Overexpression of a cytosolic pyrophosphatase (TgPPase) reveals a regulatory role of PP_i in glycolysis for Toxoplasma gondii

PP_i is a critical element of cellular metabolism as both an energy donor and as an allosteric regulator of several metabolic pathways. The apicomplexan parasite Toxoplasma gondii uses PP_i in place of ATP as an energy donor in at least two reactions: the glycolytic PP_i-dependent PFK (phosphofructokinase) and V-H⁺-PPase [vacuolar H⁺-translocating PPase (pyrophosphatase)]. In the present study, we report the cloning, expression and characterization of cytosolic TgPPase (T. gondii soluble PPase). Amino acid sequence alignment and phylogenetic analysis indicates that the gene encodes a family I soluble PPase. Overexpression of the enzyme in extracellular tachyzoites led to a 6-fold decrease in the cytosolic concentration of PP_i relative to wild-type strain RH tachyzoites. Unexpectedly, this subsequent reduction in PP_i was associated with a higher glycolytic flux in the overexpressing mutants, as evidenced by higher rates of proton and lactate extrusion. In addition to elevated glycolytic flux, TgPPase-overexpressing tachyzoites also possessed higher ATP concentrations relative to wild-type RH parasites. These results implicate PP_i as having a significant regulatory role in glycolysis and, potentially, other downstream processes that regulate growth and cell division.