Elsevier

Acta Tropica

Volume 100, Issues 1–2, November 2006, Pages 88-95
Acta Tropica

Characterization of ecto-phosphatase activities of Trypanosoma cruzi: A comparative study between Colombiana and Y strains

https://doi.org/10.1016/j.actatropica.2006.05.014Get rights and content

Abstract

The etiological agent of Chagas disease, Trypanosoma cruzi, is consisted of two phylogenetic lineages. Using live epimastigotes, in this study we have characterized ecto-phosphatase activities of two strains of T. cruzi, one (Y strain) is a member of group T. cruzi I and the other (Colombiana) is a member of group T. cruzi II. About one-third of the total ecto-phosphatase activity from the Y strain was Mg2+-dependent, but no such activity was observed with Colombiana. The level of Mg2+-independent activity was dramatically different in the two strains, with Colombiana showing more than 15-fold higher activity. Experiments using classical inhibitors of acid phosphatases, as well as inhibitors of phosphotyrosine phosphatase, showed a decrease in these phosphatase activities, with different patterns of inhibition. The Mg2+-independent activities of the Colombiana and Y strains decreased inversely with pH, varying from 6.5 to 8.0. On the other hand, the Mg2+-dependent activity of the Y strain increased concomitantly with the increase in pH in the same range.

Introduction

Chagas disease is widespread throughout Latin America where nearly 20 million people are infected by Trypanosoma cruzi and 90 million are at risk in endemic areas. A large fraction of these will die a premature death, usually of cardiac complications (Guhl et al., 2005). T. cruzi undergoes a complex life cycle, which allows the adaptation to both intermediate hosts (triatomine insects and mammals, including man). T. cruzi cell differentiation gives rise to four morphogenetic forms: epimastigote, metacyclic trypomastigote, amastigote, and bloodstream trypomastigotes. This process is highly regulated and includes significant changes in morphology, biochemical and signal transduction pathways, gene expression and structural alterations in the surface molecules of these parasites (De Souza, 2002). Therefore, studies related to the enzymes responsible for the phosphorylation and dephosphorylation of proteins, which are present on the external surface of these parasites, are extremely important.

Ecto-protein kinases have been identified in Leishmania major (Sacerdoti-Sierra and Jaffe, 1997) and ecto-phosphatase activities have been characterized in some members of the family Trypanosomatidae, such as Trypanosoma (Fernandes et al., 1997, Meyer-Fernandes et al., 1999) and Leishmania (Glew et al., 1982, Gottlieb and Dwyer, 1981, Martiny et al., 1996, Remaley et al., 1985a, Remaley et al., 1985b). Although the physiological role of the membrane-bound acid phosphatases are not well established, in trypanosomatids they are supposed to be involved with nutrition (Gottlieb and Dwyer, 1981), protection (Gottlieb and Dwyer, 1981, Remaley et al., 1985a), virulence (Furuya et al., 1998, Vannier-Santos et al., 1995), and cellular differentiation (Bakalara et al., 1995, Fernandes et al., 1997, Fernandes et al., 2003, Santos et al., 2002).

Some strains of T. cruzi are known to be more frequently associated with human disease, such as the Y strain, which is a member of lineage II (group T. cruzi I), while lineage I (group T. cruzi II) is associated with the sylvatic cycle of the parasite, such as the Colombiana strain (Tibayrenc, 1995, Souto et al., 1996, Anonymous, 1999, Kawashita et al., 2001, Cribb et al., 2004). Considering that the proteins controlling extracellular phosphorylation are thought to play a central role in the modulation of several key biochemical functions in Trypanosoma spp. (Furuya et al., 1998, Rodrigues et al., 1999, Bakalara et al., 2000, Gomes et al., 2006), our goal was to test whether the major divergence of groups T. cruzi I and T. cruzi II may be associated with distinct activities of their protein ecto phosphatases. Here we compare the biochemical characteristics of the phosphatase activities of the Y strain (group T. cruzi I) with the Colombiana strain (group T. cruzi II), using live epimastigotes.

Section snippets

Microorganisms and growth conditions

In the present work we have used culture epimastigote forms of T. cruzi Y and Colombiana strains. The parasites were grown in LIT medium, supplemented with 10% fetal calf serum, at 28 °C. Seven days after inoculation, the 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. Cellular viability was accessed, before and after incubations, by motility and cell dye exclusion

Phosphatase activity

The time course of each phosphatase activity present on the external surface of T. cruzi Y (Fig. 1, panel A) and Colombiana (Fig. 1, panel B) epimastigotes was linear for at least 1 h. The integrity of the cells was tested by Trypan blue dye exclusion and all the parasites remained without any color after this treatment, showing that there was no disruption of the parasites. The Y strain showed two different activities, a magnesium independent activity (control) and magnesium dependent one

Discussion

Parasitic protozoa within the taxon T. cruzi are derived from multiple clonal roots with two major phylogenetic lineages, defined by PCR amplification of a ribosomal RNA gene sequence, a mini-exon gene sequence and random amplified polymorphic DNA (RAPD) (Tibayrenc, 1995, Souto et al., 1996, Kawashita et al., 2001) and named as groups T. cruzi I and T. cruzi II by an assembly of experts (Anonymous, 1999). Typing data support that T. cruzi I group is relatively homogeneous, whereas it has been

Acknowledgements

This work was partially supported by grants from the Brazilian Agencies Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Financiadora de Estudos e Projetos (FINEP), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ).

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