Abstract
In this study, we performed the molecular and biochemical characterization of an ecto-enzyme present in Trypanosoma rangeli that is involved with the hydrolysis of extracellular inorganic pyrophosphate. PCR analysis identified a putative proton-pyrophosphatase (H+-PPase) in the epimastigote forms of T. rangeli. This protein was recognized with Western blot and flow cytometry analysis using an antibody against the H+-PPase of Arabidopsis thaliana. Immunofluorescence microscopy confirmed that this protein is located in the plasma membrane of T. rangeli. Biochemical assays revealed that the optimum pH for the ecto-PPase activity was 7.5, as previously demonstrated for other organisms. Sodium fluoride (NaF) and aminomethylenediphosphonate (AMDP) were able to inhibit approximately 75% and 90% of the ecto-PPase activity, respectively. This ecto-PPase activity was stimulated in a dose-dependent manner by MgCl2. In the presence of MgCl2, this activity was inhibited by millimolar concentrations of CaCl2. The ecto-PPase activity of T. rangeli decreased with increasing cell proliferation in vitro, thereby suggesting a role for this enzyme in the acquisition of inorganic phosphate (Pi). Moreover, this activity was modulated by the extracellular concentration of Pi and increased approximately two-fold when the cells were maintained in culture medium depleted of Pi. All of these results confirmed the occurrence of an ecto-PPase located in the plasma membrane of T. rangeli that possibly plays an important role in phosphate metabolism of this protozoan.
Highlights
In the Trypanosomatidae family, the genus Trypanosoma comprises digenetic flagellates that typically have insects as vectors and infect human beings and other animals as hosts
The presence of a complementary DNA (cDNA) sequence amplified by the primers designed for the sequence of the H+-PPase of T. cruzi was detected in two different inocula of T. rangeli
Following the identification of a putative H+-PPase in T. rangeli, we searched for the recognition of this protein by an antibody directed against a 326 peptide sequence of A. thaliana AVP2
Summary
In the Trypanosomatidae family, the genus Trypanosoma comprises digenetic flagellates that typically have insects as vectors and infect human beings and other animals as hosts. The parasite and its vector engage in a series of interactions resulting in oral infection; trypomastigotes differentiate into epimastigotes, which multiply in the gut, and the parasite penetrates through the epithelial cells of the digestive tract to enter into the hemocoel, where they freely multiply in the hemolymph or inside the hemocytes. The parasites complete their growth in the salivary glands, the site of metacyclogenesis [2,3,4,5,6,7,8]
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