphate; PMA, phorbol 12-myristate 13-acetate; BSA, bovine serum albumin; mpV-PIC, monoperoxo(picolinato)oxovanadate(V); bpV-PHEN, potassiumbisperoxo(1,10-phenanthroline)oxovanadate(V); PAF, platelet-activating factor; DAG, diacylglycerol; PKC, protein kinase C.
0003-9861/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 3 - 9 8 6 1 ( 0 2 ) 0 0 4 0 3 - 4
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[13], in cell surface anionogenic groups [14], in the expression of several proteinases [15], and in protein synthesis [15]. Nevertheless, little information is known about the signal transduction pathways that could be involved in the effects of DMSO on the morphology and physiology of these flagellates. Parasites respond to extracellular stimuli by signaling pathways that coordinate processes involved in the activation and/or synthesis of protein kinases, protein phosphatases, second messengers, and transcription factors, which often culminate in cellular differentiation [16,17]. Membrane-bound protein kinases and phosphatases have been characterized in several members of the family Trypanosomatidae [18–25]. The physiological role of these ecto-phosphatases has not been well established yet, although they are supposed to be involved in nutrition, protection, and cellular differentiation [20,24,26–29]. In the present work we characterized a phosphatase activity in H. samuelpessoai by biochemical and cytochemical methods. This phosphatase activity was stimulated by DMSO, which also induced the secretion and/ or shedding of H. samuelpessoai phosphatase to the extracellular medium, with a possible involvement of protein kinase C in this process.
Materials and methods Microorganism and cultivation. Parasites of the species H. samuelpessoai (CT-IOC-067) were kindly provided by Dr. Maria Auxiliadora de Sousa (Colecß~ao de Tripanossomatıdeos, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil). The flagellates were cultured under chemically defined condition [30], at 26 °C, with or without the addition of 4% DMSO [15]. DMSO was filter-sterilized (Seitz filter) before it was added to the culture medium. Two-day cultured parasites were harvested by centrifugation, washed twice with 0.9% saline, and once with 30 mM Tris–HCl/75 mM sucrose buffer, pH 6.8, and kept in the same buffer before the assays. The cell culture supernatants were filtered in a 0.22-lm membrane (Millipore) and concentrated (10-fold) by ultrafiltration in a 10,000 molecular weight cutoff Centricon microconcentrator (AMICON, Beverly, MA) [31]. Phosphatase activity. The phosphatase activity was determined measuring the rate of p-nitrophenol (p-NP) production. Intact cells were incubated for 1 h at 37 °C in 0.5 ml of a reaction mixture containing 30 mM Tris– HCl/75 mM sucrose, pH 6.8, 10.0 mM p-nitrophenylphosphate (p-NPP) as substrate and 1 mg of protein. Reactions were started by the addition of cells, cell culture supernatants, or reaction mixture supernatant (obtained from the centrifugation of the parasites at 1500g at 4 °C, after incubation in the reaction mixture
for 1 h) and stopped by the addition of 2 ml 1 N NaOH. The phosphatase activity was calculated by subtracting the nonspecific p-NPP hydrolysis measured in the absence of parasites. For determining the concentration of released p-nitrophenol, a product of p-NPP hydrolysis, the tubes were centrifuged at 1500g for 10 min and the supernatant was measured spectrophotometrically at 425 nm, using an extinction coefficient of 14:3 103 M 1 cm 1 [32]. For detection of secreted phosphatase activity, DMSO-treated and control parasites were incubated in the presence or in the absence of 4% DMSO for an additional 1 h. Then, the supernatants were collected by two centrifugation steps at 1500g for 10 min at 4 °C and assayed for phosphatase activity, as described above. To test the role of protein kinase C modulators on the secreted phosphatase activity, the living parasites were incubated in the presence of 50 ng/ml sphingosine for 60 min or 20 ng/ml phorbol 12-myristate 13-acetate (PMA) for 20 min, centrifuged, and incubated for 1 h in a fresh reaction mixture containing 4% DMSO. Then, the supernatants were collected and filtered, prior to the phosphatase activity assay. Protein concentration was determined by the method described by Lowry et al. [33], using bovine serum albumin (BSA) as standard. Cellular viability was accessed, before and after incubations, by motility and Trypan blue cell dye exclusion [34]. The viability of the parasites was not affected by the conditions used in this work. Cytochemical detection of acid phosphatase. Parasites were cultivated in the absence or in the presence of 4% DMSO, collected by centrifugation, washed in 0.9% saline, briefly fixed for 20 min at 4 °C with glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, washed in 0.1 M cacodylate buffer, pH 7.2, and in 0.1 mM Tris– maleate buffer, pH 5.0. After that, the pellet was incubated for 1 h at 37 °C in 2 mM cerium chloride, 5% sucrose, 0.1 mM Tris–acetate buffer, pH 5.0, and 2 mM sodium b-glycerophosphate, as substrate. The cells were then washed in Tris–maleate and cacodylate buffers, refixed with 2.5% glutaraldehyde diluted in 0.1 M cacodylate buffer, postfixed in 2% osmium tetroxide, dehydrated in a graded acetone series, and embedded in Epon. As a control, the same number of cells was incubated in the absence of substrate. Ultrathin sections were observed unstained in a transmission electron microscope (Zeiss CEM 900, Carl Zeiss, and Oberkochen, Germany), operated at 80 kV. Chemicals. Dimethyl sulfoxide, divalent cations, and phosphatase inhibitors were purchased from Merck (Darmstaldt, Germany). p-Nitrophenylphosphate, sodium b-glycerophosphate, EDTA, EGTA, sphingosine, and PMA were obtained from Sigma Chemical (St. Louis, MO). All other reagents were analytical grade. Statistical analysis. All experiments were performed in triplicate, with similar results obtained in three separate cell suspensions. Vmax and apparent Km for p-NPP
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were calculated using an iterative nonlinear regression program of the data to the Michaelis–Menten equation as described by Motulsky and Ransnas [35]. The data were analyzed statistically by means of Student’s t test.
Results and discussion H. samuelpessoai living parasites were able to hydrolyze p-NPP at a rate of 2:53 0:01 nmol Pi /mg of protein min. This ecto-phosphatase activity was linear with time, for at least 1 h (Fig. 1A) and with cell density (Fig. 1B) and represents a small fraction (8.4%) of the total phosphatase activity considering disrupted cells. The H. samuelpessoai phosphatase activity was measured in the pH range from 6.5 to 8.0, in which the parasites were viable throughout the time of reaction. In this pH range the phosphatase activity decreased concomitantly with the increase of pH (Fig. 1C), suggesting an acid phosphatase activity, as previously described for other trypanosomatids [20,24,28,36]. The dependence on p-NPP concentration revealed a normal Michaelis– Menten kinetics for this phosphatase activity and the values of Vmax and apparent Km for p-NPP were 3:03 0:42 nmol Pi /mg of protein min and 1:91 0:46 mM, respectively (Fig. 2). H. samuelpessoai living parasites were also able to hydrolyze b-glycerophosphate at a rate of 1:42 0:15 nmol Pi /mg of protein min (not shown). The inhibition of p-NPP hydrolysis monitored by p-NP release promoted by b-glycerophosphate (Table 1) suggests that the same enzyme use p-NPP and b-glycerophosphate as substrates. Various divalent cations, metal chelators, and phosphatase inhibitors were tested and the results are shown in Table 1. Magnesium chloride, calcium chloride, and cupric sulfate had no significant effect on H. samuelpessoai phosphatase activity. However, the addition of EDTA and EGTA inhibited this phosphatase activity. The acid phosphatase inhibitors zinc chloride, sodium fluoride, and ammonium molybdate [24] inhibited this enzyme activity by 77.4, 84.5, and 85.2%, respectively. The
A
B
Fig. 2. Influence of p-NPP concentration on the phosphatase activity of intact cells of H. samuelpessoai. The reactions were performed at room temperature for 1 h in a reaction mixture (0.5 ml) containing 30 mM Tris–HCl/75 mM sucrose buffer, pH 6.8, 1 mg protein, and several p-NPP concentrations, as seen in the abscissa. The curve represents the adjustment of kinetic parameters by nonlinear regression, using the Michaelis–Menten equation. The parasites were viable during the course of the experiments under all conditions used. The values represent the mean SE of three independent experiments, which were performed in triplicate.
phosphotyrosine phosphatase inhibitors sodium orthovanadate [monoperoxo(picolinato)oxovanadate(V)] (mpV-PIC) and [potassiumbisperoxo(1,10-phenanthroline)oxovanadate(V)] (bpV-PHEN) [37] also inhibited the enzymatic activity by 91.7, 82.7, and 71.4%, respectively. The high sensitivity to these three well-known potent phosphotyrosyl protein phosphatase inhibitors [37] suggests that this enzyme has similarities to the phosphotyrosine phosphatases present in other protozoa [22,23,38,39]. Phosphotyrosine phosphatases have been related to cellular differentiation of some trypanosomatids including T. cruzi, T. brucei, L. mexicana, and Herpetomonas muscarum muscarum [22–24]. Interestingly, H. samuelpessoai had its ecto-phosphatase activity augmented by 163% (Fig. 3A), when grown in the presence of 4% DMSO, for 48 h, which were the con-
C
Fig. 1. Time course, effects of cell density and of pH on the phosphatase activity of H. samuelpessoai. Living parasites were incubated at room temperature in a reaction mixture (0.5 ml) containing 30 mM Tris–HCl/75 mM sucrose buffer, pH 6.8, 10 mM p-nitrophenylphosphate (p-NPP) as substrate and 1 mg of protein, in different times (A), or for 1 h using increasing concentrations of cells (B), or in the same buffer adjusted to pH values between 6.5 and 8.0 (C). Data are means SE of three determinations with different cell suspensions. The parasites were viable during the course of the experiments under all conditions used. The values represent the mean SE of three independent experiments, which were performed in triplicate.
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Table 1 Influence of different compounds on the phosphatase activity of intact cells of Herpetomonas samuelpessoai Addition
Relative activity
None Ammonium molybdate (0.1 mM) Sodium orthovanadate (0.1 mM) Sodium tartrate (10 mM) Sodium fluoride (10 mM) bpV-PHEN (10 lM) mpV-PIC (10 lM) Zinc chloride (1 mM) Magnesium chloride (5 mM) Cuprum sulfate (5 mM) Calcium chloride (5 mM) EDTA (1 mM) EGTA (1 mM) b-Glycerophosphate (10 mM)
100:0 4:3 14:8 0:5 8:3 1:6 67:8 3:4 15:5 1:2 28:6 1:7 17:3 1:9 22:6 1:2 117:3 2:2 87:7 2:3 96:1 4:5 40:0 0:01 5:7 0:02 33:3 0:02
The ecto-phosphatase activity was measured in the standard assay described under Materials and methods. Phosphatase activity is expressed as a percentage of that measured under control conditions, i.e., without other additions. The phosphatase activity (2:53 0:01 nmol Pi / mg protein min) was taken as 100%. The standard errors were calculated from absolute values of three experiments, performed in triplicate, with different cell suspensions and converted to percentage of the control value. The parasites were viable during the course of the experiments under all conditions used.
A
B
Fig. 3. Effect of DMSO on phosphatase activity of intact cells (A) and cell culture supernatant (B) of H. samuelpessoai. (A) The parasites were cultured under chemically defined conditions at 26 °C for 48 h, without or with 4% DMSO. Cells were harvested by centrifugation and washed three times in 30 mM Tris–HCl/75 mM sucrose buffer, pH 6.8. (B) The cell culture supernatants of DMSO-untreated and treated cells were filtered in a 0.22-lm membrane (Millipore). The reactions were performed at room temperature for 1 h in a reaction mixture (0.5 ml) containing 30 mM Tris–HCl/75 mM sucrose buffer, pH 6.8, 1 mg protein, and 10 mM p-NPP. The parasites were viable during the course of the experiments under all conditions used. The values represent the mean SE of three independent experiments, which were performed in triplicate. Parasites and supernatants treated with DMSO had a rate of p-NPP hydrolysis significantly different from control cells (P < 0:05, Student’s t test).
ditions shown to stimulate the process of cellular differentiation in these parasites [12,13,15]. Sodium tartrate, which was shown to be a potent inhibitor of secreted acid phosphatases of L. donovani [40] and H. m. muscarum [24], inhibited about 32% of H. samuelpessoai phosphatase activity (Table 1). In fact, H. samuelpessoai flagellates presented an extracellular phosphatase activity, measured in the culture medium,
which was higher in the cell-free medium obtained from parasites grown in the presence of DMSO (1:34 0:10 nmol Pi /mg of protein min), as compared to the medium from control flagellates (0:41 0:04 nmol Pi /mg of protein min) (Fig. 3B). Accordingly, H. samuelpessoai parasites secrete several proteins to the environment. Some of these proteins present proteolytic activity, which were modulated during the differentiation process triggered by DMSO [15]. These effects induced by DMSO on the ecto-phosphatase activity of H. samuelpessoai were confirmed by cytochemical analysis, which was detected by electrondense cerium phosphate deposits, the product of the reaction between cerium chloride and the inorganic phosphate obtained from the cleavage of b-glycerophosphate by phosphatase activity. Flagellates cultured in the presence of 4% DMSO showed different patterns of cytochemical reaction, as compared to the control parasites (Fig. 4). The control system showed electrondense deposits of cerium phosphate in cytoplasmic organelles (Figs. 4A and B), resembling acidocalcisomes, as described for other protozoan parasites due to the high concentrations of inorganic phosphate present in this organelle [41]. No cerium phosphate deposits were observed on the cell membrane or in the flagellar pocket (Fig. 4A). On the other hand, in DMSO-treated parasites, small electron-dense clusters were observed in the bottom of the flagellar pocket (Fig. 4C) and on the flagellum and flagellar membranes (Figs. 4D and E). Some of those clusters were free in the flagellar pocket or bound to small membrane vesicles, suggesting shedding of the enzyme from the parasite surface (Figs. 4D and E). It is important to note that the trypanosomatid flagellar pocket is a highly specialized area of the parasite membrane that is involved in several processes, including protein secretion to the external environment [42]. Similar results were obtained when T. cruzi parasites were grown in the presence of platelet-activating factor (PAF) [36], a lipid mediator that stimulates the cellular differentiation of T. cruzi [43] and of H. m. muscarum [44]. It is remarkable the similarity between PAF effects on T. cruzi with those obtained when H. samuelpessoai were grown in the presence of DMSO, regarding the induction of a secreted phosphatase, as well as the stimulation of cell differentiation. Taken together, those data show a striking resembling pattern induced by two completely different modulators, which could mean that when cell differentiation is triggered, a similar cascade of biochemical and morphological events occurs, regardless of the stimuli to which the cells are exposed. This secreted phosphatase activity present in supernatants is a small fraction (3%) of the total phosphatase activity. The phosphatase activity observed by cytochemical analysis could be the ecto-phosphatase shed from the surface of the parasites and/or it could be an enzyme
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Fig. 4. Cytochemistry assay for localization of acid phosphatase activity of H. samuelpessoai. The trypanosomatids were grown in a chemically defined medium at 26 °C for 48 h, in the absence (A and B) or the presence of 4% DMSO (C–E). The reactions were performed at room temperature in a buffer containing Tris–acetate, pH 5.0, and using b-glycerophosphate as the substrate and cerium chloride as the capture agent. In untreated cells, electron-dense deposits of cerium phosphate are observed in cytoplasmic organelles resembling acidocalcisomes (arrows in A and B). No labeling was observed on the cell membrane or in the flagellar pocket (arrowheads in A). In DMSO-treated cells acid phosphatase activity is observed as small electron-dense clusters, in the bottom of the flagellar pocket (arrow in C) and on the flagellum and flagellar pocket membranes (D and E). Some clusters are free in the flagellar pocket or bound to small membrane vesicles, suggesting a shedding of the enzyme from the parasite surface (arrowheads in D and E). N, nucleus; F, flagellum; FP, flagellar pocket. Bars, 1 lm.
secreted into the medium, during the course of the assay. In order to test the previous hypothesis, washed parasites grown in the absence or in the presence of 4% DMSO were further incubated for 1 h in the presence of 4% DMSO. The cells were washed three times and the supernatants were obtained, employing two sequential
centrifugation steps at 1500g for 10 min and then by filtering them through a 0.22-lm-pore membrane. The phosphatase activity measured in supernatants obtained from DMSO-treated parasites was higher than the activity of supernatants from control flagellates (Fig. 5B). Interestingly, whole DMSO-treated parasites presented
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B
A
C
Fig. 5. Effect of DMSO and protein kinase C (PKC) modulators on the phosphatase activities of H. samuelpessoai. (A) Effect of DMSO on cellassociated phosphatase activity of H. samuelpessoai. The parasites were cultured under chemically defined conditions at 26 °C for 48 h, without (a) or with 4% DMSO (c) Cells were harvested by centrifugation and washed three times in 30 mM Tris–HCl/75 mM sucrose buffer, pH 6.8. Living parasites grown in the absence or in the presence of DMSO were further incubated in the presence of 4% DMSO (b, d) for 60 min, centrifuged, and washed twice in the same buffer. Then, cells were incubated for 1 h in a fresh reaction mixture containing 30 mM Tris–HCl/75 mM sucrose buffer, pH 6.8, 1 mg protein, and 10 mM p-NPP. The parasites were viable during the course of the experiments under all conditions used. (B) Effect of DMSO on released phosphatase activity of H. samuelpessoai. Living parasites grown in the absence (a) or in the presence (c) of DMSO were further incubated with 4% DMSO (b, d) for 60 min and the supernatants from those cells were assayed for phosphatase activity, as described above (A). Supernatant from control cells, further treated with 4% DMSO for 1 h, had a rate of p-NPP hydrolysis significantly different from supernatant from control cells. DMSO-treated cells, further incubated for 1 h with 4% DMSO, showed an increase in the phosphatase activity associated to the cells (d, panel A), but not in the released phosphatase activity (d, panel B) (P < 0:05, Student’s t test). (C) Effect of DMSO and the PKC modulators sphingosine and PMA on the secreted and/or released phosphatase activity of H. samuelpessoai. The living parasites were incubated in the presence of 50 ng/ml sphingosine (Sphin) for 60 min or 20 ng/ml PMA for 20 min, centrifuged, and incubated for 1 h in a fresh reaction mixture containing 4% DMSO. Then, the supernatants were collected and filtered, prior to the phosphatase activity assays. Supernatants obtained from cells incubated in the reaction mixture without any drug addition are stated as control. Parasites treated with 4% DMSO had rates of p-NPP hydrolysis significantly different from control cells, from parasites treated with PMA, and from parasites pretreated with PMA and then with DMSO (P < 0:05, Student’s t test). All the values represent the mean SE of three independent experiments, which were performed in triplicate.
a 42% increase in its membrane-bound phosphatase activity, when they were incubated for an additional hour in the presence of 4% DMSO (Fig. 5A). This DMSO effect could not be attributed to a disruption of the cells once the cellular viability and the permeability of the plasma membrane to charged small molecules (like p-NPP) were not affected under the conditions employed here, and that no lactate dehydrogenase activity was detected either in the absence or in the presence of 4% DMSO (data not shown). DMSO modulates the activity of several enzymes and is able to activate lysosomes, resulting in the release of acid phosphatases [45]. Also, DMSO elicits a biphasic increase in diacylglycerol (DAG) levels, which is the endogenous activator of protein kinase C (PKC), during
the morphological differentiation of N1E-115 neuroblastoma cells [46]. In order to investigate if DMSO effects occur through a signal transduction pathway involving PKC, two modulators of PKC, sphingosine and phorbol-esther myristate (PMA), were tested on the DMSO-induced secretion of phosphatase (Fig. 5C). The phosphatase activity detected in the supernatants showed that 50 ng/ml sphingosine, known to inhibit PKC activity, induced a secretion of a phosphatase into the extracellular medium, which was similar to the results obtained when 4% DMSO was used, although sphingosine was not able to promote any additive effect on this DMSO-induced secretion. The phosphatase activity measured in the supernatant obtained from parasites pretreated with the PKC stimulator PMA (20 ng/
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ml) was 56.4% lower than that measured in the supernatant from control flagellates (Fig. 5C). Our results also show that 20 ng/ml PMA was able to suppress the DMSO-induced phosphatase secretion (Fig. 5C). Similarly, PMA abrogated the PAF-induced phosphatase secretion in T. cruzi [36]. Interestingly, PMA stimulates a PKC activity and induces filopodium-like projections in T. cruzi [47,48], as well as modulates a secreted acid phosphatase activity in Leishmania amazonensis, which mediates the infection of mouse macrophages by these flagellates [21]. Cell surface components play a key role in the survival of protozoan parasites in hostile insect and vertebrate environments and in confrontation with host immune responses. The precise role of ecto-phosphatase is not well established, but it has been related to cell growth, providing the cell with a source of inorganic phosphate by hydrolyzing phosphomonoester metabolites [22,24,28], as well as protecting the parasite by preventing the protozoan digestion in the alimentary tract of the invertebrate host [19]. Interestingly, the prevailing pH range of the insect gut (pH 6–7), the normal habitat for Herpetomonas [1], coincides with the pH range where higher values for phosphatase activity were observed in this paper. The characterization of cell surface-located phosphatase activity is particularly interesting because of its possible role in cell-to-cell interaction and/or reception and transduction of external stimuli [21,49]. Moreover, these enzymes could be good targets for the growth control of these protozoan parasites [50]. It would be of extreme importance to further investigate the modulation of phosphatase activities by DMSO in parasites, as well as the participation of these enzymes in the invertebrate-parasite relationship.
Acknowledgments We thank Dr. Patrıcia M.L. Dutra and Dr. Claudia O. Rodrigues for helpful discussions and Ms. Celina Monteiro Abreu for technical assistance. This work was supported by the Brazilian agencies PRONEX (0885), CNPq, FAPERJ, FINEP and FUJB/UFRJ.
References [1] F.G. Wallace, Exp. Parasitol. 18 (1966) 124–193. [2] J. Angluster, M.M. Bunn, W. De Souza, J. Parasitol. 63 (1977) 922–924. [3] R.B. McGhee, W.B. Cosgrove, Microbiol. Rev. 44 (1980) 140– 173. [4] M.C.M. Souza, A.P. Reis, W.D. Silva, Z. Brener, J. Protozool. 21 (1974) 579–583. [5] R. Fucs, M.A. Barcinski, J. Parasitol. 67 (1981) 463–467. [6] R.J. Etges, Acta Trop. 50 (1992) 205–217.
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[7] P. Schneider, T.A. Glaser, Mol. Biochem. Parasitol. 58 (1993) 277–282. [8] J.P. Dedet, B. Roche, F. Pratlong, D. Cales-Quist, J. Jouannelle, J.C. Benichou, M. Huerre, T. Roy. Soc. Trop. Med. H. 89 (1995) 644–646. [9] A.M. Boisseau-Garsaud, D. Cales-Quist, N. Desbois, J. Jouannelle, A. Jouannelle, F. Pratlong, J.P. Dedet, T. Roy. Soc. Trop. Med. H. 94 (2000) 51–52. [10] Z.W. Yu, P.J. Quinn, Mol. Membr. Biol. 15 (1998) 59–68. [11] R.L. Neulieb, M.K. Neulieb, Cytobios 63 (1990) 139–165. [12] G.B. Castellanos, J. Angluster, W. De Souza, Acta Trop. 38 (1981) 29–37. [13] R.M.A. Soares, C.S. Alviano, F.C. Silva-Filho, M.J.G. Esteves, J. Angluster, W. De Souza, J. Submicrosc. Cytol. 16 (1984) 735–739. [14] R.M.A. Soares, C.S. Alviano, M.J.G. Esteves, J. Angluster, F.C. Silva-Filho, W. De Souza, Cell Biophys. 13 (1988) 29–41. [15] A.L.S. Santos, L.M. Batista, C.M. Abreu, C.S. Alviano, J. Angluster, R.M.A. Soares, Curr. Microbiol. 42 (2001) 111–116. [16] R. Derynck, E.F. Wagner, Curr. Opin. Cell Biol. 7 (1995) 843– 844. [17] M. Parsons, L. Ruben, Parasitol. Today 16 (2000) 56–62. [18] N. Sacerdoti-Sierra, C.L. Jaffe, J. Biol. Chem. 272 (1997) 30760– 30765. [19] M. Gottlieb, D.M. Dwyer, Science 212 (1981) 939–941. [20] K.H. Nagakura, H. Tachibana, Y. Kaneda, Comp. Biochem. Physiol. 81 (1985) 815–817. [21] M.A. Vannier-Santos, A. Martiny, J.R. Meyer-Fernandes, W. De Souza, Eur. J. Cell Biol. 67 (1995) 112–119. [22] E.C. Fernandes, J.R. Meyer-Fernandes, M.A.C. Silva-Neto, A.E. Vercesi, Z. Naturforsch. 52c (1997) 351–358. [23] J.R. Meyer-Fernandes, M.A.C. Silva-Neto, M.S. Soares, E.C. Fernandes, A.E. Vercesi, M.M. Oliveira, Z. Naturforsch. 54c (1999) 884–977. [24] P.M.L. Dutra, C.O. Rodrigues, J.B. Jesus, A.H.C.S. Lopes, T. Souto-Padr on, J.R. Meyer-Fernandes, Biochem. Biophys. Res. Commun. 253 (1998) 164–169. [25] P.M.L. Dutra, C.O. Rodrigues, A. Romeiro, L.A.M. Grillo, F.A. Dias, M. Attias, W. De Souza, A.H.C.S. Lopes, J.R. MeyerFernandes, Phytopathology 90 (2000) 1032–1038. [26] R.H. Glew, A.K. Saha, S. Das, A.T. Remaley, Microbiol. Rev. 52 (1988) 432–512. [27] A.T. Remaley, S. Das, P.I. Campbell, G.M. LaRocca, M.T. Pope, R.H. Glew, J. Biol. Chem. 260 (1985) 880–886. [28] N. Bakalara, A. Seyfang, C. Davis, T. Baltz, Exp. Parasitol. 81 (1995) 302–312. [29] N. Bakalara, X. Santarelli, C. Davis, T. Baltz, J. Biol. Chem. 275 (2000) 8863–8871. [30] C. Roitman, I. Roitman, H.P. Azevedo, J. Protozool. 19 (1972) 346–349. [31] A.L.S. Santos, A. Ferreira, V.A. Franco, C.S. Alviano, R.M.A. Soares, Curr. Microbiol. 39 (1999) 61–64. [32] P.M.L. Dutra, F.A. Dias, M.A.A. Santos, C.O. Rodrigues, A. Romeiro, M. Attias, W. De Souza, A.H.C.S. Lopes, J.R. MeyerFernandes, Curr. Microbiol. 43 (2001) 288–292. [33] O.H. Lowry, N.J. Rozebrough, A.L. Farr, R.J. Randall, J. Biol. Chem. 193 (1951) 265–275. [34] C.E. Peres-Sampaio, S.T. Palumbo, J.R. Meyer-Fernandes, Z. Naturforsch. 56c (2001) 820–825. [35] H.J. Motulsky, L.A. Ransnas, FASEB J. 1 (1987) 365–374. [36] C.O. Rodrigues, P.M.L. Dutra, F.S. Barros, T. Souto-Padr on, J.R. Meyer-Fernandes, A.H.C.S. Lopes, Biochem. Biophys. Res. Commun. 266 (1999) 36–42. [37] B.I. Posner, R. Faure, J.W. Burgess, A.P. Bevan, D. Lachance, G. Zhang-Sun, I.G. Fantus, J.B. Ng, D.A. Hall, B.S. Lum, J. Biol. Chem. 269 (1994) 4596–4604. [38] R.H. Glew, M.S. Czuczam, W.F. Diven, R.L. Berens, M.T. Pope, D.E. Katsoulis, Comp. Biochem. Physiol. 72 (1982) 581–590.
198
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[39] T. Furuya, L. Zhong, J.R. Meyer-Fernandes, H.G. Lu, S.N.J. Moreno, R. Docampo, Mol. Biochem. Parasitol. 92 (1998) 339– 348. [40] J.K. Lovelace, M. Gottlieb, Am. J. Trop. Med. Hyg. 35 (1986) 1121–1128. [41] R. Docampo, S.N.J. Moreno, Parasitol. Today 15 (1999) 443–448. [42] P. Webster, D.G. Russel, Parasitol. Today 9 (1993) 201–205. [43] C.O. Rodrigues, P.M.L. Dutra, T. Souto-Padr on, R.S.B. Cordeiro, A.H.C.S. Lopes, Biochem. Biophys. Res. Commun. 273 (1996) 735–740. [44] A.H.C.S. Lopes, P.M.L. Dutra, C.O. Rodrigues, M.J. Soares, J. Angluster, R.S.B. Cordeiro, J. Eukaryot. Microbiol. 44 (1997) 321–325.
[45] D.W. Misch, M.S. Misch, Proc. Natl. Acad. Sci. USA 58 (1967) 2462–2467. [46] S. Clejan, R.S. Dotson, E.W. Wolf, M.P. Corb, C.F. Ide, Exp. Cell Res. 224 (1996) 16–27. [47] M.L. G omez, L. Erijman, S. Arauzo, H.N. Torres, M.T. TellezI~ non, Mol. Biochem. Parasitol. 36 (1989) 101–108. [48] T.U. Carvalho, W. De Souza, Parasitol. Res. 74 (1987) 11–17. [49] A. Martiny, M.A. Vannier-Santos, V.M. Borges, J.R. MeyerFernandes, J. Asseruy, N.L. Cunha-e-Silva, W. De Souza, Eur. J. Cell Biol. 71 (1996) 206–215. [50] P.M.L. Dutra, F.A. Dias, M.A.A. Santos, C.O. Rodrigues, A. Romeiro, M. Attias, W. De Souza, A.H.C.S. Lopes, J.R. MeyerFernandes, Phytopathology 91 (2001) 408–414.
Secreted phosphatase activity induced by dimethyl sulfoxide in Herpetomonas samuelpessoai Andre L.S. Santos,a Thais Souto-Padr on,a Celuta S. Alviano,a Angela H.C.S. Lopes,a Rosangela M.A. Soares,a and Jose R. Meyer-Fernandesb,* a
Instituto de Microbiologia Prof. Paulo de G oes, CCS, Universidade Federal do Rio de Janeiro, Cidade Universit aria, Ilha do Fund~ ao, Rio de Janeiro, RJ 21941-590, Brazil b Departamento de Bioquımica M edica, ICB, Universidade Federal do Rio de Janeiro, CCS, Bloco H-2 andar – sala, 13 Cidade Universit aria, Ilha do Fund~ ao, Rio de Janeiro, RJ 21941-590, Brazil Received 26 March 2002, and in revised form 15 July 2002
Abstract A phosphatase activity of the trypanosomatid parasite Herpetomonas samuelpessoai was characterized using intact living cells. The effects of dimethyl sulfoxide (DMSO) on this activity were investigated. This phosphatase activity (2:53 0:01 nmol Pi /mg protein min) was linear with cell density and with time for at least 60 min. The optimum pH for the H. samuelpessoai phosphatase lies in the acid range. This phosphatase activity was inhibited by metal chelators and classical phosphatase inhibitors. A robust stimulation of the phosphatase activity was observed when the flagellates were grown in the presence of 4% DMSO, both when intact flagellates and when culture supernatant from those cells were assayed, as observed by biochemical and cytochemical analysis. We also demonstrate that DMSO induced the secretion and/or shedding of this phosphatase to the extracellular medium, with a possible involvement of protein kinase C in this process. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Herpetomonas samuelpessoai; Secreted phosphatase; Trypanosomatids; Dimethyl sulfoxide; Cell differentiation
The genus Herpetomonas, which has been extensively used as a model in physiological, biochemical, and ultrastructural studies, is composed of nonpathogenic trypanosomatids that display promastigote, paramastigote, and opisthomastigote developmental stages [1–3]. Herpetomonas samuelpessoai presents humoral and cellular cross-immunity against Trypanosoma cruzi and Leishmania sp. [4–7]. Intriguingly, a strain of Herpetomonas sp. was presumably the causative agent of a diffuse cutaneous ‘‘leishmaniasis-like’’ both in a patient infected with the human immunodeficiency virus [8] and in an immunocompetent patient [9].
*
Corresponding author. Fax: +55-21-2270-8647. E-mail address: [email protected] (J.R. Meyer-Fernandes).
Dimethyl sulfoxide (DMSO)1 is a widely used agent in cell biology. It is well known as a cryoprotectant, cell fusogen, and a permeability-enhancing agent. These applications depend, to a greater or lesser extent, on the effects of DMSO on the stability and dynamics of biomembranes [10]. Certain highly polar solvents, such as DMSO, can, at times, decrease the effects of carcinogens or some anesthetics by ‘‘cleansing’’ the membrane. On the other hand, they can interact directly with the membrane, producing effects similar to these surfactants [11]. DMSO triggers the process of cellular differentiation in H. samuelpessoai [12], which involves changes in the composition of membrane-associated polysaccharides 1 Abbreviations used: DMSO, dimethyl sulfoxide; p-NP, p-nitrophenol; p-NPP, p-nitrophenylphosphate; PMA, phorbol 12-myristate 13-acetate; BSA, bovine serum albumin; mpV-PIC, monoperoxo(picolinato)oxovanadate(V); bpV-PHEN, potassiumbisperoxo(1,10-phenanthroline)oxovanadate(V); PAF, platelet-activating factor; DAG, diacylglycerol; PKC, protein kinase C.
0003-9861/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 3 - 9 8 6 1 ( 0 2 ) 0 0 4 0 3 - 4
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[13], in cell surface anionogenic groups [14], in the expression of several proteinases [15], and in protein synthesis [15]. Nevertheless, little information is known about the signal transduction pathways that could be involved in the effects of DMSO on the morphology and physiology of these flagellates. Parasites respond to extracellular stimuli by signaling pathways that coordinate processes involved in the activation and/or synthesis of protein kinases, protein phosphatases, second messengers, and transcription factors, which often culminate in cellular differentiation [16,17]. Membrane-bound protein kinases and phosphatases have been characterized in several members of the family Trypanosomatidae [18–25]. The physiological role of these ecto-phosphatases has not been well established yet, although they are supposed to be involved in nutrition, protection, and cellular differentiation [20,24,26–29]. In the present work we characterized a phosphatase activity in H. samuelpessoai by biochemical and cytochemical methods. This phosphatase activity was stimulated by DMSO, which also induced the secretion and/ or shedding of H. samuelpessoai phosphatase to the extracellular medium, with a possible involvement of protein kinase C in this process.
Materials and methods Microorganism and cultivation. Parasites of the species H. samuelpessoai (CT-IOC-067) were kindly provided by Dr. Maria Auxiliadora de Sousa (Colecß~ao de Tripanossomatıdeos, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil). The flagellates were cultured under chemically defined condition [30], at 26 °C, with or without the addition of 4% DMSO [15]. DMSO was filter-sterilized (Seitz filter) before it was added to the culture medium. Two-day cultured parasites were harvested by centrifugation, washed twice with 0.9% saline, and once with 30 mM Tris–HCl/75 mM sucrose buffer, pH 6.8, and kept in the same buffer before the assays. The cell culture supernatants were filtered in a 0.22-lm membrane (Millipore) and concentrated (10-fold) by ultrafiltration in a 10,000 molecular weight cutoff Centricon microconcentrator (AMICON, Beverly, MA) [31]. Phosphatase activity. The phosphatase activity was determined measuring the rate of p-nitrophenol (p-NP) production. Intact cells were incubated for 1 h at 37 °C in 0.5 ml of a reaction mixture containing 30 mM Tris– HCl/75 mM sucrose, pH 6.8, 10.0 mM p-nitrophenylphosphate (p-NPP) as substrate and 1 mg of protein. Reactions were started by the addition of cells, cell culture supernatants, or reaction mixture supernatant (obtained from the centrifugation of the parasites at 1500g at 4 °C, after incubation in the reaction mixture
for 1 h) and stopped by the addition of 2 ml 1 N NaOH. The phosphatase activity was calculated by subtracting the nonspecific p-NPP hydrolysis measured in the absence of parasites. For determining the concentration of released p-nitrophenol, a product of p-NPP hydrolysis, the tubes were centrifuged at 1500g for 10 min and the supernatant was measured spectrophotometrically at 425 nm, using an extinction coefficient of 14:3 103 M 1 cm 1 [32]. For detection of secreted phosphatase activity, DMSO-treated and control parasites were incubated in the presence or in the absence of 4% DMSO for an additional 1 h. Then, the supernatants were collected by two centrifugation steps at 1500g for 10 min at 4 °C and assayed for phosphatase activity, as described above. To test the role of protein kinase C modulators on the secreted phosphatase activity, the living parasites were incubated in the presence of 50 ng/ml sphingosine for 60 min or 20 ng/ml phorbol 12-myristate 13-acetate (PMA) for 20 min, centrifuged, and incubated for 1 h in a fresh reaction mixture containing 4% DMSO. Then, the supernatants were collected and filtered, prior to the phosphatase activity assay. Protein concentration was determined by the method described by Lowry et al. [33], using bovine serum albumin (BSA) as standard. Cellular viability was accessed, before and after incubations, by motility and Trypan blue cell dye exclusion [34]. The viability of the parasites was not affected by the conditions used in this work. Cytochemical detection of acid phosphatase. Parasites were cultivated in the absence or in the presence of 4% DMSO, collected by centrifugation, washed in 0.9% saline, briefly fixed for 20 min at 4 °C with glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, washed in 0.1 M cacodylate buffer, pH 7.2, and in 0.1 mM Tris– maleate buffer, pH 5.0. After that, the pellet was incubated for 1 h at 37 °C in 2 mM cerium chloride, 5% sucrose, 0.1 mM Tris–acetate buffer, pH 5.0, and 2 mM sodium b-glycerophosphate, as substrate. The cells were then washed in Tris–maleate and cacodylate buffers, refixed with 2.5% glutaraldehyde diluted in 0.1 M cacodylate buffer, postfixed in 2% osmium tetroxide, dehydrated in a graded acetone series, and embedded in Epon. As a control, the same number of cells was incubated in the absence of substrate. Ultrathin sections were observed unstained in a transmission electron microscope (Zeiss CEM 900, Carl Zeiss, and Oberkochen, Germany), operated at 80 kV. Chemicals. Dimethyl sulfoxide, divalent cations, and phosphatase inhibitors were purchased from Merck (Darmstaldt, Germany). p-Nitrophenylphosphate, sodium b-glycerophosphate, EDTA, EGTA, sphingosine, and PMA were obtained from Sigma Chemical (St. Louis, MO). All other reagents were analytical grade. Statistical analysis. All experiments were performed in triplicate, with similar results obtained in three separate cell suspensions. Vmax and apparent Km for p-NPP
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were calculated using an iterative nonlinear regression program of the data to the Michaelis–Menten equation as described by Motulsky and Ransnas [35]. The data were analyzed statistically by means of Student’s t test.
Results and discussion H. samuelpessoai living parasites were able to hydrolyze p-NPP at a rate of 2:53 0:01 nmol Pi /mg of protein min. This ecto-phosphatase activity was linear with time, for at least 1 h (Fig. 1A) and with cell density (Fig. 1B) and represents a small fraction (8.4%) of the total phosphatase activity considering disrupted cells. The H. samuelpessoai phosphatase activity was measured in the pH range from 6.5 to 8.0, in which the parasites were viable throughout the time of reaction. In this pH range the phosphatase activity decreased concomitantly with the increase of pH (Fig. 1C), suggesting an acid phosphatase activity, as previously described for other trypanosomatids [20,24,28,36]. The dependence on p-NPP concentration revealed a normal Michaelis– Menten kinetics for this phosphatase activity and the values of Vmax and apparent Km for p-NPP were 3:03 0:42 nmol Pi /mg of protein min and 1:91 0:46 mM, respectively (Fig. 2). H. samuelpessoai living parasites were also able to hydrolyze b-glycerophosphate at a rate of 1:42 0:15 nmol Pi /mg of protein min (not shown). The inhibition of p-NPP hydrolysis monitored by p-NP release promoted by b-glycerophosphate (Table 1) suggests that the same enzyme use p-NPP and b-glycerophosphate as substrates. Various divalent cations, metal chelators, and phosphatase inhibitors were tested and the results are shown in Table 1. Magnesium chloride, calcium chloride, and cupric sulfate had no significant effect on H. samuelpessoai phosphatase activity. However, the addition of EDTA and EGTA inhibited this phosphatase activity. The acid phosphatase inhibitors zinc chloride, sodium fluoride, and ammonium molybdate [24] inhibited this enzyme activity by 77.4, 84.5, and 85.2%, respectively. The
A
B
Fig. 2. Influence of p-NPP concentration on the phosphatase activity of intact cells of H. samuelpessoai. The reactions were performed at room temperature for 1 h in a reaction mixture (0.5 ml) containing 30 mM Tris–HCl/75 mM sucrose buffer, pH 6.8, 1 mg protein, and several p-NPP concentrations, as seen in the abscissa. The curve represents the adjustment of kinetic parameters by nonlinear regression, using the Michaelis–Menten equation. The parasites were viable during the course of the experiments under all conditions used. The values represent the mean SE of three independent experiments, which were performed in triplicate.
phosphotyrosine phosphatase inhibitors sodium orthovanadate [monoperoxo(picolinato)oxovanadate(V)] (mpV-PIC) and [potassiumbisperoxo(1,10-phenanthroline)oxovanadate(V)] (bpV-PHEN) [37] also inhibited the enzymatic activity by 91.7, 82.7, and 71.4%, respectively. The high sensitivity to these three well-known potent phosphotyrosyl protein phosphatase inhibitors [37] suggests that this enzyme has similarities to the phosphotyrosine phosphatases present in other protozoa [22,23,38,39]. Phosphotyrosine phosphatases have been related to cellular differentiation of some trypanosomatids including T. cruzi, T. brucei, L. mexicana, and Herpetomonas muscarum muscarum [22–24]. Interestingly, H. samuelpessoai had its ecto-phosphatase activity augmented by 163% (Fig. 3A), when grown in the presence of 4% DMSO, for 48 h, which were the con-
C
Fig. 1. Time course, effects of cell density and of pH on the phosphatase activity of H. samuelpessoai. Living parasites were incubated at room temperature in a reaction mixture (0.5 ml) containing 30 mM Tris–HCl/75 mM sucrose buffer, pH 6.8, 10 mM p-nitrophenylphosphate (p-NPP) as substrate and 1 mg of protein, in different times (A), or for 1 h using increasing concentrations of cells (B), or in the same buffer adjusted to pH values between 6.5 and 8.0 (C). Data are means SE of three determinations with different cell suspensions. The parasites were viable during the course of the experiments under all conditions used. The values represent the mean SE of three independent experiments, which were performed in triplicate.
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Table 1 Influence of different compounds on the phosphatase activity of intact cells of Herpetomonas samuelpessoai Addition
Relative activity
None Ammonium molybdate (0.1 mM) Sodium orthovanadate (0.1 mM) Sodium tartrate (10 mM) Sodium fluoride (10 mM) bpV-PHEN (10 lM) mpV-PIC (10 lM) Zinc chloride (1 mM) Magnesium chloride (5 mM) Cuprum sulfate (5 mM) Calcium chloride (5 mM) EDTA (1 mM) EGTA (1 mM) b-Glycerophosphate (10 mM)
100:0 4:3 14:8 0:5 8:3 1:6 67:8 3:4 15:5 1:2 28:6 1:7 17:3 1:9 22:6 1:2 117:3 2:2 87:7 2:3 96:1 4:5 40:0 0:01 5:7 0:02 33:3 0:02
The ecto-phosphatase activity was measured in the standard assay described under Materials and methods. Phosphatase activity is expressed as a percentage of that measured under control conditions, i.e., without other additions. The phosphatase activity (2:53 0:01 nmol Pi / mg protein min) was taken as 100%. The standard errors were calculated from absolute values of three experiments, performed in triplicate, with different cell suspensions and converted to percentage of the control value. The parasites were viable during the course of the experiments under all conditions used.
A
B
Fig. 3. Effect of DMSO on phosphatase activity of intact cells (A) and cell culture supernatant (B) of H. samuelpessoai. (A) The parasites were cultured under chemically defined conditions at 26 °C for 48 h, without or with 4% DMSO. Cells were harvested by centrifugation and washed three times in 30 mM Tris–HCl/75 mM sucrose buffer, pH 6.8. (B) The cell culture supernatants of DMSO-untreated and treated cells were filtered in a 0.22-lm membrane (Millipore). The reactions were performed at room temperature for 1 h in a reaction mixture (0.5 ml) containing 30 mM Tris–HCl/75 mM sucrose buffer, pH 6.8, 1 mg protein, and 10 mM p-NPP. The parasites were viable during the course of the experiments under all conditions used. The values represent the mean SE of three independent experiments, which were performed in triplicate. Parasites and supernatants treated with DMSO had a rate of p-NPP hydrolysis significantly different from control cells (P < 0:05, Student’s t test).
ditions shown to stimulate the process of cellular differentiation in these parasites [12,13,15]. Sodium tartrate, which was shown to be a potent inhibitor of secreted acid phosphatases of L. donovani [40] and H. m. muscarum [24], inhibited about 32% of H. samuelpessoai phosphatase activity (Table 1). In fact, H. samuelpessoai flagellates presented an extracellular phosphatase activity, measured in the culture medium,
which was higher in the cell-free medium obtained from parasites grown in the presence of DMSO (1:34 0:10 nmol Pi /mg of protein min), as compared to the medium from control flagellates (0:41 0:04 nmol Pi /mg of protein min) (Fig. 3B). Accordingly, H. samuelpessoai parasites secrete several proteins to the environment. Some of these proteins present proteolytic activity, which were modulated during the differentiation process triggered by DMSO [15]. These effects induced by DMSO on the ecto-phosphatase activity of H. samuelpessoai were confirmed by cytochemical analysis, which was detected by electrondense cerium phosphate deposits, the product of the reaction between cerium chloride and the inorganic phosphate obtained from the cleavage of b-glycerophosphate by phosphatase activity. Flagellates cultured in the presence of 4% DMSO showed different patterns of cytochemical reaction, as compared to the control parasites (Fig. 4). The control system showed electrondense deposits of cerium phosphate in cytoplasmic organelles (Figs. 4A and B), resembling acidocalcisomes, as described for other protozoan parasites due to the high concentrations of inorganic phosphate present in this organelle [41]. No cerium phosphate deposits were observed on the cell membrane or in the flagellar pocket (Fig. 4A). On the other hand, in DMSO-treated parasites, small electron-dense clusters were observed in the bottom of the flagellar pocket (Fig. 4C) and on the flagellum and flagellar membranes (Figs. 4D and E). Some of those clusters were free in the flagellar pocket or bound to small membrane vesicles, suggesting shedding of the enzyme from the parasite surface (Figs. 4D and E). It is important to note that the trypanosomatid flagellar pocket is a highly specialized area of the parasite membrane that is involved in several processes, including protein secretion to the external environment [42]. Similar results were obtained when T. cruzi parasites were grown in the presence of platelet-activating factor (PAF) [36], a lipid mediator that stimulates the cellular differentiation of T. cruzi [43] and of H. m. muscarum [44]. It is remarkable the similarity between PAF effects on T. cruzi with those obtained when H. samuelpessoai were grown in the presence of DMSO, regarding the induction of a secreted phosphatase, as well as the stimulation of cell differentiation. Taken together, those data show a striking resembling pattern induced by two completely different modulators, which could mean that when cell differentiation is triggered, a similar cascade of biochemical and morphological events occurs, regardless of the stimuli to which the cells are exposed. This secreted phosphatase activity present in supernatants is a small fraction (3%) of the total phosphatase activity. The phosphatase activity observed by cytochemical analysis could be the ecto-phosphatase shed from the surface of the parasites and/or it could be an enzyme
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Fig. 4. Cytochemistry assay for localization of acid phosphatase activity of H. samuelpessoai. The trypanosomatids were grown in a chemically defined medium at 26 °C for 48 h, in the absence (A and B) or the presence of 4% DMSO (C–E). The reactions were performed at room temperature in a buffer containing Tris–acetate, pH 5.0, and using b-glycerophosphate as the substrate and cerium chloride as the capture agent. In untreated cells, electron-dense deposits of cerium phosphate are observed in cytoplasmic organelles resembling acidocalcisomes (arrows in A and B). No labeling was observed on the cell membrane or in the flagellar pocket (arrowheads in A). In DMSO-treated cells acid phosphatase activity is observed as small electron-dense clusters, in the bottom of the flagellar pocket (arrow in C) and on the flagellum and flagellar pocket membranes (D and E). Some clusters are free in the flagellar pocket or bound to small membrane vesicles, suggesting a shedding of the enzyme from the parasite surface (arrowheads in D and E). N, nucleus; F, flagellum; FP, flagellar pocket. Bars, 1 lm.
secreted into the medium, during the course of the assay. In order to test the previous hypothesis, washed parasites grown in the absence or in the presence of 4% DMSO were further incubated for 1 h in the presence of 4% DMSO. The cells were washed three times and the supernatants were obtained, employing two sequential
centrifugation steps at 1500g for 10 min and then by filtering them through a 0.22-lm-pore membrane. The phosphatase activity measured in supernatants obtained from DMSO-treated parasites was higher than the activity of supernatants from control flagellates (Fig. 5B). Interestingly, whole DMSO-treated parasites presented
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B
A
C
Fig. 5. Effect of DMSO and protein kinase C (PKC) modulators on the phosphatase activities of H. samuelpessoai. (A) Effect of DMSO on cellassociated phosphatase activity of H. samuelpessoai. The parasites were cultured under chemically defined conditions at 26 °C for 48 h, without (a) or with 4% DMSO (c) Cells were harvested by centrifugation and washed three times in 30 mM Tris–HCl/75 mM sucrose buffer, pH 6.8. Living parasites grown in the absence or in the presence of DMSO were further incubated in the presence of 4% DMSO (b, d) for 60 min, centrifuged, and washed twice in the same buffer. Then, cells were incubated for 1 h in a fresh reaction mixture containing 30 mM Tris–HCl/75 mM sucrose buffer, pH 6.8, 1 mg protein, and 10 mM p-NPP. The parasites were viable during the course of the experiments under all conditions used. (B) Effect of DMSO on released phosphatase activity of H. samuelpessoai. Living parasites grown in the absence (a) or in the presence (c) of DMSO were further incubated with 4% DMSO (b, d) for 60 min and the supernatants from those cells were assayed for phosphatase activity, as described above (A). Supernatant from control cells, further treated with 4% DMSO for 1 h, had a rate of p-NPP hydrolysis significantly different from supernatant from control cells. DMSO-treated cells, further incubated for 1 h with 4% DMSO, showed an increase in the phosphatase activity associated to the cells (d, panel A), but not in the released phosphatase activity (d, panel B) (P < 0:05, Student’s t test). (C) Effect of DMSO and the PKC modulators sphingosine and PMA on the secreted and/or released phosphatase activity of H. samuelpessoai. The living parasites were incubated in the presence of 50 ng/ml sphingosine (Sphin) for 60 min or 20 ng/ml PMA for 20 min, centrifuged, and incubated for 1 h in a fresh reaction mixture containing 4% DMSO. Then, the supernatants were collected and filtered, prior to the phosphatase activity assays. Supernatants obtained from cells incubated in the reaction mixture without any drug addition are stated as control. Parasites treated with 4% DMSO had rates of p-NPP hydrolysis significantly different from control cells, from parasites treated with PMA, and from parasites pretreated with PMA and then with DMSO (P < 0:05, Student’s t test). All the values represent the mean SE of three independent experiments, which were performed in triplicate.
a 42% increase in its membrane-bound phosphatase activity, when they were incubated for an additional hour in the presence of 4% DMSO (Fig. 5A). This DMSO effect could not be attributed to a disruption of the cells once the cellular viability and the permeability of the plasma membrane to charged small molecules (like p-NPP) were not affected under the conditions employed here, and that no lactate dehydrogenase activity was detected either in the absence or in the presence of 4% DMSO (data not shown). DMSO modulates the activity of several enzymes and is able to activate lysosomes, resulting in the release of acid phosphatases [45]. Also, DMSO elicits a biphasic increase in diacylglycerol (DAG) levels, which is the endogenous activator of protein kinase C (PKC), during
the morphological differentiation of N1E-115 neuroblastoma cells [46]. In order to investigate if DMSO effects occur through a signal transduction pathway involving PKC, two modulators of PKC, sphingosine and phorbol-esther myristate (PMA), were tested on the DMSO-induced secretion of phosphatase (Fig. 5C). The phosphatase activity detected in the supernatants showed that 50 ng/ml sphingosine, known to inhibit PKC activity, induced a secretion of a phosphatase into the extracellular medium, which was similar to the results obtained when 4% DMSO was used, although sphingosine was not able to promote any additive effect on this DMSO-induced secretion. The phosphatase activity measured in the supernatant obtained from parasites pretreated with the PKC stimulator PMA (20 ng/
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ml) was 56.4% lower than that measured in the supernatant from control flagellates (Fig. 5C). Our results also show that 20 ng/ml PMA was able to suppress the DMSO-induced phosphatase secretion (Fig. 5C). Similarly, PMA abrogated the PAF-induced phosphatase secretion in T. cruzi [36]. Interestingly, PMA stimulates a PKC activity and induces filopodium-like projections in T. cruzi [47,48], as well as modulates a secreted acid phosphatase activity in Leishmania amazonensis, which mediates the infection of mouse macrophages by these flagellates [21]. Cell surface components play a key role in the survival of protozoan parasites in hostile insect and vertebrate environments and in confrontation with host immune responses. The precise role of ecto-phosphatase is not well established, but it has been related to cell growth, providing the cell with a source of inorganic phosphate by hydrolyzing phosphomonoester metabolites [22,24,28], as well as protecting the parasite by preventing the protozoan digestion in the alimentary tract of the invertebrate host [19]. Interestingly, the prevailing pH range of the insect gut (pH 6–7), the normal habitat for Herpetomonas [1], coincides with the pH range where higher values for phosphatase activity were observed in this paper. The characterization of cell surface-located phosphatase activity is particularly interesting because of its possible role in cell-to-cell interaction and/or reception and transduction of external stimuli [21,49]. Moreover, these enzymes could be good targets for the growth control of these protozoan parasites [50]. It would be of extreme importance to further investigate the modulation of phosphatase activities by DMSO in parasites, as well as the participation of these enzymes in the invertebrate-parasite relationship.
Acknowledgments We thank Dr. Patrıcia M.L. Dutra and Dr. Claudia O. Rodrigues for helpful discussions and Ms. Celina Monteiro Abreu for technical assistance. This work was supported by the Brazilian agencies PRONEX (0885), CNPq, FAPERJ, FINEP and FUJB/UFRJ.
References [1] F.G. Wallace, Exp. Parasitol. 18 (1966) 124–193. [2] J. Angluster, M.M. Bunn, W. De Souza, J. Parasitol. 63 (1977) 922–924. [3] R.B. McGhee, W.B. Cosgrove, Microbiol. Rev. 44 (1980) 140– 173. [4] M.C.M. Souza, A.P. Reis, W.D. Silva, Z. Brener, J. Protozool. 21 (1974) 579–583. [5] R. Fucs, M.A. Barcinski, J. Parasitol. 67 (1981) 463–467. [6] R.J. Etges, Acta Trop. 50 (1992) 205–217.
197
[7] P. Schneider, T.A. Glaser, Mol. Biochem. Parasitol. 58 (1993) 277–282. [8] J.P. Dedet, B. Roche, F. Pratlong, D. Cales-Quist, J. Jouannelle, J.C. Benichou, M. Huerre, T. Roy. Soc. Trop. Med. H. 89 (1995) 644–646. [9] A.M. Boisseau-Garsaud, D. Cales-Quist, N. Desbois, J. Jouannelle, A. Jouannelle, F. Pratlong, J.P. Dedet, T. Roy. Soc. Trop. Med. H. 94 (2000) 51–52. [10] Z.W. Yu, P.J. Quinn, Mol. Membr. Biol. 15 (1998) 59–68. [11] R.L. Neulieb, M.K. Neulieb, Cytobios 63 (1990) 139–165. [12] G.B. Castellanos, J. Angluster, W. De Souza, Acta Trop. 38 (1981) 29–37. [13] R.M.A. Soares, C.S. Alviano, F.C. Silva-Filho, M.J.G. Esteves, J. Angluster, W. De Souza, J. Submicrosc. Cytol. 16 (1984) 735–739. [14] R.M.A. Soares, C.S. Alviano, M.J.G. Esteves, J. Angluster, F.C. Silva-Filho, W. De Souza, Cell Biophys. 13 (1988) 29–41. [15] A.L.S. Santos, L.M. Batista, C.M. Abreu, C.S. Alviano, J. Angluster, R.M.A. Soares, Curr. Microbiol. 42 (2001) 111–116. [16] R. Derynck, E.F. Wagner, Curr. Opin. Cell Biol. 7 (1995) 843– 844. [17] M. Parsons, L. Ruben, Parasitol. Today 16 (2000) 56–62. [18] N. Sacerdoti-Sierra, C.L. Jaffe, J. Biol. Chem. 272 (1997) 30760– 30765. [19] M. Gottlieb, D.M. Dwyer, Science 212 (1981) 939–941. [20] K.H. Nagakura, H. Tachibana, Y. Kaneda, Comp. Biochem. Physiol. 81 (1985) 815–817. [21] M.A. Vannier-Santos, A. Martiny, J.R. Meyer-Fernandes, W. De Souza, Eur. J. Cell Biol. 67 (1995) 112–119. [22] E.C. Fernandes, J.R. Meyer-Fernandes, M.A.C. Silva-Neto, A.E. Vercesi, Z. Naturforsch. 52c (1997) 351–358. [23] J.R. Meyer-Fernandes, M.A.C. Silva-Neto, M.S. Soares, E.C. Fernandes, A.E. Vercesi, M.M. Oliveira, Z. Naturforsch. 54c (1999) 884–977. [24] P.M.L. Dutra, C.O. Rodrigues, J.B. Jesus, A.H.C.S. Lopes, T. Souto-Padr on, J.R. Meyer-Fernandes, Biochem. Biophys. Res. Commun. 253 (1998) 164–169. [25] P.M.L. Dutra, C.O. Rodrigues, A. Romeiro, L.A.M. Grillo, F.A. Dias, M. Attias, W. De Souza, A.H.C.S. Lopes, J.R. MeyerFernandes, Phytopathology 90 (2000) 1032–1038. [26] R.H. Glew, A.K. Saha, S. Das, A.T. Remaley, Microbiol. Rev. 52 (1988) 432–512. [27] A.T. Remaley, S. Das, P.I. Campbell, G.M. LaRocca, M.T. Pope, R.H. Glew, J. Biol. Chem. 260 (1985) 880–886. [28] N. Bakalara, A. Seyfang, C. Davis, T. Baltz, Exp. Parasitol. 81 (1995) 302–312. [29] N. Bakalara, X. Santarelli, C. Davis, T. Baltz, J. Biol. Chem. 275 (2000) 8863–8871. [30] C. Roitman, I. Roitman, H.P. Azevedo, J. Protozool. 19 (1972) 346–349. [31] A.L.S. Santos, A. Ferreira, V.A. Franco, C.S. Alviano, R.M.A. Soares, Curr. Microbiol. 39 (1999) 61–64. [32] P.M.L. Dutra, F.A. Dias, M.A.A. Santos, C.O. Rodrigues, A. Romeiro, M. Attias, W. De Souza, A.H.C.S. Lopes, J.R. MeyerFernandes, Curr. Microbiol. 43 (2001) 288–292. [33] O.H. Lowry, N.J. Rozebrough, A.L. Farr, R.J. Randall, J. Biol. Chem. 193 (1951) 265–275. [34] C.E. Peres-Sampaio, S.T. Palumbo, J.R. Meyer-Fernandes, Z. Naturforsch. 56c (2001) 820–825. [35] H.J. Motulsky, L.A. Ransnas, FASEB J. 1 (1987) 365–374. [36] C.O. Rodrigues, P.M.L. Dutra, F.S. Barros, T. Souto-Padr on, J.R. Meyer-Fernandes, A.H.C.S. Lopes, Biochem. Biophys. Res. Commun. 266 (1999) 36–42. [37] B.I. Posner, R. Faure, J.W. Burgess, A.P. Bevan, D. Lachance, G. Zhang-Sun, I.G. Fantus, J.B. Ng, D.A. Hall, B.S. Lum, J. Biol. Chem. 269 (1994) 4596–4604. [38] R.H. Glew, M.S. Czuczam, W.F. Diven, R.L. Berens, M.T. Pope, D.E. Katsoulis, Comp. Biochem. Physiol. 72 (1982) 581–590.
198
A.L.S. Santos et al. / Archives of Biochemistry and Biophysics 405 (2002) 191–198
[39] T. Furuya, L. Zhong, J.R. Meyer-Fernandes, H.G. Lu, S.N.J. Moreno, R. Docampo, Mol. Biochem. Parasitol. 92 (1998) 339– 348. [40] J.K. Lovelace, M. Gottlieb, Am. J. Trop. Med. Hyg. 35 (1986) 1121–1128. [41] R. Docampo, S.N.J. Moreno, Parasitol. Today 15 (1999) 443–448. [42] P. Webster, D.G. Russel, Parasitol. Today 9 (1993) 201–205. [43] C.O. Rodrigues, P.M.L. Dutra, T. Souto-Padr on, R.S.B. Cordeiro, A.H.C.S. Lopes, Biochem. Biophys. Res. Commun. 273 (1996) 735–740. [44] A.H.C.S. Lopes, P.M.L. Dutra, C.O. Rodrigues, M.J. Soares, J. Angluster, R.S.B. Cordeiro, J. Eukaryot. Microbiol. 44 (1997) 321–325.
[45] D.W. Misch, M.S. Misch, Proc. Natl. Acad. Sci. USA 58 (1967) 2462–2467. [46] S. Clejan, R.S. Dotson, E.W. Wolf, M.P. Corb, C.F. Ide, Exp. Cell Res. 224 (1996) 16–27. [47] M.L. G omez, L. Erijman, S. Arauzo, H.N. Torres, M.T. TellezI~ non, Mol. Biochem. Parasitol. 36 (1989) 101–108. [48] T.U. Carvalho, W. De Souza, Parasitol. Res. 74 (1987) 11–17. [49] A. Martiny, M.A. Vannier-Santos, V.M. Borges, J.R. MeyerFernandes, J. Asseruy, N.L. Cunha-e-Silva, W. De Souza, Eur. J. Cell Biol. 71 (1996) 206–215. [50] P.M.L. Dutra, F.A. Dias, M.A.A. Santos, C.O. Rodrigues, A. Romeiro, M. Attias, W. De Souza, A.H.C.S. Lopes, J.R. MeyerFernandes, Phytopathology 91 (2001) 408–414.