TY - JOUR
T1 - A vacuolar-type H+-pyrophosphatase governs maintenance of functional acidocalcisomes and growth of the insect and mammalian forms of Trypanosoma brucei
AU - Lemercier, Guillaume
AU - Dutoya, Sandrine
AU - Luo, Shuhong
AU - Ruiz, Felix A.
AU - Rodrigues, Claudia O.
AU - Baltz, Théo
AU - Docampo, Roberto
AU - Bakalara, Norbert
N1 - Copyright:
Copyright 2008 Elsevier B.V., All rights reserved.
PY - 2002/10/4
Y1 - 2002/10/4
N2 - 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 × 105 cells/ml corresponding to a cell density reduction of five times for bloodstream form stationary phase growth.
AB - 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 × 105 cells/ml corresponding to a cell density reduction of five times for bloodstream form stationary phase growth.
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U2 - 10.1074/jbc.M204744200
DO - 10.1074/jbc.M204744200
M3 - Article
C2 - 12121996
AN - SCOPUS:0037020160
VL - 277
SP - 37369
EP - 37376
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 40
ER -