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Different secreted phosphatase activities in Leishmania amazonensis

Anne C.S. Fernandes, Deivid C. Soares, Elvira M. Saraiva, José R. Meyer-Fernandes, Thaïs Souto-Padrón
DOI: http://dx.doi.org/10.1111/1574-6968.12080 117-128 First published online: 1 March 2013

Abstract

Leishmania has strong acid phosphatase activity on the external surface of the plasma membrane and secreted into the extracellular milieu. Secreted acid phosphatase (sAcP), which is the most abundant secreted protein of Leishmania, is also a virulence factor that plays a role in vertebrate infection and survival in sand flies. In this study, we characterized the secreted phosphatase activities in Leishmania amazonensis. Both acidic and alkaline secreted phosphatase activities were observed with β-glycerophosphate and p-nitrophenyl phosphate (p-NPP) hydrolysis and were inhibited with sodium tartrate and sodium orthovanadate. Cytochemical labeling revealed a significant difference in the localization of the electron-dense precipitates depending on the substrate. β-Glycerophosphate electron-dense precipitates were concentrated on both the cell surface and flagellar pocket, whereas p-NPP labeling occurred primarily within intracellular organelles. Orthovanadate-treated metacyclic promastigotes were less infective and were confined to a tight parasitophorous vacuole (PV), which is not characteristic of this Leishmania species. Based on the results, we characterized the presence of different secreted phosphatase activities in L. amazonensis, the influence of the substrate in cytochemical labeling, and the potential involvement of secreted phosphatase activity in both PV maturation and amastigote survival.

Keywords
  • Leishmania amazonensis
  • secreted phosphatase activities
  • cytochemistry
  • macrophages

Introduction

Leishmania are a group of protozoan pathogens that cause a variety of diseases ranging from self-healing cutaneous disease to severe (and lethal if untreated) visceral leishmaniasis (Alvar et al., 2012; Antinori et al., 2012; Soong et al., 2012). Leishmania are digenetic protozoan that display distinct morphological and functional forms, existing as both motile flagellate promastigotes that live in the alimentary tract of the sandfly vector and nonflagellate intracellular amastigotes living inside the parasitophorous vacuole (PV) within the macrophages of the vertebrate host (Vannier-Santos et al., 2002). Both environments — the intestinal gut and the PV — are extremely aggressive because the pH is far from neutral, and many hydrolytic enzymes are present. Leishmania, as with other pathogenic microorganisms, have developed survival strategies that ensure their ability to survive and proliferate in adverse environments. The analysis of various species and developmental forms of Leishmania has indicated that glycoconjugates [including Lipophosphoglucan (LPG) and Protophosphoglucan (PPG)] and metalloproteases are the primary molecules involved in parasite survival, inhibition of lysis via the complement pathway and recognition by mannose-binding proteins (MBP), the inhibition of phagosome–endosome fusion, the inhibition of host lysosomal enzymes, the alteration of host signal transduction, and the inhibition of nitric oxide and cytokine production (Cunningham, 2002; Cosentino-Gomes & Meyer-Fernandes, 2011; Gomes et al., 2011).

Trypanosomatids, such as Trypanosoma and Leishmania, have the ability to hydrolyze organophosphates; this reaction is catalyzed by different protein phosphatases that are classified either as ecto-/extracytoplasmic and found on the cell surface or as secreted/extracellular isolated from the culture media (Escalona-Montaño et al., 2010; Szoor, 2010; Cosentino-Gomes & Meyer-Fernandes, 2011; Gomes et al., 2011). Both protein phosphorylation and dephosphorylation are important post-translational modifications for all known eukaryotic organisms. These processes regulate various signaling pathways involved in the control of cell cycle and differentiation, cell–cell and cell–substrate interactions, cell motility, ion channel, immune response, and basic metabolism (Hunter, 1995). In pathogenic microorganisms, membrane-bound and secreted kinase and phosphatase activities are directly associated with the degree of virulence (Cunningham, 2002).

The ecto- and secreted phosphatase activities in several Leishmania species have been studied for many years by different groups that have defined their optimum pH levels, substrate and inhibitor specificities, molecular weight, cellular localization, and resistance to proteolytic degradation (Bates & Dwyer, 1987; Bates et al., 1989, 1990; De Almeida-Amaral et al., 2006; Heneberg, 2009, 2012). Secreted acid phosphatases are constitutively released by promastigote forms of all Leishmania species except L. major. In Leishmania, the function of phosphatases can be related to the survival of the parasite inside the host cell (Barbieri et al., 1990). This enzyme inhibits the production of superoxide anions by neutrophils and macrophages indicating the involvement of this enzyme with an important virulence factor of this parasite (Bates & Dwyer, 1987; Katakura & Kobayashi, 1988; Ellis et al., 1998). Although marked differences were observed in both the levels and molecular weights of the secreted acid phosphatases released by different species, stocks, and developmental forms, each type shares similar immunological epitopes (Doyle & Dwyer, 1993). In the present study, we characterized distinct secreted phosphatase activities in L. amazonensis based on ultrastructural localization, substrate affinity, and susceptibility to inhibitors. Moreover, we addressed the role that phosphatases play in Leishmania infection of macrophages and intracellular development. Trypanosomatids have been used for many years in our laboratory as a model for studies of cell biology of protozoa due to their own structural and physiological characteristics. In particular, we use the Leishmania amazonensis due to relevance and morbidity in the New World not only causing cutaneous leishmaniasis but also the diffuse and visceral manifestation of the disease (Barral et al., 1991).

Materials and methods

Parasites

The MHOM/BR/75/Josefa strain of L. amazonensis was used in this study. Promastigotes were cultured in Schneider's insect medium (Sigma) supplemented with 10% fetal calf serum (FCS) at 26 °C for 72 h. The procyclic forms were harvested from 1- to 2-day logarithmic phase cultures. The metacyclic promastigotes were purified from 5- to 6-day stationary cultures using a Ficoll gradient as previously described by Späth & Beverley (2001). Leishmania amazonensis amastigotes were isolated as previously described (Saraiva et al., 1983; Wanderley et al., 2006). Briefly, amastigotes isolated from BALB/c mouse footpad lesions, which had been inoculated 4–6 weeks prior with 1 × 106 promastigotes, were obtained by dissection and homogenization in saline buffer under sterile conditions. The parasites were centrifuged, washed, and then quantified via microscopic analysis. Parasite viability was monitored based on motility and propidium iodide labeling using flow cytometry. Parasite viability was not affected under the conditions employed here.

Preparation of culture supernatants

The parasites were incubated (1 × 108 cells mL−1) in RPMI medium without serum for 1 h at 26 °C. The culture aliquots were centrifuged at 1500 g to remove the cells, and the resulting supernatants were filtered through a 0.22-µm membrane.

Measurement of phosphatase activity

Phosphatase activity was determined spectrophotometrically by measuring the rate of p-nitrophenol hydrolysis (p-NP, p-NPP hydrolysis products). Both intact cells (2.0 × 107 cells mL−1) and the supernatant (50 µL) were incubated at 25 °C for 60 min in a reaction mixture (0.5 mL) containing 5 mM p-NPP as the substrate in a 50 mM Mes–Hepes–Tris buffer (pH range 4.0–9.0). The reaction was started by the addition of cells and stopped by the addition of 1.0 mL 1 M NaOH. The control cells, which were added after the interruption of the reaction, were used as blank. To determine the concentration of released p-NPP, the tubes were centrifuged at 1500 g for 15 min at room temperature, and the supernatants were then analyzed using a spectrophotometer at 425 nm. We also tested sodium β-glycerophosphate as a substrate. In this case, the hydrolytic activities were analyzed spectrophotometrically by measuring the released inorganic phosphate (Pi) from these substrates under the same conditions described above (Fiske & Subbarow, 1925). Phosphatase activity was measured by subtracting the values for nonspecific hydrolysis and that present in conditioned medium. The values obtained for phosphatase activity using p-NPP as substrate with either p-NP or Pi were exactly the same. The values shown represent the average value ± SE of three independent experiments.

Inhibition assays

Both the ecto membrane-bound and secreted phosphatase activities of L. amazonensis were analyzed in an incubation medium containing 5 mM β-glycerophosphate or p-NPP as substrates and inhibitors of phosphatase activity, sodium tartrate and sodium orthovanadate (10 mM), and sodium fluoride, ammonium molybdate, zinc chloride, and levamisole (1 mM). The inhibitors were added to the incubation medium immediately before the addition of either parasites or culture supernatant. The phosphatase activities of intact parasites and cell-free culture supernatants in the absence of inhibitors, 9.95 ± 0.25 nmol h−1 10−7 cells and 83.66 ± 0.25 nmol h−1 10−7 cells, respectively, were defined as 100%.

Flow cytometry analysis of cell viability

Untreated promastigote and amastigote forms, those incubated for 3 h in culture medium and those treated with orthovanadade (10 mM) at a final concentration of 1 × 106 cells mL−1, were incubated with 15 µg mL−1 PI for 15 min at 26 °C. Cells were kept on ice until data acquisition and analysis with a FACSCalibur Flow Cytometer (Becton-Dickinson, Franklin Lakes, NJ) equipped with CellQuest software (Joseph Trotter, Scripps Research Institute, San Diego, CA). A total of 10 000 events were acquired in the region previously established to correspond to the parasites. Data were obtained from at least three independent experiments.

Parasite–macrophage interactions

Thioglycolate (3%)-stimulated macrophages isolated from female BALB/c mice were harvested in RPMI medium and plated on 13-mm2 coverslips in 24-well plates, and 5 × 105 cells were allowed to adhere for 30 min at 37 °C in a 5% CO2 atmosphere on coverslips placed in 24-well culture plates. Next, nonadherent cells were removed, and the adherent macrophages were washed twice with PBS (pH 7.2) and cultured for 24 h in RPMI medium supplemented with 10% FBS for 24 h at 37 °C in a 5% CO2 atmosphere. Both untreated and orthovanadate (10 mM for 1 h at 26 °C)-treated promastigotes were allowed to interact with the macrophages at a 5:1 ratio for 1 h in RPMI medium supplemented with 10% FBS, pH 6.0. Next, the coverslips were washed, fixed in Bouin, and stained by Giemsa to determine the percentage of infected macrophages. Some coverslips were cultured for up to 24 h in the presence of either RPMI medium or supernatant obtained as previously described. The coverslips were washed, fixed, and stained as previously described, and the percentage of infected macrophages was determined by counting 200 cells in triplicate coverslips. Macrophages used in this investigation were obtained following the guidelines for animal experimentation provided by the USA National Institute of Health, and the experimental protocol was approved by the Centro de Ciências da Saúde (Universidade Federal do Rio de Janeiro) ethical committee for animal experimentation.

Cytochemical detection of phosphatase activities

Promastigotes were first fixed in 1% glutaraldehyde and 3.7% sucrose in 0.1 M cacodylate buffer (pH 7.2) for 10 min at 4 °C. Next, they were washed once in 0.1 M cacodylate buffer (pH 7.2) containing 3.7% sucrose and then incubated for 10 min in either 0.1 M sodium acetate buffer (pH 5.0) or Tris-maleate buffer (pH 8.0) containing 5% sucrose. The cells were then incubated for 1 h at 37 °C under constant agitation in medium (2 mM sodium β-glycerophosphate or p-NPP (used as substrates) and either 2 mM cerium chloride, 5% sucrose, and 0.1 M sodium acetate buffer (pH 5.0) for acid phosphatase detection or Tris-maleate buffer (pH 8.0) containing 5% sucrose for alkaline phosphatase detection). The control cells were incubated either in reaction medium without substrate or in reaction medium containing either 10 mM sodium tartrate or sodium orthovanadate as an enzyme inhibitor. After incubation in the cytochemical medium, the cells were washed in Tris-maleate and sodium cacodylate buffers; fixed again for 1 h in 2.5% glutaraldehyde, 3.7% sucrose, and 0.1 M cacodylate buffer at room temperature; postfixed in OsO4; dehydrated in a graded acetone series; and embedded in PolyBed resin (Tokumitsu & Fishman, 1983; Robinson, 1985). The unstained ultrathin sections were analyzed using an FEI Morgagni transmission electron microscope.

Graphics and statistical analysis

All graphs and statistical analyses were generated in GraphPad Prism 5.0. All data are presented as the mean value ± the SE of the mean. The means were determined from three independent assays. Standard error bars were not shown where the error range is smaller than the symbol site. P value ≤ 0.05 was considered significant.

Results

The detection of acid phosphatase activities in the promastigotes and amastigotes of L. amazonensis

Previous data (De Almeida-Amaral et al., 2006) demonstrated the presence of acid phosphatase activity on the surface of intact procyclic L. amazonensis promastigotes using p-NPP as a substrate. In the same study, it was shown that this activity was concentrated on the parasite cell surface with an ecto-phosphatase, as the acid phosphatase activity in the supernatant of the parasites examined using the same parameters was not significant. Previous studies dedicated to the analysis of acid phosphatase activity in various trypanosomatids using cytochemical techniques have consistently shown the presence of strong reactivity localized to the membrane of the cell body and within the flagellar pocket, which is primarily observed in Leishmania (Pimenta & De Souza, 1986; Dutra et al., 1998; Rodrigues et al., 1999). The difference between the two studies was the enzyme substrate used; most of the electron microscopy studies involving Leishmania have used β-glycerophosphate as the substrate for the cytochemical detection of acid phosphatases (Pimenta & De Souza, 1986; Dutra et al., 1998; Rodrigues et al., 1999). Therefore, we analyzed both the ecto- and secreted phosphatase activities in all developmental forms of L. amazonensis using p-NPP and β-glycerophosphate as substrates considering that the secreted phosphatase activity had already been described in L. donovani amastigotes (Ellis et al., 1998). We observed that the p-NPP substrate was more specific for the detection of phosphatase activity on the surface (ecto-phosphatase) of intact parasites (Fig. 1a), whereas β-glycerophosphate was preferentially recognized by the secreted form of the enzyme (Fig. 1b). Procyclic promastigotes had a relatively increased response to the two substrates followed by metacyclics. Flow cytometry analysis of propidium iodide labeling indicated that phosphatase secretion does not result in cell damage (Fig. 1c).

Comparative analysis of the phosphatase activities of Leishmania amazonensis in the presence of different substrates. The phosphatase activity of both intact parasites (2 × 107 cells mL−1) (a) and those secreted in the culture medium (approximately 0.9784 mg ptn mL−1) (b) was assayed for 1 h at 26 °C in the presence of 5 mM β-glycerophosphate (■) or p-NPP () in buffer containing 116.0 mM NaCl, 5.4 mM KCl, 5.5 mM glucose, and 50.0 mM Hepes–Tris, pH 7.0. The results are expressed as nmol of inorganic phosphate (a) or p-NP (b). (c) The figure shows the viability of procyclic, metacyclic, and amastigote forms incubated with propidium iodide and analyzed by flow cytometry. No significant differences were observed between the viability of control cells and those incubated for 3 h in culture medium (3 hpi). The values reported represent the average mean of three independent experiments, and the results displayed in the bar graphs represent the mean values ± SE.

The effect of pH on secreted phosphatase activity

Upon identifying the differences in the activity of the secreted form of the phosphatase of L. amazonensis in relation to the substrate, we evaluated the influence of pH on this activity. The supernatants of the procyclic and metacyclic promastigotes and amastigotes were assayed in the presence of both p-NPP and β-glycerophosphate. The supernatant of the procyclic promastigotes was the only sample that had residual activity for the p-NPP substrate, which did not change with varying pH levels. Regarding the enzymatic activities obtained with β-glycerophosphate, we found that promastigotes had at least two different secreted phosphatase activity both acid and alkaline secreted phosphatase activities. Of the promastigotes, the procyclic promastigotes had the highest activity levels at both pH 5.0 (119 nmol Pixh−1 10−8 cell) and pH 9.0 (122 nmol Pixh−1 10−8 cell) and very low activity levels (2.33 nmol Pixh−1 10−8 cell) at pH 7.5 (Fig. 2a). Different from that observed in procyclics, significant levels of acidic phosphatase activity were observed in metacyclic promastigotes between pH 6.5 and pH 7.0 (∼35 nmol Pixh−1 10−8 cell), which decreased at pH 7.5 (20.3 nmol Pixh−1 10−8 cell) and increased at pH 8.0 (37.7 nmol Pixh−1 10−8 cell; the highest value) (Fig. 2b). These data demonstrate that promastigotes had acid and alkaline secreted phosphatases, in which the pH optimum varied with the developmental forms (Fig. 2c). Ecto-phosphatase activity was not detected in amastigotes. A significant, but reduced secreted phosphatase activity (7.54 nmol Pixh−1 10−8 cell) was observed at pH 6.5. It decreased with increasing pH and was not detected in pH 7.5 and 8.0 (data not shown).

The characterization of the secreted phosphatase activities of Leishmania amazonensis. (a–c) The effect of pH on secreted phosphatase activity. The supernatant of the procyclic (a) and metacyclic promastigotes (b), which were obtained as described previously, was assayed for 1 h at 26 °C at pH values ranging from 4.0 to 9.0 in the presence of 5 mM βglycerophosphate in 116.0 mM NaCl, 5.4 mM KCl, 5.5 mM glucose, and 50.0 mM Mes–Hepes–Tris buffer. In this pH range, the cells were viable throughout the course of reaction. (c) Comparative analysis of the levels of secreted acid and alkaline phosphatase activity obtained from the three different developmental forms of L. amazonensis at different pHs. The reactions were performed in the presence of 5 mM β-glycerophosphate in the same incubation media described above and at the indicated pH values; the results are expressed as nmol of Pi. The values reported in each graph represent the mean ± SE of the average value of at least three independent experiments, which where performed in triplicate.

The acid and alkaline secreted phosphatase activity time courses were analyzed at pH 6.5 and 8.5. β-Glycerophosphate was used as the substrate, and the phosphatase activities were quantified at regular time intervals from 10 to 120 min at 26 °C (Fig. 3). After a 10-min incubation of the procyclic promastigotes in culture medium, we found that the acid phosphatase activity at pH 6.5 was higher than that observed at pH 8.5. The level of secreted acid phosphatase activity increased significantly up to 20 min and increased more to 1 h, representing a value approximately 80% greater than the observed in 10 min. After 1 h, we observed a gradual decrease in activity with increasing time, in last times. There were no significant changes in secreted acid and alkaline phosphatase activity from 90 min to 120 min. However, the secreted alkaline phosphatase displayed significantly different kinetics. It increased slightly from 10 to 20 min and reached a maximum value after 60 min of incubation (Fig. 3).

The time course of acid (black line) and alkaline (dashed line) secreted phosphatase activity of Leishmania amazonensis. The supernatant of the procyclic promastigotes, which was obtained as described in the Materials and Methods section, was incubated in reaction medium containing 116.0 mM NaCl, 5.4 mM KCl, 5.5 mM glucose, 50.0 mM Hepes–Tris buffer (pH 6.5 or 8.5), and 5 mM sodium β-glycerophosphate. The values reported represent the mean ± SE of the average of at least three independent experiments, which were performed in triplicate.

The effects of phosphatase Inhibitors

The effect of different phosphatase inhibitors on both the ecto- and secreted phosphatase activities in procyclic and metacyclic promastigotes of L. amazonensis in both acidic (pH 6.5) and alkaline (pH 8.5) conditions are summarized in Table 1. The inhibitors used were as follows: sodium tartrate, sodium fluoride (NaF), sodium molybdate, zinc chloride (ZnCl2), and levamisole. The acid and alkaline ecto-phosphatase activities of the different developmental forms of L. amazonensis, which were obtained in the presence of p-NPP, showed different levels of sensitivity to sodium tartrate. We observed that the acid and alkaline activities of the procyclic promastigotes were only partially inhibited (∼22%), while that detected in metacyclic forms were completely blocked by tartrate. When β-glycerophosphate was used as substrate for the detection of ecto-phosphatase activity, we observed that the percentage of inhibition achieved in the presence of tartrate, NaF, molybdate, ZnCL2, and levamisole varied from ∼70% to 100%. However, the alkaline ecto-phosphatase activities observed in both procyclics and metacyclics were less sensitive to the effect of levamisole. Ecto-phosphatase activities on amastigotes of L. amazonensis assayed in the presence of both p-NPP and β-glycerophosphate were significantly (from 71 to 100%) inhibited in the presence of sodium tartrate (data not shown).

View this table:
Table 1

Effect of inhibitors on the phosphatase activity of Leishmania amazonensis

Phosphatase activity — percentage of inhibition
Acid (pH 6.5)Alkaline (pH 8.5)
PhosphatasesInhibitorsProMetaProMeta
Ecto-phosphataseSodium tartrate (10 mM)74.4 (23.6)100 (100)92.3 (22.4)100 (100)
Sodium fluoride (1 mM)7210090100
Ammonium molybdate (1 mM)10010010090
Zinc chloride (1 mM)70100100100
Levamisole (1 mM)71.87930.630
Sodium tartrate (10 mM)97.493.599.892.9
Secreted phosphataseNaF (1 mM)94.16495.8100
Ammonium molybdate (1 mM)94.310080100
Zinc chloride (1 mM)99.410083.675
Levamisole (1 mM)95.760100100
  • Values of inhibition in the presence of β-glycerophosphate as substrate.

  • Values of inhibition in the presence of p-NPP as substrate.

The effect of inhibitors on the acid and alkaline secreted phosphatase activity of L. amazonensis was assayed in procyclic and metacyclic promastigotes in the presence of β-glycerophosphate as substrate (Table 1). Both phosphatase activities were significantly blocked by the different inhibitors. The values of the rate of inhibition ranged from 60 to 100%.

Phosphatase cytochemistry

Because we verified that the phosphatase activities in both the intact parasite and the culture medium had different specificities with respect to the substrate, we examined the cytochemical localization of these activities using both p-NPP and β-glycerophosphate as substrates. With β-glycerophosphate as the substrate, we observed that a majority of the cells displayed an intense and homogeneous staining pattern on the cell body, flagellar pocket, and flagellar membranes (Fig. 4ac). Reaction products were also observed within the flagellar pocket and on the vesicle membranes (Fig. 4b). In the cytoplasm, electron-dense precipitate was observed in the ER, distributed throughout the cell body and in the tubules and vesicles located in the region of the flagellar pocket (Fig. 4d). Intense staining was also observed within the multivesicular tubules (Fig. 4a). The cytochemical staining obtained using p-NPP as a substrate showed a distribution pattern that was significantly different from that observed using β-glycerophosphate as a substrate. Intense staining of intracellular compartments, such as the ER, Golgi, and multivesicular tubules, was observed (Fig. 4eg). Labeling of the cell body, flagellar pocket, and flagellum membranes was not homogeneous and much less intense than that observed with β-glycerophosphate. In addition to the weak cell surface labeling, promastigotes also had weak phosphatase activity in the flagellar pocket (Fig. 4e).

Cytochemical detection of phosphatase activities in the procyclic promastigotes of Leishmania amazonensis. Acid (a–g) and alkaline (h, i) phosphatase activities were observed in incubation media containing β-glycerophosphate (a–d, h) and p-NPP as substrates (e–g, i). Longitudinal (a) and cross sections (c) of the procyclic forms showing the reaction product on the surface (black arrows) and within the flagellar pocket (arrowhead). Note that significant amounts of reaction product were found on the flagellar membrane (white arrow) and in some cytoplasmic compartments localized at the posterior region of the parasite (asterisk). (b) A higher magnification of the flagellar pocket region showing the intense reaction on the flagellar pocket membrane (black arrows) and on the membrane of small vesicles present in the pocket (white arrows). (d) The anterior end of the parasite showing the reaction product in several compartments near the flagellar pocket (arrows). (e–g) The reaction product was concentrated in intracellular organelles, such as the multivesicular tubules (arrows in e and g) and the Golgi complex (arrowheads in e and f). Patches of reaction product were observed on the cell body and flagellar membranes (white arrows in e). (h–i) The detection of alkaline phosphatase activity in the presence of β-glycerophosphate (h) and p-NPP (i). Note the presence of a thicker electron-dense layer when β-glycerophosphate was used as the substrate (small arrow in h). The inhibition of acid (j, k) and alkaline (l, m) phosphatase activity with the addition of incubation media with 10 mM sodium tartrate (j, l) or 10 mM sodium orthovanadate (k, m). Note the intense reduction in the electron-dense precipitates in all images. Bars: a–d = 300 nm; e–g, j, and l = 500 nm; h, i, k, and m = 1 µm.

β-Glycerophosphate was also the best substrate for the detection of the surface and secreted alkaline phosphatase activities. The deposition of the reaction product was observed on both the whole cell body membrane and the flagellum. The electron-dense precipitates were also observed in the flagellar pocket (Fig. 4h). With p-NPP as the substrate, the reaction was weaker but had the same localization pattern (Fig. 4i). The intracellular labeling observed with the two different substrates was weak and concentrated in both the Golgi and the compartments within the anterior region of the cell body located near the flagellar pocket.

In the detection of both acidic and alkaline phosphatase cytochemical staining, no reaction product was observed when the substrates were omitted or when sodium tartrate and sodium orthovanadate were added to the incubation medium (Fig. 4jk).

Phosphatase function in both phagocytosis and the infection process

Having established the presence of secreted phosphatases, we next tested the functional role that acid phosphate activity plays in macrophage infection. Infection of macrophages by both control- and sodium orthovanadate-treated metacyclic promastigotes was evaluated by determining the endocytic index (EI) after 1- and 24-h infections (Fig. 5). After the 1-h infection, a large number of infected macrophages, some with more than three parasites, were observed in the Giemsa-stained coverslips (Fig. 5a). An accentuated EI reduction (63%) was observed when the macrophages were incubated in the presence of the sodium orthovanadate-treated parasites (Fig. 5d and f). After the 24-h infection with the control metacyclic promastigotes, we observed macrophages with large vacuoles containing amastigote forms, which is characteristic of L. amazonensis infection (Fig. 5b and C). The same was not observed when the orthovanadate-treated parasites were used (Fig. 5e). Apparently, the characteristic PVs were not present, and an accentuated reduction in EI was found when compared with the 1-h infection results. These observations suggest that some of the orthovanadate-treated parasites did not survive the infection process. Additionally, we performed infection assays as previously described in the presence of a conditioned medium as a source of additional secreted acid phosphatase activity (55.06 nmol Pixh−1 10−8 cell). In this case, an increase of 56% in the EI value was observed after the 1-h infection (Fig. 5f). After the 24-h infection, we observed the same result with the infected macrophages as was observed with the untreated controls in which the amastigotes were found inside large vacuoles (Fig. 5c and f). Interestingly, we did not observe multiplication, as the EI values recorded after the 1- and 24-h infection did not vary. This suggests that, although the internalized metacyclic forms in this experimental condition differentiated into amastigotes, they did not multiply inside the PV. With the parasite–macrophage interaction in the presence of conditioned medium plus sodium orthovanadate, we observed a reduction in EI at approximately 86% and 95% for the 1- and 24-h infections, respectively, which was associated with the values obtained exclusively in the presence of the conditioned medium (Fig. 5f). In Fig. 5 was observed that both membrane-associated and secreted phosphatases are involved in the macrophage infection and intracellular development, as there was a significant increase in the infection with the addition of secreted phosphatases present in the conditioned medium. Here, both secreted and membrane enzymes seem to act synergistically.

The involvement of secreted phosphatases in the infection and intracellular survival of Leishmania amazonensis. Both control (a, b, and f) and orthovanadate-treated (d–f) metacyclic promastigotes were incubated in the presence of murine macrophages, and the cells were fixed after 1 h (a, d, and f □) and 24 h (b–c, e, and f), Giemsa-stained, analyzed by light microscopy (a–e), and counted to determine the endocytic index (EI = % infected macrophage (MØ) × average parasites/MØ after 24 h). (a) After a 1-h interaction, most macrophages were infected, and some had up to five parasites that were either adhered or internalized (arrow). (b) After a 24-h infection with control parasites, the macrophages had many intracellular amastigotes within large vacuoles (small arrows). (c) After a 24-h infection in the presence of conditioned medium, the macrophages also had parasites inside large vacuoles (arrows). After a 1-h (d) and 24-h (e) infection with orthovanadate-treated parasites, there were few infected macrophages; in those with intracellular parasites, large vacuoles were not observed (small arrows in E). The comparative analysis of EI under different experimental conditions (f). Orthovanadate treatment significantly reduced EI. The addition of secreted phosphatases present in the conditioned medium increased the EI by approximately 50%. However, the presence of additional secreted phosphatases failed to reverse the inhibition caused by orthovanadate treatment. The letters represent the statistical analysis, in which the different letters represent significant differences among the analyzed groups. P ≤ 0.05. The results reported in the bar graphs represent the mean values ± SE of the average of at least three independent experiments, which where performed in triplicate. (g) The figure shows the viability of metacyclic forms incubated with propidium iodide and analyzed by flow cytometry. No significant differences were observed between the viability of control parasites and those incubated in the presence of vanadate for 3 h in culture medium (3 hpi).

Discussion

Phosphorylation and dephosphorylation processes regulate virtually all aspects of cellular life (Hunter, 1995). They are coupled processes in which the lack of control produces serious consequences on normal cellular function. It has been shown that these two important enzymatic activities are also critical for the interaction between cells, particularly for pathogen–host cell interactions (Kneipp et al., 2004; Gomes et al., 2011). Kinases and proteases on the cell surface and secreted by some protozoa (Katakura & Kobayashi, 1988; Singla et al., 1992; Heneberg, 2012; Lambertz et al., 2012; Lim et al., 2012) are considered to be important virulence factors.

In a previous study, Gottlibe & Dwyer (1981) demonstrated the presence of acid phosphatase activity on the outer surface of L. donovani promastigotes. This activity was also detected in Golgi-derived intracellular vesicles that fuse with endosomal compartments, which are transferred along the endocytic pathway to the lysosomes. A high level of acid phosphatase activity in Leishmania was confirmed by Glew et al. (1982), who also showed that promastigotes have low levels of other acid hydrolases. The surface-bound acid phosphatases were further divided into three distinct groups according to molecular weight, resistance to sodium tartrate, and substrate affinity (Remaley et al., 1985). In addition to those activities described as either intracellular or membrane-bound, various studies regarding Leishmania described an intense acidic phosphatase activity in the culture medium (Gottlieb & Dwyer, 1982; Lovelace & Gottlieb, 1986; Doyle & Dwyer, 1993; Shakarian & Dwyer, 2000; Escalona-Montaño et al., 2010). Recent studies have shown that promastigotes and amastigotes of different pathogenic species secrete sAcP via an unconventional mechanism that involves the release of exosome-like vesicles; however, the possibility that the enzymes are released via a shedding mechanism cannot be ignored (Silverman et al., 2010).

Most studies focused on the description of phosphatase activities in Leishmania have only reported the observation of acid activities (Gottlieb & Dwyer, 1982; De Almeida-Amaral et al., 2006). In the present study, we provide the first characterization of ecto- and secreted alkaline phosphatase activities in L. amazonensis.

At physiological pH levels, the phosphatases of L. amazonensis function as previously shown in L. donovani, with p-NPP and β-glycerophosphate as the best substrates for ecto- and secreted phosphatase activities, respectively (Gottlieb & Dwyer, 1982). For the analysis of phosphatase activity at different pH, only the β-glycerophosphate was used due to the low detection in the presence of p-NPP. Even using β-glycerophosphate as a substrate for the detection of ecto-phosphatase activity, we observed acidic and alkaline activities only in procyclic promastigotes with relatively similar activity values. As might be expected, the inhibition of ecto-phosphatase activity was not significant in the presence of sodium tartrate, which is more specific for the secreted form. In the presence of orthovanadate, our results were similar to those previously published by De Almeida-Amaral et al. (2006).

When we analyzed the secreted phosphatase activity of the different developmental forms of L. amazonensis we observed, for L. donovani, that β-glycerophosphate was the best substrate, which is in contrast to the observations of Gottlieb & Dwyer (1982). Procyclic promastigotes had the highest levels of both acid and alkaline enzyme activity. However, the secreted activity observed in the presence of p-NPP was significantly reduced and did not vary with pH, thereby suggesting a different origin from that observed in the presence of β-glycerophosphate. The time courses of both the acidic and alkaline secreted phosphatases were distinct, thereby suggesting that the enzyme in acidic form acts via a different and more rapid release mechanism.

The secreted activity levels with alkaline characteristics were higher in the procyclic promastigotes that survive and multiply in the intestinal tract during and after a blood meal. It has been shown that the pH of the intestinal tract of Lutzomyia, a New World vector of Leishmania, is alkalinized to pH 8.5 after a blood meal (Santos et al., 2008, 2011). Interestingly, the procyclic promastigote stage differentiates directly from the ingested amastigotes and adheres to the intestinal tract (Pimenta et al., 1992). After blood meal digestion, the intestinal pH level returns to pH 6.0, which is the original pH of an unfed midgut, and it has been hypothesized that this acidification could be associated with metacyclic differentiation (Santos et al., 2008). We believe that phosphatases may play a role in parasite development in the intestinal environment; specifically, the alkaline phosphatase for procyclic forms and the acid phosphatase for the metacyclic forms. Amastigotes, the intracellular form, had secreted alkaline phosphatase activity levels much lower than that of procyclic promastigote.

Because the activity levels of the ecto- and secreted phosphatases measured in the presence of p-NPP and β-glycerophosphate were quite different, we used both substrates in the ultrastructural cytochemistry assays. In previous studies, the same substrate was used to determine enzymatic activity, but the ultrastructural localization generated conflicting results (Gottlieb & Dwyer, 1982). In L amazonensis procyclic promastigotes, we noted that the electron-dense precipitate observed when β-glycerophosphate was used as substrate was much more intense and preferably localized to the cell surface, on the flagellar pocket membrane, the single place of secretion in Leishmamia, on the membrane of small vesicles found in the flagellar pocket or budding from the parasite surface as previously observed by Gottlibe & Dwyer (1981) and Pimenta & De Souza (1986). Cytochemical staining observed with p-NPP was more evident in the intracellular compartments, whereas the pattern of surface labeling was heterogeneous as previously observed in the bloodstream forms of Trypanosoma congolense (Tosomba et al., 1996).

The outcome of Leishmania infection depends on both host and pathogen factors. It has been suggested that Leishmania acid phosphatase plays a role in the parasite–macrophage-binding process and subsequent infection (Vannier-Santos et al., 1995). The interaction between L. amazonensis and phagocytic cells is a critical step in the pathogenesis of this parasite, and the role that ecto- and secreted phosphatases play in this process remains under investigation. Previous studies gave the first evidence of the influence of AcP in the L. amazonensis–macrophage interaction involving PKC activation in the early stage of a 60-min interaction (Vannier-Santos et al., 1995). Because the intracellular life cycle of Leishmania involves several steps beyond the early processes of adhesion and internalization, we extended the analysis to a total period of 24 h postinfection. We wanted to understand whether the previous inhibition of an acidic phosphatase secreted by the parasite influenced parasite differentiation into the amastigote stage and whether those forms could survive within the PV. Vanadate treatment significantly reduced the EI even in the presence of increased concentrations of secreted phosphatase. After a 24-h infection, profound modification was observed in the PV and in the number of amastigote inside of host cell, which became tight in contrast with the control cells. The development and maturation of the PV orchestrated by Leishmania depend on direct interaction with late endosomes and lysosomes. It has also been shown in L. donovani and L. major that sAcP leaves the PV and becomes concentrated in compartments distributed throughout the cytoplasm of infected macrophages (McCall & Matlashewski, 2010). The role that host cell kinases and phosphatases play in the process of PV maturation has been characterized in Mycobacterium tuberculosis, Salmonella, and Coxiella burnetti (Hussain et al., 2010; Thi et al., 2012), thereby suggesting the involvement of microorganism-secreted phosphatases in both PV biogenesis and bacteria growth via an unknown mechanism (Hussain et al., 2010). In the present study, the pre-inhibition of metacyclic promastigote sAcP with vanadate caused a similar effect to that described by Hussain et al. (2010) in PV and parasite development, thereby suggesting that L. amazonensis phosphatases also mediate PV maturation.

In this report, we characterized the varying expression levels of the secreted acidic and alkaline phosphatases in the different developmental forms of L amazonensis. These proteins actively participate in parasite–host cell interactions and seem to influence both the formation of the PV and parasite survival.

Acknowledgements

We thank Venício Féo da Veiga and Tarcísio Corrêa for valuable technical assistance. This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ).

References

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