RESUMO
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 x 10(5) cells/ml corresponding to a cell density reduction of five times for bloodstream form stationary phase growth.
