A Pyrophosphatase Regulating Polyphosphate Metabolism in Acidocalcisomes Is Essential for Trypanosoma brucei Virulence in Mice*

We report the functional characterization of a soluble pyrophosphatase (TbVSP1), which localizes to acidocalcisomes, a vesicular acidic compartment of Trypanosoma brucei. Depending on the pH and the cofactors Mg2+ or Zn2+, both present in the compartment, the enzyme hydrolyzes either inorganic pyrophosphate (PPi) (kcat = 385 s–1) or tripolyP (polyP3) and polyphosphate (polyP) of 28 residues (polyP28) with kcat values of 52 and 3.5 s–1, respectively. An unusual N-terminal domain of 160 amino acids, containing a putative calcium EF-hand-binding domain, is involved in protein oligomerization. Using double-stranded RNA interference methodology, we produced an inducible bloodstream form (BF) deficient in the TbVSP1 protein (BFiVSP1). The long-chain polyP levels of these mutants were reduced by 60%. Their phenotypes revealed a deficient polyP metabolism, as indicated by their defective response to phosphate starvation and hyposmotic stress. BFiVSP1 did not cause acute virulent infection in mice, demonstrating that TbVSP1 is essential for growth of bloodstream forms in the mammalian host.

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