Ammonium production during hypo-osmotic stress leads to alkalinization of acidocalcisomes and cytosolic acidification in Trypanosoma cruzi

Osmotic swelling of Trypanosoma cruzi epimastigotes resulted in alkalinization of acidocalcisomes, as revealed by changes in acridine orange fluorescence of intact cells. Concomitant with these changes, intracellular ammonium levels increased while extracellular ammonium levels decreased significantly. Hypo-osmotic stress also resulted in cytosolic acidification. The observed changes in intracellular pH (pH_i) were independent of extracellular calcium, and other ions concentration. Taken together, these results are consistent with a stimulation of ammonium production upon hypo-osmotic stress and its accumulation in acidocalcisomes resulting in their alkalinization, which might be responsible for polyphosphate hydrolysis and osmotic changes in the organelles.     

A proton pumping pyrophosphatase in the Golgi apparatus and plasma membrane vesicles of Trypanosoma cruzi

The proton pumping pyrophosphatase (H⁺-PPase) is an enzyme that has been identified in membranes of plant vacuoles, in the Golgi complex of plants and Chlamydomonas reinhardtii, and more recently in acidocalcisomes of different trypanosomatids and apicomplexan parasites. Immunofluorescence and immunoelectron microscopy studies using antibodies against the plant enzyme also suggested a plasma membrane localization in different stages of Trypanosoma cruzi. In this report we provide immunogold electron microscopy evidence of the presence of the H⁺-PPase in the Golgi complex and plasma membrane of epimastigotes of T. cruzi. Pyrophosphate promoted acidification of plasma membrane vesicles as determined using acridine orange. This activity was stimulated by K⁺ ions, inhibited by the pyrophosphate analogs imidodiphosphate (IDP) and aminomethylenediphosphonate (AMDP) by KF, NaF and DCCD, and it had different responses to ions and inhibitors as compared with the activity present in acidocalcisomes. Surface localization of the H⁺-PPase was confirmed by experiments using biotinylation of cell surface proteins and immunoprecipitation with antibodies against H⁺-PPase. Taken together, these results are consistent with the presence of a functional H⁺-PPase in the plasma membrane of these parasites.     

A proton pumping pyrophosphatase in acidocalcisomes of Herpetomonas sp

Acidocalcisomes are acidic calcium storage organelles found in several microorganisms. They are characterized by their acidic nature, high electron density, high content of polyphosphates and several cations. Electron microscopy contrast tuned images of Herpetomonas sp. showed the presence of several electron dense organelles ranging from 100 to 300 nm in size. In addition, X-ray element mapping associated with energy-filtering transmission electron microscopy showed that most of the cations, namely Na, Mg, P, K, Fe and Zn, are located in their matrix. Using acridine orange as an indicator dye, a pyrophosphate-driven H⁺ uptake was measured in cells permeabilized by digitonin. This uptake has an optimal pH of 6.5–6.7 and was inhibited by sodium fluoride (NaF) and imidodiphosphate (IDP), two H⁺-pyrophosphatase inhibitors. H⁺ uptake was not promoted by ATP. Addition of 50 μM Ca²⁺ induced the release of H⁺, suggesting the presence of a Ca²⁺/H⁺ countertransport system in the membranes of the acidic compartments. Na⁺ was unable to release protons from the organelles. The pyrophosphate-dependent H⁺ uptake was dependent of ion K⁺ and inhibited by Na⁺ Herpetomonas sp. immunolabeled with monoclonal antibodies raised against a Trypanosoma cruzi V-H⁺-pyrophosphatase shows intense fluorescence in cytoplasmatic organelles of size and distribution similar to the electron-dense vacuoles.

Together, these results suggest that the electron dense organelles found in Herpetomonas sp. are homologous to the acidocalcisomes described in other trypanosomatids. They possess a vacuolar H⁺-pyrophosphatase and a Ca²⁺/H⁺ antiport. However, in contrast to the other trypanosomatids so far studied, we were not able to measure any ATP promoted H⁺ transport in the acidocalcisomes of this parasite.