Shuhong Luo

Acidocalcisomes and a vacuolar H+-pyrophosphatase in malaria parasites.

Plasmodium berghei trophozoites were loaded with the fluorescent calcium indicator, fura-2 acetoxymethyl ester, to measure their intracellular Ca(2+) concentration ([Ca(2+)](i)). [Ca(2+)](i) was increased in the presence of the sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase inhibitor, thapsigargin. Trophozoites also possess a significant amount of Ca(2+) stored in an acidic compartment. This was indicated by: (1) the increase in [Ca(2+)](i) induced by bafilomycin A(1), nigericin, monensin, or the weak base, NH(4)Cl, in the nominal absence of extracellular Ca(2+), and (2) the effect of ionomycin, which cannot take Ca(2+) out of acidic organelles and was more effective after alkalinization of this compartment by addition of bafilomycin A(1), nigericin, monensin, or NH(4)Cl. Inorganic PP(i) promoted the acidification of a subcellular compartment in cell homogenates of trophozoites. The proton gradient driven by PP(i) collapsed by addition of the K(+)/H(+) exchanger, nigericin, and eliminated by the PP(i) analogue, aminomethylenediphosphonate (AMDP). Both PP(i) hydrolysis and proton transport were dependent upon K(+), and Na(+) caused partial inhibition of these activities. PP(i) hydrolysis was sensitive in a dose-dependent manner to AMDP, imidodiphosphate, sodium fluoride, dicyclohexylcarbodi-imide and to the thiol reagent, N-ethylmaleimide. Immunofluorescence microscopy using antibodies raised against conserved peptide sequences of a plant vacuolar pyrophosphatase (V-H(+)-PPase) suggested that the proton pyrophosphatase is located in intracellular vacuoles and the plasma membrane of trophozoites. AMDP caused an increase in [Ca(2+)](i) in the nominal absence of extracellular Ca(2+). Ionomycin was more effective in releasing Ca(2+) from this acidic intracellular compartment after treatment of the cells with AMDP. Taken together, these results suggest the presence in malaria parasites of acidocalcisomes with similar characteristics to those described in trypanosomatids and Toxoplasma gondii, and the colocalization of the V-H(+)-PPase and V-H(+)-ATPase in these organelles.

A plasma membrane-type Ca2+-ATPase co-localizes with a vacuolar H+-pyrophosphatase to acidocalcisomes of Toxoplasma gondii

Ca2+‐ATPases are likely to play critical roles in the biochemistry of Toxoplasma gondii, since these protozoa are obligate intracellular parasites and the Ca2+ concentration in their intracellular location is three orders of magnitude lower than in the extracellular medium. Here, we report the cloning and sequencing of a gene encoding a plasma membrane‐type Ca2+‐ATPase (PMCA) of T.gondii (TgA1). The predicted protein (TgA1) exhibits 32–36% identity to vacuolar Ca2+‐ATPases of Trypanosoma cruzi, Saccharomyces cerevisiae, Entamoeba histolytica and Dictyostelium discoideum. Sequencing of both cDNA and genomic DNA from T.gondii indicated that TgA1 contains two introns near the C‐terminus. A hydropathy profile of the protein suggests 10 transmembrane domains. TgA1 suppresses the Ca2+ hypersensitivity of a mutant of S.cerevisiae that has a defect in vacuolar Ca2+ accumulation. Indirect immunofluorescence and immunoelectron microscopy analysis indicate that TgA1 localizes to the plasma membrane and co‐localizes with the vacuolar H+‐pyrophosphatase to intracellular vacuoles identified morphologically and by X‐ray microanalysis as the acidocalcisomes. This vacuolar‐type Ca2+‐ATPase could play an important role in Ca2+ homeostasis in T.gondii.

Oxidative Phosphorylation and Rotenone-insensitive Malate- and NADH-Quinone Oxidoreductases in Plasmodium yoelii yoelii Mitochondria in Situ*

Respiration, membrane potential, and oxidative phosphorylation of mitochondria of Plasmodium yoelii yoelii trophozoites were assayed in situ after permeabilization with digitonin. ADP induced an oligomycin-sensitive transition from resting to phosphorylating respiration in the presence of oxidizable substrates. A functional respiratory chain was demonstrated. In addition, the ability of the parasite to oxidize exogenous NADH, as well as the insensitivity of respiration to rotenone and its sensitivity to flavone, suggested the presence of an alternative NADH-quinone (NADH-Q) oxidoreductase. Rotenone-insensitive respiration and membrane potential generation in the presence of malate suggested the presence of a malate-quinone oxidoreductase. These results are in agreement with the presence of genes in P. yoelii encoding for proteins with homology to NADH-Q oxidoreductases of bacteria, plant, fungi, and protozoa and malate-quinone oxidoreductases of bacteria. The complete inhibition of respiration by antimycin A and cyanide excluded the presence of an alternative oxidase as described in other parasites. An uncoupling effect of fatty acids was partly reversed by bovine serum albumin and GTP but was unaffected by carboxyatractyloside. These results provide the first biochemical evidence of the presence of an alternative NADH-Q oxidoreductase and a malate-quinone oxidoreductase and confirm the operation of oxidative phosphorylation in malaria parasites.

A plant-like vacuolar H+-pyrophosphatase in Plasmodium falciparum

Inorganic pyrophosphate promoted the acidification of a subcellular compartment in cell homogenates of Plasmodium falciparum trophozoites. The proton gradient driven by pyrophosphate was collapsed by addition of NH4Cl or the K+/H+ exchanger nigericin and eliminated by the pyrophosphate analog aminomethylenediphosphonate. Pyrophosphatase activity was dependent upon K+, and partially inhibited by Na+. The presence of a plant‐like vacuolar H+‐translocating pyrophosphatase (V‐H+‐PPase) was confirmed using antibodies raised against conserved peptide sequences of the enzyme, which cross reacted with a protein band of 76.5 kDa. Immunofluorescence microscopy using these antibodies showed a general fluorescence over the whole parasites and intracellular bright spots suggesting a vesicular and plasma membrane localization. Together, these results indicate the presence in P. falciparum of a V‐H+‐PPase of similar characteristics to those of the enzyme from plants.

The acidocalcisome Ca2+-ATPase (TgA1) of Toxoplasma gondii is required for polyphosphate storage, intracellular calcium homeostasis and virulence

A large proportion of intracellular Ca2+ in Toxoplasma gondii tachyzoites is stored within acidocalcisomes. These organelles are characterized by their acidic nature and high calcium and polyphosphate (polyP) content. The activity of a Ca2+/H+‐ATPase named TgA1 may be important for the accumulation of Ca2+ in these organelles. This enzyme belongs to a group of plasma membrane Ca2+‐ATPase (PMCA) that lack a calmodulin‐binding domain and have vacuolar localization. To investigate the role of this enzyme, we have generated T. gondii mutants deficient in TgA1 through gene disruption. Proliferation of these mutants decreased dramatically because of deficient cell invasion. In addition, these cells had reduced virulence in a mouse model. Biochemical analysis revealed that the cell polyP content was drastically reduced, and the basal calcium levels were increased and unstable. Microneme secretion under the conditions of stimulation by ionophores was altered. Complementation of null mutants with TgA1 restored most functions. In summary, these results establish a link between TgA1, calcium homeostasis, polyP storage and virulence.

Role for a P-type H+-ATPase in the acidification of the endocytic pathway of Trypanosoma cruzi

Previous studies in Trypanosoma cruzi, the etiologic agent of Chagas disease, have resulted in the cloning and sequencing of a pair of tandemly linked genes (TcHA1 and TcHA2) that encode P (phospho-intermediate form)-type H+-ATPases with homology to fungal and plant proton-pumping ATPases. In the present study, we demonstrate that these pumps are present in the plasma membrane and intracellular compartments of three different stages of T. cruzi. The main intracellular compartment containing these ATPases in epimastigotes was identified as the reservosome. This identification was achieved by immunofluorescence assays and immunoelectron microscopy showing their co-localization with cruzipain, and by subcellular fractionation and detection of their activity. ATP-dependent proton transport by isolated reservosomes was sensitive to vanadate and insensitive to bafilomycin A1, which is in agreement with the localization of P-type H+-ATPases in these organelles. Analysis by confocal immunofluorescence microscopy revealed that epitope-tagged TcHA1-Ty1 and TcHA2-Ty1 gene products are localized in the reservosomes, whereas the TcHA1-Ty1 gene product is additionally present in the plasma membrane. Immunogold electron microscopy showed the presence of the H+-ATPases in other compartments of the endocytic pathway such as the cytostome and endosomal vesicles, suggesting that in contrast with most cells investigated until now, the endocytic pathway of T. cruzi is acidified by a P-type H+-ATPase.

Polyphosphate content and fine structure of acidocalcisomes of Plasmodium falciparum.

Although acidocalcisomes have been well characterized morphologically in other apicomplexan parasites, no such characterization has been done in Plasmodium spp. Here, we report that Plasmodium falciparum merozoites possess electron-dense organelles rich in phosphorus and calcium, as detected by X-ray microanalysis of intact cells, which are similar to the acidocalcisomes of other apicomplexans, but of more irregular form. In agreement with these results malaria parasites possess large amounts of short- and long-chain polyphosphate (polyP), which are associated with acidocalcisomes in other organisms. PolyP levels were highest in the trophozoite stage of the parasite. Treatment of isolated trophozoites with chloroquine resulted in a significant hydrolysis of polyP. Taken together, these results provide evidence that acidocalcisomes from Plasmodium falciparum do not differ significantly from acidocalcisomes of other apicomplexan parasites.

Overexpression of a Zn2+-sensitive Soluble Exopolyphosphatase from Trypanosoma cruzi Depletes Polyphosphate and Affects Osmoregulation

We report the cloning, expression, purification, and characterization of the Trypanosoma cruzi exopolyphosphatase (TcPPX). The product of this gene (TcPPX), has 383 amino acids and a molecular mass of 43.1 kDa. TcPPX differs from most exopolyphosphatases in its preference for short-chain polyphosphate (poly P). Heterologous expression of TcPPX in Escherichia coli produced a functional enzyme that had a neutral optimum pH and was dramatically inhibited by low concentrations of Zn2+, high concentrations of basic amino acids (lysine and arginine), and heparin. TcPPX is a processive enzyme and does not hydrolyze ATP, pyrophosphate, or p-nitrophenyl phosphate, although it hydrolyzes guanosine 5′-tetraphosphate very efficiently. Overexpression of TcPPX resulted in a dramatic decrease in total short-chain poly P and partial decrease in long-chain poly P. This was accompanied by a delayed regulatory volume decrease after hyposmotic stress. These results support the role of poly P in T. cruzi osmoregulation.

A Malaria Parasite-encoded Vacuolar H+-ATPase Is Targeted to the Host Erythrocyte

The asexual development of malaria parasites inside the erythrocyte is accompanied by changes in the composition, structure, and function of the host cell membrane and cytoplasm. The parasite exports a membrane network into the host cytoplasm and several proteins that are inserted into the erythrocyte membrane, although none of these proteins has been shown to have enzymatic activity. We report here that a functional malaria parasite-encoded vacuolar (V)-H+-ATPase is exported to the erythrocyte and localized in membranous structures and in the plasma membrane of the infected erythrocyte. This localization was determined by separation of parasite and erythrocyte membranes and determination of enzyme marker activities and by immunofluorescence microscopy assays using antibodies against the B subunit of the malarial V-H+-ATPase and erythrocyte (spectrins) and parasite (merozoite surface protein 1) markers. Our results suggest that this pump has a role in the maintenance of the intracellular pH (pHi) of the infected erythrocyte. Our results also indicate that although the pHi maintained by the V-H+-ATPase is important for maximum uptake of small metabolites at equilibrium, it does not appear to affect transport across the erythrocyte membrane and is, therefore, not involved in the previously described phenomenon of increased permeability of infected erythrocytes that is sensitive to chloride channel inhibitors (new permeation pathway). This constitutes the first report of the presence of a functional enzyme of parasite origin in the plasma membrane of its host.

Molecular Characterization of Trypanosoma brucei P-type H+-ATPases

Previous studies in Trypanosoma brucei have shown that intracellular pH homeostasis is affected by inhibitors of H+-ATPases, suggesting a major role for these pumps in this process (Vander-Heyden, N., Wong, J., and Docampo, R., (2000) Biochem. J. 346, 53-62). Here, we report the cloning and sequencing of three genes (TbHA1, TbHA2, and TbHA3) present in the genome of T. brucei that encode proteins with homology to fungal and plant P-type proton-pumping ATPases. Northern and Western blot analyses revealed that these genes are up-regulated in procyclic trypomastigotes. TbHA1, TbHA2, and TbHA3 complemented a Saccharomyces cerevisiae strain deficient in P-type H+-ATPase activity, providing genetic evidence for their function. Indirect immunofluorescence analysis showed that TbHA proteins are localized mainly in the plasma membrane of procyclic forms and in the plasma membrane and flagellum of bloodstream forms. T. brucei H+-ATPase genes were functionally characterized using double-stranded RNA interference methodology. The induction of double-stranded RNA (RNA interference) caused growth inhibition, which was more accentuated in procyclic forms and when expression of all TbHA proteins was decreased. Knockdown of TbHA1 and TbHA3, but not of TbHA2, resulted in cells with a lower steady-state pHi and a slower rate of pHi recovery from acidification. No evidence was found of an intracellular P-type H+-ATPase activity. These results establish that T. brucei H+-ATPases are plasma membrane enzymes essential for parasite viability.