Giardia lamblia: Characterization of ecto-phosphatase activities

This work is dedicated to Leopoldo De Meis on his 70th birthday
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Abstract

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 p-nitrophenylphosphatase activity. We also observed a positive correlation between the ecto-phosphatase activity and the capacity to encystation of G. lamblia trophozoites.

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 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, Meyer-Fernandes 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).

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 ecto-phosphatase 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.

Section snippets

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

Results

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

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 external stimuli (Collopy-Junior et al., 2006, Furuya et al., 1998, Kiffer-Moreira et al., 2007, Kneipp et al., 2004). Cellular responses to extracellular stimuli (e.g.,

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

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