A vacuolar-type H+-pyrophosphatase governs maintenance of functional acidocalcisomes and growth of the insect and mammalian forms of Trypanosoma brucei.
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.
Laboratoire de Parasitologie Moléculaire, Bâtiment 3A, Unite Mixté Réchérche-Centre National de la Recherche Scientifique 5016, 146, rue Leo Saignat, 33076 Bordeaux, France.
Guillaume
G
Lemercier
Sandrine
S
Dutoya
Shuhong
S
Luo
Felix A
FA
Ruiz
Claudia O
CO
Rodrigues
Théo
T
Baltz
Roberto
R
Docampo
Norbert
N
Bakalara
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D003594:Cytoplasmic Granules / Q000201:enzymology*; D017931:DNA Primers; D011756:Diphosphates / Q000378:metabolism; D017353:Gene Deletion; D007313:Insecta / Q000254:growth & development*; D007700:Kinetics; D008322:Mammals; D008969:Molecular Sequence Data; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012330:RNA, Double-Stranded / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000469:parasitology; D014617:Vacuoles / Q000201:enzymology
D017931:DNA Primers; D011756:Diphosphates; D012330:RNA, Double-Stranded; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases