Experimental Parasitology 121 (2009) 15–21
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Giardia lamblia: Characterization of ecto-phosphatase activities Juliana Natal Amazonas a, Daniela Cosentino-Gomes a, Aline Werneck-Lacerda a, Ana Acácia de Sá Pinheiro a, Adriana Lanfredi-Rangel b, Wanderley De Souza c, José R. Meyer-Fernandes a,* a
Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, CCS, Bloco H, sala H2-013, Cidade Universitária, Ilha do Fundão, 21941-590 Rio de Janeiro, Brazil Centro de Pesquisas Gonçalo Muniz, Fundação Oswaldo Cruz (FIOCRUZ), Salvador, Bahia, BA, Brazil c Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, CCS, Bloco C, Cidade Universitária, Ilha do Fundão, 21941-590 Rio de Janeiro, Brazil b
a r t i c l e
i n f o
Article history: Received 16 May 2008 Received in revised form 16 September 2008 Accepted 18 September 2008 Available online 26 September 2008 This work is dedicated to Leopoldo De Meis on his 70th birthday Keywords: Giardia lamblia Ecto-phosphatase Phospho-tyrosine phosphatase
a b s t r a c t Ecto-phosphatase activities of Giardia lamblia were characterized in intact cells, which are able to hydrolyze the artificial substrate p-nitrophenylphosphate (p-NPP) to p-nitrophenol (p-NP) at a rate of 8.4 ± 0.8 nmol p-NP/h/107 cells. The ecto-phosphatase activities were inhibited at high pH as well as by classical inhibitors of acid phosphatases, such as sodium fluoride and sodium molybdate and by inorganic phosphate, the final product of the reaction. Experiments using a classical inhibitor of phosphotyrosine phosphatase, sodium orthovanadate, also showed that the ecto-phosphatase activity was inhibited in a dose-dependent manner. Different phosphorylated amino acids were used as substrates for the G. lamblia ecto-phosphatase activities the highest rate of phosphate release was achieved using phosphotyrosine. Not only p-NPP hydrolysis but also phosphotyrosine hydrolysis was inhibited by sodium orthovanadate. Phosphotyrosine but not phospho-serine or phospho-threonine inhibited the pnitrophenylphosphatase activity. We also observed a positive correlation between the ecto-phosphatase activity and the capacity to encystation of G. lamblia trophozoites. Ó 2008 Elsevier Inc. All rights reserved.
1. Introduction The protozoan Giardia lamblia is one of the most important human enteric parasites, which during its life cycle presents a parasitic stage represented by the vegetative trophozoite and a resistant form, the cyst, which the host eliminates with feces and which is responsible for transmission. Giardiasis has been recently included in the WHO Neglected Diseases Initiative (Savioli et al., 2006). Giardia lamblia is considered a primitive eukaryotic cell because it lacks organelles such as mitochondria, peroxisomes, and a typical Golgi apparatus (Adam, 2001). The secretory and endocytic pathways are affected by the same ancient organelle complex, the peripheral vesicles, which are located below the plasma membrane (Gaechter et al., 2008; Lanfredi-Rangel et al., 1998; McCaffery et al., 1994; Nash, 2002). Infection is initiated by the ingestion of the cyst form, followed by excystation and colonization of the gut mucosa by Giardia trophozoites. The parasite displays distinct tissue tropism, so this infection is restricted to the small intestine, where Giardia attaches to the mucosal surface, exerting pathological effects (Müller and Allmen, 2005). Attachment of the parasite to the substratum is thought to be mediated by the ventral adhesive disk. However, this mechanism may not account for the selective colonization of the
* Corresponding author. Fax: +55 21 2270 8647. E-mail address:
[email protected] (J.R. Meyer-Fernandes). 0014-4894/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2008.09.010
proximal small intestine (Müller and Allmen, 2005). Therefore, recognition and adherence must be mediated by specific host and parasite membrane determinants. Membrane-bound lectins are believed to mediate several specific cell–cell interactions, including those between parasite and host cells. In Giardia, lectins have been proposed to mediate attachment of trophozoites (Lev et al., 1986) and to cause microvillus shortening (Farthing et al., 1986). Nevertheless, variant specific proteins (VSPs) have been described as a major surface component presenting an important role in antigenic variation of the trophozoite infection (Kulakeva et al., 2006). The plasma membrane contains enzymes whose active sites face the external medium rather than the cytoplasm. The activity of these enzymes, referred to as ectoenzymes, can be measured using intact cells (Meyer-Fernandes, 2002). Membrane-bound acid phosphatase activities have been characterized in some species of the family Trypanosomatidae, such as Trypanosoma spp. (Bakalara et al., 2000; Fernandes et al., 1997; Gomes et al., 2006; MeyerFernandes et al., 1999), Herpetomonas muscarum muscarum (Dutra et al., 1998), Leishmania donovani (Glew et al., 1982; Remaley et al., 1985), and Leishmania amazonensis (De Almeida-Amaral et al., 2006). Although the physiological role of the membrane-bound acid phosphatases has not been well established, they are supposed to be involved with nutrition (Gottlieb and Dwyer, 1981), escape (Martiny et al., 1999; Remaley et al., 1985; Saha et al., 1985), virulence (Furuya et al., 1998; Katakura and Kobayashi, 1988; Singla et al., 1992), and cell differentiation (Bakalara et al., 1995).
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Protein phosphorylation represents a major mechanism in the control of biological phenomena in most organisms. Phosphorylation–dephosphorylation of serine, threonine, and tyrosine residues triggers conformational changes that modulate protein biological properties (Hunter, 1995). The signaling regulation of stimulus–response coupling during differentiation and proliferation is largely mediated by protein phosphorylation in eukaryotes (Hunter, 1995), including protozoa parasites (Parsons et al., 1993). The presence of phosphotyrosyl protein phosphatases has been described in various tissues and cells which are also active toward low molecular weight, nonprotein phosphoesters such as alkyl and aryl phosphates, including p-nitrophenylphosphate (p-NPP) (Lau et al., 1989). The presence of protein tyrosine phosphatase activities in L. donovani (Cool and Blum, 1993), Trypanosoma brucei (Bakalara et al., 2000; Fernandes et al., 2003) and Trypanosoma cruzi (Furuya et al., 1998; Meyer-Fernandes et al., 1999) has been demonstrated. In this work, we report the presence of membrane-bound ectophosphatase activities on the cell surface of living G. lamblia trophozoites able to hydrolyze phosphorylated amino acids. We also show a positive correlation between the ecto-phosphatase activity and the capacity of G. lamblia trophozoites to encyst. 2. Materials and methods 2.1. Microorganisms Trophozoites of the Portland-1 (P1) and WB strains of G. lamblia (Meyer, 1976) were cultivated in TYI-S-33 medium supplemented with 10% heat-inactivated bovine serum and 0.1% bovine bile at 37 °C (Keister, 1983). Subcultures were made twice a week and vials containing cells that had grown for 72 h (late exponential phase) were incubated on ice at 4 °C for 15 min. The free parasites were harvested by centrifugation at 500g for 7 min, washed three times and kept in 116.0 mM NaCl, 5.4 mM KCl, 5.5 mM D-glucose, 30.0 mM Hepes–Tris buffer, pH 7.2. Cellular viability was assessed, before and after incubations, by motility and Trypan blue dye exclusion (Pinheiro et al., 2006). The viability of cells was not affected under the conditions employed here. The cells were induced in vitro to encyst under conditions that are characteristic of the human intestinal lumen (Gillin et al., 1989) as described by Abel et al. (2001). Cells were collected after 2, 4, 8, 16, 20, 24, 28, and 30 h by chilling and scraping using a rubber policeman and, then centrifuged for 10 min at 4 °C. For evaluation of the cyst formation, G. lamblia Trophozoites were induced to encyst for 24 h at 37 °C in the absence or in the presence of 0.1 or 1.0 mM of sodium orthovanadate and fixed in a solution containing 2% paraformaldehyde in a phosphate buffer saline, pH 7.4. 1500 cells were counted of each experiment.
employed above (Lowry and Lopez, 1946). The values obtained for p-nitrophenylphosphatase activity using these methods were exactly the same. The values shown represent averages SE of three independent experiments. 2.3. ATPase measurements The ATPase activity was determined under the same conditions employed to phosphatase activity by measuring the hydrolysis of [k-32P]ATP (104 Bq/nmol ATP) (Guilherme et al., 1991). The experiments were started by the addition of living cells and terminated by the addition of 1.0 mL of ice-cold 25% charcoal in 0.1 M HCl. The tubes were then centrifuged at 1500g for 10 min at 4 °C. Aliquots (0.5 mL) of the supernatants containing the released inorganic phosphate (32Pi) were transferred to scintillation vials containing 9.0 mL of scintillation fluid. ATPase activity was calculated by subtracting the non-specific ATP hydrolysis measured in the absence of cells. 2.4. Inhibition assays Phosphatase activity of intact cells of G. lamblia was analyzed with a specific inhibitor of protein tyrosine phosphatase (sodium orthovanadate). We also analyzed the effects of ammonium molybdate and sodium fluoride (inhibitors of acid phosphatases), as well as inorganic phosphate (natural product of the phosphatase activities). The phosphatase activity in the absence of inhibitors (8.4 ± 0.8 nmol p-NP/h/107 cells) was taken to be 100%. 2.5. Reagents All reagents were purchased from E. Merck (São Paulo, Brazil) or Sigma–Aldrich (Sigma Co., St. Louis, MO). Deionized distilled water from a MilliQ resin system (Millipore Corp., Bedford, MA) was used in the preparation of all solutions including substrates and inhibitors. 2.6. Statistical analysis All experiments were performed in triplicates, with similar results obtained in at least three separate cell suspensions. Data were analyzed by means of ANOVA One-way followed by the Turkey test using the Sigma Stat computer software (SYSTAT Software Inc. San Jose, CA, USA). Ki values for the inhibitors were calculated using a computerized non-linear regression program (Sigma Plot 2000— Jandel Scientific Software, 1986–2000, San Diego, CA, USA) on the data.
2.2. Phosphatase measurements
3. Results
The phosphatase activity was measured by the rate of p-nitrophenol (p-NP) production. Intact cells were incubated for 1 h at 30 °C in 0.5 ml of reaction mixture containing 116.0 mM NaCl, 5.4 mM KCl, 5.5 mM glucose, 30.0 mM Hepes–Tris buffer pH 7.0, 5.0 mM p-nitrophenylphosphate (p-NPP) as substrate and 3 107 cells/mL. The reaction was initiated by the addition of cells and stopped by the addition of 1.0 ml 1 N NaOH. Controls in which cells were added after interruption of the reaction were used as blanks. To determine the concentration of released p-NP the tubes were centrifuged at 1500g for 15 min and the supernatants were measured spectrophotometrically at 425 nm, using an extinction coefficient of 14.3 103 M1 cm1. We also tested phosphoamino acids as substrates. In this case, the hydrolytic activities were spectrophotometrically analyzed by measuring the released inorganic phosphate from these substrates, under the same conditions
The time course of phosphatase activity present on the external surface of G. lamblia was linear for at least 1 h (Fig. 1A). Similarly, in assay to determine the influence of cell density on the ecto-phosphatase activity, it was observed that this activity was directly proportional to the number of cells (Fig. 1B). At pH 7.2, intact cells were able to hydrolyze p-NPP at a rate of 8.4 ± 0.8 nmol p-NP/h/ 107 cells. To check the possibility that the observed p-NPP hydrolyzed was the result of secreted soluble enzymes, as seen in other parasites (Rodrigues et al., 1999; Dutra et al., 2001; Santos et al., 2002) cells were incubated in the absence of p-NPP. Subsequently, the suspension was centrifuged to remove cells and the supernatant was assayed for phosphatase activity. This supernatant failed to show p-NPP hydrolysis (data not shown). These data also rule out the possibility that the phosphatase activity described here could be derived from lysed G. lamblia trophozoites.
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Fig. 1. Time course and cell density dependence of the ecto-phosphatase activity of G. lamblia trophozoites. Intact cells were incubated at 30 °C in a reaction medium (final volume: 0.5 mL) containing 116 mM NaCl, 5.4 mM KCl, 5.5 mM D-glucose and 30.0 mM Hepes, pH 7.2, 5.0 mM p-NPP as substrate and 3 107 cells/mL, either for different times (A) or for 60 min with increasing concentrations of cells (B). Data are means ± SE of three experiments with different cell suspensions.
In the pH range of 5.6–8.4, in which trophozoites were alive throughout the time course of the reaction, the phosphatase activity reached its maximum at pH 5.6, decreasing concomitantly with the increase of pH (Fig. 2). This acidic characteristic of the enzyme was corroborated by an observed insensitivity to levamizole (1 mM), an alkaline phosphatase inhibitor (Van Belle, 1976) (Table 1). Although the optimum pH for this enzyme is in the acidic range, all experiments were carried out at pH 7.2 because that is the ideal pH for cellular development (Keister, 1983). At pH 7.2, neither EDTA, a metal chelator, nor and divalent cations, such as, Ca2+, Mg2+, Mn2+, Sr2+, Co2+ or Cd2+ had any effect on the ecto-phosphatase activity (data not shown). Sodium tartrate, a secreted phosphatase inhibitor (Dutra et al., 2000; Santos et al., 2002), had no effect on the phosphatase activity (Table 1). The inhibition by two different acid phosphatase inhibitors (Pinheiro et al., 2007) is shown in Fig. 3. The ecto-phosphatase activity was strongly inhibited by sodium fluoride (NaF) and sodium molybdate (Na2MoO4) in a dose-dependent manner, with Ki values of 76.63 ± 8.57 and 12.15 ± 1.24 lM, respectively. In addition, sodium orthovanadate (Na3VO4), an inhibitor of acid and of phospho-tyrosine phosphatase (De Almeida-Amaral et al., 2006), and inorganic phosphate, the final product of biological reactions catalyzed by phosphatases, also inhibited the ecto-phosphatase activity with Ki values of 0.42 ± 0.05 lM and 2.66 ± 0.36 mM, respectively (Fig. 3). Living G. lamblia were also able to hydrolyze phospho-tyrosine, phospho-serine and phospho-threonine at ratios of 10.3 ± 1.1,
6.8 ± 0.7, and 6.6 ± 0.8 nmol Pi/h/107 cells, respectively (Fig. 4). However, as with p-NPP, only phospho-tyrosine hydrolysis was inhibited by sodium orthovanadate (Fig. 4). These results suggest that at least two phosphatase activities are present on the G. lamblia surface, implying that the same enzyme was active against both p-NPP and phospho-tyrosine. To test this hypothesis, inhibition studies were performed. As shown in Fig. 5, the hydrolysis of p-NPP was inhibited by phospho-tyrosine in a dose-dependent manner but not by phospho-serine and phospho-threonine. Two different strains of G. lamblia were analyzed with respect to ecto-phosphatase activity. The WB strain, which present high capacity for encystation, also present higher ecto-phosphatase activity than the P1 strain (data not shown). Accordingly, the phosphatase activity increased during the time course of encystation (Fig. 6). This increase was not observed for the ecto-ATPase activity (Fig. 6) also present on the external surface of G. lamblia (Pinheiro et al., 2008). Interestingly, 1 mM sodium orthovanadate, an inhibitor for ecto-phosphatase activity did not modify the proliferation of G. lamblia trophozoites (data not shown) but inhibited the encystation process (Fig. 7). 4. Discussion Little is known about the functionality of membrane-bound enzymes in living cells and their possible role in the process of host–parasite interactions. The detection of cell surface-located phosphatase activity is particularly interesting due to its possible role in cell–cell interaction or reception and transduction of exter-
Table 1 Effect of different agents on phosphatase activity present on the surface of Giardia lamblia
Fig. 2. Effect of pH on the ecto-phosphatase activity of G. lamblia trophozoites. Intact cells were incubated in reaction medium containing 116 mM NaCl, 5.4 mM KCl, 5.5 mM D-glucose, 5.0 mM p-NPP, and 10.0 mM Mes–Hepes–Tris buffer adjusted with HCl and NaOH to pH values from 5.6 to 8.4. In this pH range, cells were viable throughout the course of the reaction. It was not possible to observe maximal cellular viability below or above this pH range. Data are means SE of three experiments with different cell suspensions.
Addition
% of activity
None Levamizole (1 mM) Tartrate (10 mM) Sodium fluoride (10 mM) Ammonium molybdate (1 mM) Sodium orthovanadate (1 mM) Pi (10 mM)
100.0 ± 4.7 94.9 ± 4.9 89.0 ± 4.6 17.7 ± 0.9* 10.5 ± 0.8* 4.4 ± 0.2* 17.0 ± 0.8*
Reactions were performed at 30 oC in a medium (final volume: 0.2 mL) containing 116 mM NaCl, 5.4 mM KCl, 5.5 mM D-glucose, 50 mM Hepes, pH 7.2, 5 mM p-NPP and 3 107 cells/mL in the absence or presence of other additions, as shown in the first column of the table. Phosphatase activity (8.57 ± .0.4 nmol Pi/h/107 cells) was taken to be 100%. The results shown are representative of at least three independent experiments. * Denotes significant differences (p < 0.05) after comparison with the control (no addition).
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Fig. 3. Effect of different phosphatase inhibitors on the ecto-phosphatase activity of G. lamblia trophozoites. Intact cells were incubated for 60 min at 30 oC in the same reaction medium described in Fig. 1, in the absence or presence of increasing concentrations of NaF (A), Na2MoO4 (B), Na3VO4 (C) and inorganic phosphate (D). Data are means ± SE of three experiments with different cell suspensions.
nal stimuli (Collopy-Junior et al., 2006; Furuya et al., 1998; KifferMoreira et al., 2007; Kneipp et al., 2004). Cellular responses to extracellular stimuli (e.g., parasite adhesion) can evoke signaling pathways including protein phosphorylation/dephosphorylation. It was recently shown that fungal ecto-phosphatases are involved in the binding of fungi to epithelial cells (Collopy-Junior et al., 2006; Kiffer-Moreira et al., 2007; Kneipp et al., 2004). This paper reports the presence of ecto-phosphatase activities on the external surface of G. lamblia trophozoites. Under the conditions employed here G. lamblia does not secret phosphatases into the extracellular medium as observed in other microorganisms
Fig. 4. Substrate specificity for ecto-phosphatase activities in the absence (black bars) or in the presence of 1 mM sodium orthovanadate (blank bars). Intact G. lamblia trophozoite cells were incubated for 60 min at 30 oC in the same reaction medium described in Fig. 1, with 5 mM of one of the substrates p-nitrophenylphosphate (p-NPP), phospho-tyrosine (P-Tyr), phospho-serine (P-Ser) or phosphothreonine (P-Thr). Data are means ± SE of three experiments with different cell suspensions. *Denotes significant differences (p < 0.05) after comparison with the control (no sodium orthovanadate).
(Rodrigues et al., 1999; Dutra et al., 2001; Santos et al., 2002). This conclusion is based on the observations that trophozoites were insensitive to sodium tartrate, an inhibitor of secreted phosphatases (Dutra et al., 2000, 2001; Santos et al., 2002), and that secreted proteins failed to hydrolyze p-NPP (data not shown). However, our experiments do not rule out the possibility that the phosphatase activity could be released in vivo once in contact with mammalian cells, or that it could even be translocated into these cells, as suggested in the case of Yersinia tyrosine phosphatase (Bliska et al., 1993). The most obvious artifact that could lead to the hydrolysis of substrates added to intact cells is the presence of lysed cells or their disruption under the assay conditions used. The following observations seemed to exclude this hypothesis: (i)
Fig. 5. Influence of increasing concentrations of phospho-amino acids on p-NPP hydrolysis catalyzed by G. lamblia trophozoites. Intact cells were incubated for 60 min at 30 oC in the same reaction medium described in Fig. 1, in the absence or presence of increasing concentrations of P-Tyr (d), P-Ser (j) or P-Thr (N). Data are means ± SE of three experiments with different cell suspensions.
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Fig. 6. Time course of ecto-phosphatase and ecto-ATPase activities of living WB strain G. lamblia trophozoites during encystation process in vitro. Trophozoites were incubated in a culture encystation medium for the times shown in abscissa. Intact cells were incubated for 60 min at 30 oC in the same reaction medium described in Fig. 1. Ecto-phosphatase (closed circles) and ecto-ATPase (open circles) activities are expressed as percentages of the control (no encystation conditions, 100% = 21.3 ± 2.8 nmol p-NP/h/107 cells for ecto-phosphatase activity and 100% = 6.5 ± 0.7 nmol Pi/h/107 cells for ecto-ATPase activity). Data are means ± SE of three experiments with different cell suspensions.
Fig. 7. Effect of sodium orthovanadate on encystation process of WB strain G. lamblia trophozoites. Trophozoites were incubated in a culture encystation medium for 24 h at 37 oC in the absence or in the presence of 0.1 and 1 mM of sodium orthovanadate. Data are means ± SE of three experiments with different cell suspensions. *Denotes significant differences (p < 0.05) after comparison with the control (no sodium orthovanadate). **Denote significant differences (p < 0.01) after comparison with the control (no sodium orthovanadate).
trophozoites were viable during the incubation periods, according to eosin dye exclusion assays; (ii) the p-NPPase activity was linear with time, suggesting that eventual cell disruption during the course of incubation did not add appreciably to the total activity; (iii) cell disruption would also be required for any activity dependent on lysosomal enzymes extruded during the assay period, which would also show a different kinetics, as pointed out and (iv) there was also no detectable activity in the supernatants of cells incubated for 60 min at 30o C in the assay buffer. The physiological role of ecto-phosphatases is not well established in protozoa parasites. However, it has been suggested that these enzyme activities are involved in the pathogenic mechanism of vaginitis and urethritis caused by the extracellular parasite Trichomonas vaginalis (De Jesus et al., 2006, 2002; Rendón-Maldonado et al., 1998) and in host infection caused by some trypanosomatids (Furuya et al., 1998; Singla et al., 1992; Zhong et al., 1998). In addition, it has been described that some protein phosphatases, such as the receptor protein tyrosine phosphatase (RPTP), were shown to have an important role in the process of hemophilic cell–cell adhesion (Fischer et al., 1991). We suggest that ecto-phosphatase activities present on the membrane surface may have a physiological role in the trophozoite stages for the encystation of this parasite (Fig. 6). This ecto-phosphatase dispose of the catalytic site externally, is active within the range of physiological pH and is
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able to hydrolyze p-NPP. In G. lamblia an acid phosphatase was localized on the vacuoles adjacent to the plasma membrane (Feely and Dyer, 1987). Recently, it was shown that the dephosphorylation of cyst wall proteins by a secreted lysosomal acid phosphatase is essential for excystation of G. lamblia (Slavin et al., 2002). Sequencing of the Giardia genome has provided a survey for several kinds of transduction molecules including several tyrosine phosphatases. (Andreeva and Kutuzov, 2008). Interestingly, among the protein tyrosine phosphatases present in the G. lamblia genome, a protein tyrosine phosphatase-like protein (PTPL) is shown to be upregulated during encystation (http://giardiadb.org). We can speculate that this membrane-bound phosphatase, which externally exposes the catalytic site, could represent a virulence marker for G. lamblia, as was previously proposed for promastigote forms of Leishmania sp. (Martiny et al., 1999; Singla et al., 1992) and for extracellular parasites T. vaginalis (De Jesus et al., 2006) and Entamoeba histolytica (Pinheiro et al., 2007). The giardial differentiation into a cystic form which enables the parasite to survive in the environment and infect a new host, relies on the assembly of the extracellular cyst wall (CW). During the parasite differentiation, the encystation specific secretory vesicles (ESVs) are also formed in order to transport the cyst wall contents to the cellular membrane (Reiner et al., 1990; Lanfredi-Rangel et al., 2003). The CW composition and mechanisms of assembly are not completely understood. Among the components of the Giardia cyst wall are two closely related proteins (CWP1 and CWP2) (Luján et al., 1995). It was described the presence of CW proteins related a leucine-rich repeat (Luján et al., 1995; Sun et al., 2003) and it has been reported that CWP2 is a key regulator of ESV formation and an aggregation factor for CWP1 and CWP3 (Gottig et al., 2006). The post translational process of CWP has been described in the ESV pathway by several enzymatic activities including a kinase-independent phosphatase processing CWP1 and CWP2 (Lauwaet et al., 2007). The increase of ecto-phosphatase activity during this stage of development can be related to the role of ESVs discharge on the cell surface, improving the relationship of the membrane and the CW formation. As described previously, trophozoites incubated in encystation medium for 24 h were able to assemble the CW components externally. The incubation of those encysting cells with sodium orthovanadate, a specific phosphotyrosine phosphatase inhibitor, demonstrated that encysting cells were not able to assembly the cyst wall components externally (Fig. 7). Giardial encystation has been studied from morphological, cell biological, biochemical and molecular viewpoints (Lauwaet et al., 2007). The present investigation brings to light additional biochemical data, which may be useful for understanding the pathogenesis and encystation process of G. lamblia in relation to its host cells. Acknowledgments We acknowledge the excellent technical assistance of Fabiano Ferreira Esteves and Rosangela Rosa de Araújo, and Dr. Martha Sorenson (Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro) for revising the English. This work was supported by grants from the Brazilian Agencies Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Fundação de Amparo á Pesquisa do Estado da Bahia (FAPESB). References Abel, E.S., Davids, B.J., Robles, L.D., Loflin, C.E., Gillin, F.D., Chakrabarti, R., 2001. Possible role of protein kinase A in cell motility and excystation of the early
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