Abstract
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.
Highlights
There is considerable interest in developing novel chemotherapeutic approaches against Trypanosoma cruzi, the etiologic agent of Chagas’ disease that remains an important health problem in Mexico and Central and South America [1]
Cruzi; values for short chain polyP were in the mM range and considerably higher in epimastigotes, which have mM amounts of long chain polyP
The lack of detection of other polyPs suggests that the short chain polyPs present in the different stages are too small to be recognized by toluidine blue [25]
Summary
Culture Methods—T. cruzi amastigotes and trypomastigotes (Y strain) were obtained from the culture medium of L6E9 myoblasts as we have described before [15]. 8 –10) are from single representative experiments, with data points showing mean Ϯ S.E. Acidocalcisomal Synthesis and Degradation of PolyP—To investigate polyP synthesis by isolated acidocalcisomes, the isolated fraction (100 g of protein) [5] was incubated for 5 min at 37 °C in Buffer A containing 0.1 mM PPi. ATP (1 mM) was added, and the preparation was incubated for different times at 37 °C, after which 500 l of guanidine isothiocyanate lysis buffer (for long chain polyP determination) or 300 l of ice-cold 0.5 M HClO4 (for short chain polyP determination) was added, and polyP was extracted and quantified as described above. Final supernatants and pellets were used for measurement of tetrapolyphosphatase activity at different pH and CaCl2 concentrations
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