Ca2+ entry at the plasma membrane and uptake by acidic stores is regulated by the activity of the V-H+ -ATPase in Toxoplasma gondii.

Ca2+ is a universal intracellular signal that regulates many cellular functions. In Toxoplasma gondii, the controlled influx of extracellular and intracellular Ca2+ into the cytosol initiates a signaling cascade that promotes pathogenic processes like tissue destruction and dissemination. In this work, we studied the role of proton transport in cytosolic Ca2+ homeostasis and the initiation of Ca2+ signaling. We used a T. gondii mutant of the V-H+ -ATPase, a pump previously shown to transport protons to the extracellular medium, and to control intracellular pH and membrane potential and we show that proton gradients are important for maintaining resting cytosolic Ca2+ at physiological levels and for Ca2+ influx. Proton transport was also important for Ca2+ storage by acidic stores and, unexpectedly, the endoplasmic reticulum. Proton transport impacted the amount of polyphosphate (polyP), a phosphate polymer that binds Ca2+ and concentrates in acidocalcisomes. This was supported by the co-localization of the vacuolar transporter chaperone 4 (VTC4), the catalytic subunit of the VTC complex that synthesizes polyP, with the V-ATPase in acidocalcisomes. Our work shows that proton transport regulates plasma membrane Ca2+ transport and control acidocalcisome polyP and Ca2+ content, impacting Ca2+ signaling and downstream stimulation of motility and egress in T. gondii.

NUDIX hydrolases with inorganic polyphosphate exo- and endopolyphosphatase activities in the glycosome, cytosol and nucleus of Trypanosoma brucei.

Trypanosoma brucei, a protist parasite that causes African trypanosomiasis or sleeping sickness, relies mainly on glycolysis for ATP production when in its mammalian host. Glycolysis occurs within a peroxisome-like organelle named the glycosome. Previous work from our laboratory reported the presence of significant amounts of inorganic polyphosphate (polyP), a polymer of three to hundreds of orthophosphate units, in the glycosomes and nucleoli of T. brucei In this work, we identified and characterized the activity of two Nudix hydrolases (NHs), T. brucei Nudix hydrolase (TbNH) 2 and TbNH4, one located in the glycosomes and the other in the cytosol and nucleus, respectively, which can degrade polyP. We found that TbNH2 is an exopolyphosphatase with higher activity on short chain polyP, while TbNH4 is an endo- and exopolyphosphatase that has similar activity on polyP of various chain sizes. Both enzymes have higher activity at around pH 8.0. We also found that only TbNH2 can dephosphorylate ATP and ADP but with lower affinity than for polyP. Our results suggest that NHs can participate in polyP homeostasis and therefore may help control polyP levels in glycosomes, cytosol and nuclei of T. brucei.

Pyrophosphate Stimulates the Phosphate-Sodium Symporter of Trypanosoma brucei Acidocalcisomes and Saccharomyces cerevisiae Vacuoles.

Inorganic pyrophosphate (PPi) is a by-product of biosynthetic reactions and has bioenergetic and regulatory roles in a variety of cells. Here we show that PPi and other pyrophosphate-containing compounds, including polyphosphate (polyP), can stimulate sodium-dependent depolarization of the membrane potential and Pi conductance in Xenopus oocytes expressing a Saccharomyces cerevisiae or Trypanosoma brucei Na+/Pi symporter. PPi is not taken up by Xenopus oocytes, and deletion of the TbPho91 SPX domain abolished its depolarizing effect. PPi generated outward currents in Na+/Pi-loaded giant vacuoles prepared from wild-type or pho91Δ yeast strains expressing TbPHO91 but not from the pho91Δ strains. Our results suggest that PPi, at physiological concentrations, can function as a signaling molecule releasing Pi from S. cerevisiae vacuoles and T. brucei acidocalcisomes.IMPORTANCE Acidocalcisomes, first described in trypanosomes and known to be present in a variety of cells, have similarities with S. cerevisiae vacuoles in their structure and composition. Both organelles share a Na+/Pi symporter involved in Pi release to the cytosol, where it is needed for biosynthetic reactions. Here we show that PPi, at physiological cytosolic concentrations, stimulates the symporter expressed in either Xenopus oocytes or yeast vacuoles via its SPX domain, revealing a signaling role of this molecule.

5-Diphosphoinositol pentakisphosphate (5-IP7) regulates phosphate release from acidocalcisomes and yeast vacuoles.

Acidocalcisomes of Trypanosoma brucei and the acidocalcisome-like vacuoles of Saccharomyces cerevisiae are acidic calcium compartments that store polyphosphate (polyP). Both organelles possess a phosphate-sodium symporter (TbPho91 and Pho91p in T. brucei and yeast, respectively), but the roles of these transporters in growth and orthophosphate (Pi) transport are unclear. We found here that Tbpho91-/- trypanosomes have a lower growth rate under phosphate starvation and contain larger acidocalcisomes that have increased Pi content. Heterologous expression of TbPHO91 in Xenopus oocytes followed by two-electrode voltage clamp recordings disclosed that myo-inositol polyphosphates stimulate both sodium-dependent depolarization of the oocyte membrane potential and Pi conductance. Deletion of the SPX domain in TbPho91 abolished this stimulation. Inositol pyrophosphates such as 5-diphosphoinositol pentakisphosphate generated outward currents in Na+/Pi-loaded giant vacuoles prepared from WT or from TbPHO91-expressing pho91Δ strains but not from the pho91Δ yeast strains or from the pho91Δ strains expressing PHO91 or TbPHO91 with mutated SPX domains. Our results indicate that TbPho91 and Pho91p are responsible for vacuolar Pi and Na+ efflux and that myo-inositol polyphosphates stimulate the Na+/Pi symporter activities through their SPX domains.

Inorganic polyphosphate interacts with nucleolar and glycosomal proteins in trypanosomatids.

Inorganic polyphosphate (polyP) is a polymer of three to hundreds of phosphate units bound by high-energy phosphoanhydride bonds and present from bacteria to humans. Most polyP in trypanosomatids is concentrated in acidocalcisomes, acidic calcium stores that possess a number of pumps, exchangers, and channels, and are important for their survival. In this work, using polyP as bait we identified > 25 putative protein targets in cell lysates of both Trypanosoma cruzi and Trypanosoma brucei. Gene ontology analysis of the binding partners found a significant over-representation of nucleolar and glycosomal proteins. Using the polyphosphate-binding domain (PPBD) of Escherichia coli exopolyphosphatase (PPX), we localized long-chain polyP to the nucleoli and glycosomes of trypanosomes. A competitive assay based on the pre-incubation of PPBD with exogenous polyP and subsequent immunofluorescence assay of procyclic forms (PCF) of T. brucei showed polyP concentration-dependent and chain length-dependent decrease in the fluorescence signal. Subcellular fractionation experiments confirmed the presence of polyP in glycosomes of T. brucei PCF. Targeting of yeast PPX to the glycosomes of PCF resulted in polyP hydrolysis, alteration in their glycolytic flux and increase in their susceptibility to oxidative stress.

Dynamic nuclear polarization facilitates monitoring of pyruvate metabolism in Trypanosoma brucei.

Dynamic nuclear polarization provides sensitivity improvements that make NMR a viable method for following metabolic conversions in real time. There are now many in vivo applications to animal systems and even to diagnosis of human disease. However, application to microbial systems is rare. Here we demonstrate its application to the pathogenic protozoan, Trypanosoma brucei, using hyperpolarized 13C1 pyruvate as a substrate and compare the parasite metabolism with that of commonly cultured mammalian cell lines, HEK-293 and Hep-G2. Metabolic differences between insect and bloodstream forms of T. brucei were also investigated. Significant differences are noted with respect to lactate, alanine, and CO2 production. Conversion of pyruvate to CO2 in the T. brucei bloodstream form provides new support for the presence of an active pyruvate dehydrogenase in this stage.

The inositol pyrophosphate synthesis pathway in Trypanosoma brucei is linked to polyphosphate synthesis in acidocalcisomes.

Inositol pyrophosphates are novel signaling molecules possessing high-energy pyrophosphate bonds and involved in a number of biological functions. Here, we report the correct identification and characterization of the kinases involved in the inositol pyrophosphate biosynthetic pathway in Trypanosoma brucei: inositol polyphosphate multikinase (TbIPMK), inositol pentakisphosphate 2-kinase (TbIP5K) and inositol hexakisphosphate kinase (TbIP6K). TbIP5K and TbIP6K were not identifiable by sequence alone and their activities were validated by enzymatic assays with the recombinant proteins or by their complementation of yeast mutants. We also analyzed T. brucei extracts for the presence of inositol phosphates using polyacrylamide gel electrophoresis and high-performance liquid chromatography. Interestingly, we could detect inositol phosphate (IP), inositol 4,5-bisphosphate (IP2 ), inositol 1,4,5-trisphosphate (IP3 ), and inositol hexakisphosphate (IP6 ) in T. brucei different stages. Bloodstream forms unable to produce inositol pyrophosphates, due to downregulation of TbIPMK expression by conditional knockout, have reduced levels of polyphosphate and altered acidocalcisomes. Our study links the inositol pyrophosphate pathway to the synthesis of polyphosphate in acidocalcisomes, and may lead to better understanding of these organisms and provide new targets for drug discovery.