Inorganic polyphosphate (polyP) has been identified and measured in different stages of Trypanosoma cruzi. Millimolar levels (in terms of P i residues) in chains of less than 50 residues long, and micromolar levels in chains of about 700 -800 residues long, were found in different stages of T. cruzi. Analysis of purified T. cruzi acidocalcisomes indicated that polyPs were preferentially located in these organelles. This was confirmed by visualization of polyPs in the acidocalcisomes using 4,6-diamidino-2-phenylindole. A rapid increase (within 2-4 h) in the levels of short and long chain polyPs was detected during trypomastigote to amastigote differentiation and during the lag phase of growth of epimastigotes (within 12-24 h). Levels rapidly decreased after the epimastigotes resumed growth. Short and long chain polyP levels rapidly decreased upon exposure of epimastigotes to hypo-osmotic or alkaline stresses, whereas levels increased after hyperosmotic stress. Ca 2؉ release from acidocalcisomes by a combination of ionophores (ionomycin and nigericin) was associated with the hydrolysis of short and long chain polyPs. In agreement with these results, acidocalcisomes were shown to contain polyphosphate kinase and exopolyphosphatase activities. Together, these results suggest a critical role for these organelles in the adaptation of the parasite to environmental changes.

Inorganic pyrophosphate promoted the acidification of an intracellular compartment in permeabilized procyclic trypomastigotes of Trypanosoma brucei, as measured by acridine orange uptake. The proton gradient generated by pyrophosphate was collapsed by addition of nigericin or NH 4 Cl. Pyrophosphate-driven proton translocation was stimulated by potassium ions and inhibited by KF, by the pyrophosphate analogs imidodiphosphate and aminomethylenediphosphonate (AMDP), and by the thiol reagent p-hydroxymercuribenzoate at concentrations similar to those that inhibit the plant vacuolar H ؉ -pyrophosphatase (PPase). The proton translocation activity had a pH optimum around 7.5 and was partially inhibited by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (10 M) and unaffected by bafilomycin A 1 (40 nM), concanamycin A (5 nM), sodium ovanadate (500 M), oligomycin (1 M), N-ethylmaleimide (100 M), and KNO 3 . AMDP-sensitive pyrophosphate hydrolysis was detected in both procyclic and bloodstream trypomastigotes. Measurements of acridine orange uptake in permeabilized procyclic trypomastigotes in the presence of different substrates and inhibitors suggested the presence of H ؉ -ATPase, H ؉ -PPase, and (ADP-dependent) H ؉ /Na ؉ antiport activity in the same compartment. Separation of bloodstream and procyclic trypomastigote extracts on Percoll gradients yielded fractions that contained H ؉ -PPase (both stages) and H ؉ /Na ؉ exchanger (procyclics) activities but lacked markers for mitochondria, glycosomes, and lysosomes. The organelles in these fractions were identified by electron microscopy and X-ray microanalysis as acidocalcisomes (electron-dense vacuoles). These results provide further evidence for the unique nature of acidocalcisomes in comparison with other, previously described, organelles.

Acidocalcisomes are acidic calcium storage compartments described in several unicellular eukaryotes, including trypanosomatid and apicomplexan parasites, algae, and slime molds. In this work, we report that the volutin granules of Agrobacterium tumefaciens possess properties similar to the acidocalcisomes. Transmission electron microscopy revealed that each intracellular granule was surrounded by a membrane. X-ray microanalysis of the volutin granules showed large amounts of phosphorus, magnesium, potassium, and calcium. Calcium in the volutin granules increased when the bacteria were incubated at high extracellular calcium concentration. Immunofluorescence and immunoelectron microscopy, using antisera raised against peptide sequences conserved in the A. tumefaciens proton pyrophosphatase, indicated localization in intracellular vacuoles. Purification of the volutin granules using iodixanol density gradients indicated a preferential localization of the pyrophosphatase activity in addition to high concentrations of phosphate, pyrophosphate, short-and long-chain polyphosphate, but lack of markers of the plasma membrane. The pyrophosphatase activity was potassium-insensitive and inhibited by the pyrophosphate analogs, amynomethylenediphosphonate and imidodiphosphate, by dicyclohexylcarbodiimide, and by the thiol reagent N-ethylmaleimide. Polyphosphate was also localized to the volutin granules by 4,6-diamino-2-phenylindole staining. The organelles were acidic, as demonstrated by staining with LysoSensor blue DND-167, a dye especially used to detect very acidic compartments in cells, and cycloprodigiosin, a compound isolated from a marine bacterium that has been shown to uncouple proton pyrophosphatase activity acting as a chloride/proton symport. The results suggest that acidocalcisomes arose before the prokaryotic and eukaryotic lineages diverged.

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Vacuolar proton pyrophosphatases (V-H؉ -PPases) are electrogenic proton pumps found in many organisms of considerable industrial, environmental, and clinical importance. V-H ؉ -PPases of several parasites were shown to be associated with acidic vacuoles named acidocalcisomes, which contain polyphosphate and calcium. In this work we functionally characterized a Trypanosoma brucei V-H ؉ -PPase gene by using double-stranded RNA interference methodology to produce inducible V-H ؉ -PPase-deficient strains of procyclic and bloodstream forms (PFiVP1 and BFiVP1). Acidocalcisomes of these mutated parasites lost acidity and contained 90% less polyphosphate. PFiVP1 did not release calcium after the addition of nigericin, and its total acidity was reduced by 70%. This mutant also failed to stabilize its intracellular pH on exposure to external basic pH >7.4 and recovered from intracellular acidification at a slower rate and to a more acidic final intracellular pH. In the absence of T. brucei V-H ؉ -PPase expression, PFiVP1 and BFiVP1 grew at a slower rate with doubling times of 27 h instead of 15 h, and 10 h instead of 7.5 h, respectively. Moreover, BFiVP1 could not grow over 5 ؋ 10 5 cells/ml corresponding to a cell density reduction of five times for bloodstream form stationary phase growth.Intracellular acidic vacuoles containing polyphosphate (polyP), 1 initially called volutin or polyP bodies, have been described in bacteria, algae, yeast, and protozoa (1). In trypanosomatids, these polyP vacuoles were called acidocalcisomes (2) and shown to be electron dense and contain large concentrations of PP i , calcium, magnesium, and other elements (3). Similar organelles have been identified in apicomplexan parasites (4, 5) as well as in the green algae, Chlamydomonas reinhardtii (6) and the slime mold, Dictyostelium discoideum (7). Acidocalcisomes were postulated to play an important role in the regulation of both cytosolic Ca 2ϩ concentration and intracellular pH (pH i ). For example, polyP hydrolysis (8) and activation of the Na ϩ /H ϩ antiporter (9, 10) were postulated to protect the cells against alkaline pH stress and increase the intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ). In addition, these organelles possess a vacuolar-type H ϩ -translocating pyrophosphatase (V-H ϩ -PPase) (11), which is an electrogenic proton pump initially discovered in photosynthetic bacteria and plants (12). It has been shown to be associated with the plasma membrane or vacuoles in plants and with the chromatophore membranes of Rhodospirullum rubrum. The biochemical function of this enzyme in plants and unicellular eukaryotes is to couple hydrolysis of the high energy phosphate bond of PP i with H ϩ translocation from the cytosol to acidify the plant vacuole (tonoplast) or the acidocalcisome, respectively (10, 13). Working on isolated acidocalcisomes, Rodrigues et al. (14) demonstrated that the Trypanosoma brucei H ϩ -PPase was able to generate a membrane potential by PP i -dependent proton uptake. Moreover, Scott et al. (11,15) showed tha...