Regulatory volume decrease in Trypanosoma cruzi involves amino acid efflux and changes in intracellular calcium.

A regulatory volume decrease (RVD) in response to hyposmotic stress has been characterized in different life-cycle stages of Trypanosoma cruzi. Hyposmotic stress initially caused swelling, but this was rapidly reversed by a compensatory volume reversal that was essentially complete by 5 min. Volume recovery was associated with an amino acid efflux that accounted for approximately 50% of the regulatory volume decrease in all three life-cycle stages. The amino acid efflux was selective for neutral and anionic amino acids, but excluded cationic amino acids. Acidocalcisomes contained an amino acid pool over four times more concentrated than whole-cell levels, but about 90% of this was composed of Arg and Lys, so involvement of this pool in amino acid efflux was ruled out. Hyposmotic stress induced a rise in intracellular calcium that was dependent on influx of calcium across the plasma membrane, since chelation of extracellular calcium abolished the response. Influx of calcium was confirmed by demonstration of manganese-mediated quenching of intracellular fura-2 fluorescence and partial inhibition of the rise in calcium by calcium channel blockers. Manipulation of intra- and extracellular calcium levels had minor effects on the initial rate of amino acid efflux and no effect on the rate of volume recovery.

The polyphosphate bodies of Chlamydomonas reinhardtii possess a proton-pumping pyrophosphatase and are similar to acidocalcisomes.

Acidocalcisomes are acidic calcium storage compartments described initially in trypanosomatid and apicomplexan parasites. In this work, we describe organelles with properties similar to acidocalcisomes in the green alga Chlamydomonas reinhardtii. Nigericin and NH(4)Cl released (45)Ca(2+) from preloaded permeabilized cells, suggesting the incorporation of a significant amount of this cation into an acidic compartment. X-ray microanalysis of the electron-dense vacuoles or polyphosphate bodies of C. reinhardtii showed large amounts of phosphorus, magnesium, calcium, and zinc. Immunofluorescence microscopy, using antisera raised against a peptide sequence of the vacuolar type proton pyrophosphatase (H(+)-PPase) of Arabidopsis thaliana which is conserved in the C. reinhardtii enzyme, indicated localization in the plasma membrane, in intracellular vacuoles, and the contractile vacuole where it colocalized with the vacuolar proton ATPase (V-H(+)-ATPase). Purification of the electron-dense vacuoles using iodixanol density gradients indicated a preferential localization of the H(+)-PPase and the V-H(+)-ATPase activities in addition to high concentrations of PP(i) and short and long chain polyphosphate, but lack of markers for mitochondria and chloroplasts. In isolated electron-dense vacuoles, PP(i)-driven proton translocation was stimulated by potassium ions and inhibited by the PP(i) analog aminomethylenediphosphonate. Potassium fluoride, imidodiphosphate, N,N'-dicyclohexylcarbodiimide, and N-ethylmaleimide also inhibited PP(i) hydrolysis in the isolated organelles in a dose-dependent manner. These results indicate that the electron-dense vacuoles of C. reinhardtii are very similar to acidocalcisomes with regard to their chemical composition and the presence of proton pumps. Polyphosphate was also localized to the contractile vacuole by 4',6-diamidino-2-phenylindole staining, suggesting, with the immunochemical data, a link between these organelles and the acidocalcisomes.

The sterol composition of Trypanosoma cruzi changes after growth in different culture media and results in different sensitivity to digitonin-permeabilization.

Respiration, oxidative phosphorylation. and the corresponding changes in membrane potential (deltapsi) of Trypanosoma cruzi epimastigotes grown either in liver infusion-tryptose (LIT) or brain heart infusion (BHI) culture medium were assayed in situ using digitonin to render their plasma membrane permeable to succinate, ADP, safranine O, and other small molecules. When the cells were permeabilized with 64 microM digitonin, a concentration previously used with epimastigotes, the ability of the cells grown in LIT medium to sustain oxidative phosphorylation was demonstrated by the detection of an oligomycin-sensitive decrease in mitochondrial membrane potential induced by ADP. In contrast, the cells grown in BHI medium were not able to sustain a stable membrane potential and did not respond to ADP addition. Analyses of oxygen consumption by these permeabilized cells indicated that the rate of basal respiration, which was similar in both cell types, was significantly decreased by 64 microM digitonin. Addition of ADP to the permeabilized cells grown in LIT medium promoted an oligomycin-sensitive transition from resting to phosphorylating respiration in contrast to the cells grown in BHI medium, whose respiration decreased steadily and did not respond either to ADP or CCCP. Titration of the cells grown in BHI medium with different digitonin concentrations indicated that their mitochondria have higher sensitivity to digitonin than those grown in LIT medium. Analysis of the sterol composition of epimastigotes grown in the two different media showed a higher percentage of cholesterol in total and mitochondrial extracts of epimastigotes grown in BHI medium as compared to those grown in LIT medium, suggesting the involvement of this sterol in their increased sensitivity to digitonin-permeabilization.

Bisphosphonates are potent inhibitors of Trypanosoma cruzi farnesyl pyrophosphate synthase.

We report the cloning and sequencing of a gene encoding the farnesyl pyrophosphate synthase of Trypanosoma cruzi. The protein (T. cruzi farnesyl pyrophosphate synthase, TcFPPS) is an attractive target for drug development, since the growth of T. cruzi is inhibited by carbocation transition state/reactive intermediate analogs of its substrates, the nitrogen-containing bisphosphonates currently in use in bone resorption therapy. The protein predicted from the nucleotide sequence of the gene has 362 amino acids and a molecular mass of 41.2 kDa. Several sequence motifs found in other FPPSs are present in TcFPPS. Heterologous expression of TcFPPS in Escherichia coli produced a functional enzyme that was inhibited by the nitrogen-containing bisphosphonates alendronate, pamidronate, homorisedronate, and risedronate but was less sensitive to the non-nitrogen-containing bisphosphonate etidronate, which, unlike the nitrogen-containing bisphosphonates, does not affect parasite growth. The protein contains a unique 11-mer insertion located near the active site, together with other sequence differences that may facilitate the development of novel anti-Chagasic agents.

Recent developments in the chemotherapy of Chagas disease.

Chagas disease remains an important health problem in the Americas. Advances are being made in parts of South America in blocking transmission from insect vectors or blood transfusion, but more effective chemotherapy is needed for the millions who are already infected. This is especially important since recent results have indicated that treatment is beneficial for the elimination of the chronic course of the disease. The rational development of new drugs depends on the identification of differences between human metabolism and that of the causative parasite, Trypanosoma cruzi. Recent developments in the study of the basic biochemistry of the parasite have allowed the identification of novel targets for chemotherapy, such as sterol metabolism, protein prenylation, proteases, and phospholipid metabolism, and these are the subject of this review.

(31)P NMR of apicomplexans and the effects of risedronate on Cryptosporidium parvum growth.

High-resolution 303.6 MHz (31)P NMR spectra have been obtained of perchloric acid extracts of Plasmodium berghei trophozoites, Toxoplasma gondii tachyzoites, and Cryptosporidium parvum oocysts. Essentially complete resonance assignments have been made based on chemical shifts and by coaddition of authentic reference compounds. Signals corresponding to inorganic pyrophosphate were detected in all three species. In T. gondii and C. parvum, additional resonances were observed corresponding to linear triphosphate as well as longer chain polyphosphates. Spectra of P. berghei and T. gondii also indicated the presence of phosphomonoesters and nucleotide phosphates. We also report that the pyrophosphate analog drug, risedronate (used in bone resorption therapy), inhibits the growth of C. parvum in a mouse xenograft model. When taken together, our results indicate that all the major disease-causing apicomplexan parasites contain extensive stores of condensed phosphates and that as with Plasmodium falciparum and T. gondii, the pyrophosphate analog drug risedronate is an inhibitor of C. parvum cell growth.

TcSCA complements yeast mutants defective in Ca2+ pumps and encodes a Ca2+-ATPase that localizes to the endoplasmic reticulum of Trypanosoma cruzi.

Intracellular Ca(2+) in Trypanosoma cruzi is mainly located in an acidic compartment named the acidocalcisome, which among other pumps and exchangers possesses a plasma membrane-type Ca(2+)-ATPase. Evidence for an endoplasmic reticulum-located Ca(2+) uptake has been more elusive and based on indirect results. Here we report the cloning and sequencing of a gene encoding a sarcoplasmic-endoplasmic reticulum-type Ca(2+)-ATPase from T. cruzi. The protein (TcSCA) predicted from the nucleotide sequence of the gene has 1006 amino acids and a molecular mass of 109.7 kDa. Several sequence motifs found in sarcoplasmic-endoplasmic reticulum-type Ca(2+)-ATPases were present in TcSCA. Expression of TcSCA in yeast mutants deficient in the Golgi and vacuolar Ca(2+) pumps (pmr1 pmc1 cnb 1) restored growth on EGTA. Membranes were isolated from the pmr1 pmc1 cnb1 mutant transformed with TcSCA, and it was found that the TcSCA polypeptide formed a Ca(2+)-dependent and hydroxylamine-sensitive (32)P-labeled phosphoprotein of 110 kDa in the presence of [gamma-(32)P]ATP. Cyclopiazonic acid, but not thapsigargin, blocked this phosphoprotein formation. Transgenic parasites expressing constructs of TcSCA with green fluorescent protein exhibited co-localization of TcSCA with the endoplasmic reticulum proteins BiP and calreticulin. An endoplasmic reticulum location was also found in amastigotes and trypomastigotes using a polyclonal antibody against a COOH-terminal region of the protein. The ability of TcSCA to restore growth of mutant pmr1 pmc1 cnb 1 on medium containing Mn(2+) suggests that TcSCA may also regulate Mn(2+) homeostasis by pumping Mn(2+) into the endoplasmic reticulum of T. cruzi.

The acidocalcisome.

The acidocalcisome is an electron-dense acidic organelle which contains a matrix of pyrophosphate and polyphosphates with bound calcium and other cations. Its limiting membrane possesses a number of pumps and exchangers for the uptake and release of these elements. The acidocalcisome does not belong to the endocytic pathway and may represent a branch of the secretory pathway in trypanosomatids and apicomplexan parasites. The acidocalcisome is possibly involved in polyphosphate and cation storage and in adaptation of these microoganisms to environmental stress.

Rapid changes in polyphosphate content within acidocalcisomes in response to cell growth, differentiation, and environmental stress in Trypanosoma cruzi.

Inorganic polyphosphate (polyP) has been identified and measured in different stages of Trypanosoma cruzi. Millimolar levels (in terms of P(i) residues) in chains of less than 50 residues long, and micromolar levels in chains of about 700--800 residues long, were found in different stages of T. cruzi. Analysis of purified T. cruzi acidocalcisomes indicated that polyPs were preferentially located in these organelles. This was confirmed by visualization of polyPs in the acidocalcisomes using 4',6-diamidino-2-phenylindole. A rapid increase (within 2--4 h) in the levels of short and long chain polyPs was detected during trypomastigote to amastigote differentiation and during the lag phase of growth of epimastigotes (within 12--24 h). Levels rapidly decreased after the epimastigotes resumed growth. Short and long chain polyP levels rapidly decreased upon exposure of epimastigotes to hypo-osmotic or alkaline stresses, whereas levels increased after hyperosmotic stress. Ca(2+) release from acidocalcisomes by a combination of ionophores (ionomycin and nigericin) was associated with the hydrolysis of short and long chain polyPs. In agreement with these results, acidocalcisomes were shown to contain polyphosphate kinase and exopolyphosphatase activities. Together, these results suggest a critical role for these organelles in the adaptation of the parasite to environmental changes.

Bisphosphonates inhibit the growth of Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, and Plasmodium falciparum: a potential route to chemotherapy.

We have investigated the effects in vitro of a series of bisphosphonates on the proliferation of Trypanosoma cruzi, Trypanosoma brucei rhodesiense, Leishmania donovani, Toxoplasma gondii, and Plasmodium falciparum. The results show that nitrogen-containing bisphosphonates of the type used in bone resorption therapy have significant activity against parasites, with the aromatic species having in some cases nanomolar or low-micromolar IC(50) activity values against parasite replication (e.g. o-risedronate, IC(50) = 220 nM for T. brucei rhodesiense; risedronate, IC(50) = 490 nM for T. gondii). In T. cruzi, the nitrogen-containing bisphosphonate risedronate is shown to inhibit sterol biosynthesis at a pre-squalene level, most likely by inhibiting farnesylpyrophosphate synthase. Bisphosphonates therefore appear to have potential in treating parasitic protozoan diseases.

Bisphosphonates derived from fatty acids are potent growth inhibitors of Trypanosoma cruzi.

We have investigated the effect of a series of bisphosphonates derived from fatty acids against Trypanosoma cruzi proliferation in in vitro assays. Some of these drugs proved to be potent inhibitors against the intracellular form of the parasite exhibiting IC50 values at the low micromolar level. As bisphosphonates are FDA clinically approved for treatment of bone resorption, their potential innocuousness makes them good candidates to control tropical diseases.

Bisphosphonates as chemotherapeutic agents against trypanosomatid and apicomplexan parasites.

Trypanosomatid and apicomplexan parasites remain an important health problem in developing countries. Advances are being made in parts of the world in blocking transmission from insect vectors, but more effective chemotherapy is urgently needed. This is especially important since development of resistance is a growing problem. The rational development of new drugs depends on the identification of differences between human metabolism and that of the parasites. Recent developments in the study of the basic biochemistry of these parasites have resulted in the discovery that bisphosphonates, drugs widely used in the treatment of benign and malignant diseases characterized by increased bone resorption, could have a role as lead antiparasitic agents.

Cloning and functional expression of a gene encoding a vacuolar-type proton-translocating pyrophosphatase from Trypanosoma cruzi.

Acidocalcisomes are acidic Ca(2+)-storage organelles found in trypanosomatids that are similar to organelles known historically as volutin granules. Acidification of these organelles is driven in part by a vacuolar H(+)-pyrophosphatase (V-H(+)-PPase), an enzyme that is also present in plant vacuoles and in some bacteria. Here, we report the cloning and sequencing of a gene encoding the acidocalcisomal V-H(+)-PPase of Trypanosoma cruzi. The protein (T. cruzi pyrophosphatase, TcPPase) predicted from the nucleotide sequence of the gene has 816 amino acids and a molecular mass of 85 kDa. Several sequence motifs found in plant V-H(+)-PPases were present in TcPPase, explaining its sensitivity to N-ethylmaleimide and N,N'-dicyclohexylcarbodi-imide. Heterologous expression of the cDNA encoding TcPPase in the yeast Saccharomyces cerevisiae produced a functional enzyme. Phylogenetic analysis of the available V-H(+)-PPase sequences indicates that TcPPase is nearer to the vascular plant cluster and the branch containing Chara, a precursor to land plants, than to any of the other pyrophosphatase sequences included in the analysis. The apparent lack of such a V-H(+)-PPase in mammalian cells may provide a target for the development of new drugs.

The fine structure of acidocalcisomes in Trypanosoma cruzi.

Trypanosoma cruzi survives in vertebrate and invertebrate hosts and has developed mechanisms that allow it to adapt to changes in the microenvironment such as temperature, pH, and ionic composition. Most of its calcium is concentrated in an organelle named the acidocalcisome, which is acidified by a (V-H+)-adenosine triphosphatase and has H+/Ca2+ counter-transportation for calcium uptake. In this work, acidocalcisomes were examined using different transmission electron microscopy techniques. In thin sections of different stages, acidocalcisomes presented a circular shape with an electron-dense inclusion containing P3-, Ca2+, Na+, Mg2+, K+, and Zn2+. They could be distinguished from gold-labeled albumin-containing reservosomes in whole epimastigotes, and a morphometric analysis showed higher amounts of these organelles in amastigotes as compared with epimastigotes and trypomastigotes. It is possible that this variation in the amount of acidocalcisomes in the different evolutive stages could reflect adaptation mechanisms used by the parasite to survive and multiply in different environmental conditions.

31P NMR spectroscopy of Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major. Evidence for high levels of condensed inorganic phosphates.

High resolution (31)P nuclear magnetic resonance spectra at 303.6 MHz (corresponding to a (1)H resonance frequency of 750 MHz) have been obtained of perchloric acid extracts of Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major, the causative agents of African sleeping sickness, Chagas' disease, and leishmaniasis. Essentially complete assignments have been made based on chemical shifts and by direct addition of authentic reference compounds. The results indicate the presence of high levels of short chain condensed polyphosphates: di-, tri-, tetra-, and pentapolyphosphate. (31)P NMR spectra of purified T. brucei, T. cruzi, and L. major acidocalcisomes, calcium and phosphorus storage organelles, indicate that polyphosphates are abundant in these organelles and have an average chain length of 3.11-3.39 phosphates. In the context of the recent discovery of several pyrophosphate-utilizing enzymes in trypanosomatids, the presence of these inorganic polyphosphates implies a critical role for these molecules in these parasites and a potential new route to chemotherapy.

Design and synthesis of aryloxyethyl thiocyanate derivatives as potent inhibitors of Trypanosoma cruzi proliferation.

As a part of our project directed at the search of new chemotherapeutic agents against American trypanosomiasis (Chagas' disease), several drugs possessing the 4-phenoxyphenoxy skeleton and other closely related structures employing the thiocyanate moiety as polar end group were designed, synthesized, and evaluated as antiproliferative agents against Trypanosoma cruzi, the parasite responsible for this disease. These thiocyanate analogues were envisioned bearing in mind the potent activity shown by 4-phenoxyphenoxyethyl thiocyanate (compound 8) taken as lead drug. This compound had previously proved to be an extremely active growth inhibitor against T. cruzi with IC(50) values ranging from the very low micromolar level in epimastigotes to the low nanomolar level in the intracellular form of the parasite. Of the designed compounds, the ethyl thiocyanate drugs connected to nonpolar skeletons, namely, arylthio, 2,4-dichlorophenoxy, ortho-substituted aryloxy, and 2-methyl-4-phenoxyphenoxy (compounds 15, 34, 47, 52, 72, respectively), were shown to be very potent antireplicative agents against T. cruzi. On the other hand, conformationally restricted analogues as well as branched derivatives at the aliphatic side chain were shown to be moderately active against T. cruzi growth. The biological activity of drugs bearing the thiocyanate group correlated quite well with the activity exhibited by their normal precursors, the tetrahydropyranyl ether derivatives, when bonded to the same nonpolar skeleton. Compounds having the tetrahydropyranyl moeity as polar end were proportionally much less active than sulfur-containing derivatives in all cases. Drugs 47 and 72 also resulted to be very active against the amastigote form of the parasite growing in myoblasts; however, they were slightly less active than the lead drug 8. On the other hand, compounds 34 and 52 were almost devoid of activity against myoblasts. Surprisingly, the dithio derivative 15 was toxic for myoblasts.

Characterization of isolated acidocalcisomes of Trypanosoma cruzi.

The acidocalcisome is an acidic calcium store in trypanosomatids with a vacuolar-type proton-pumping pyrophosphatase (V-H(+)-PPase) located in its membrane. In this paper, we describe a new method using iodixanol density gradients for purification of the acidocalcisome from Trypanosoma cruzi epimastigotes. Pyrophosphatase assays indicated that the isolated organelle was at least 60-fold purified compared with the large organelle (10,000 x g) fraction. Assays for other organelles generally indicated no enrichment in the acidocalcisome fraction; glycosomes were concentrated 5-fold. Vanadate-sensitive ATP-driven Ca(2+) uptake (Ca(2+)-ATPase) activity was detectable in the isolated acidocalcisome, but ionophore experiments indicated that it was not acidic. However, when pyrophosphate was added, the organelle acidified, and the rate of Ca(2+) uptake increased. Use of the indicator Oxonol VI showed that V-H(+)-PPase activity generated a membrane potential. Use of sulfate or nitrate in place of chloride in the assay buffer did not affect V-H(+)-PPase activity, but there was less activity with gluconate. Organelle acidification was countered by the chloride/proton symport cycloprogidiosin. No vacuolar H(+)-ATPase activity was detectable in isolated acidocalcisomes. However, immunoblots showed the presence of at least a membrane-bound V-H(+)-ATPase subunit, while experiments employing permeabilized epimastigotes suggested that vacuolar H(+)-ATPase and V-H(+)-PPase activities are present in the same Ca(2+)-containing compartment.

Presence of a Na(+)/H(+) exchanger in acidocalcisomes of Leishmania donovani and their alkalization by anti-leishmanial drugs.

Acidocalcisomes are acidic vacuoles present in trypanosomatids that contain most of the cellular calcium. The data presented here demonstrate that Leishmania donovani acidocalcisomes possess a Na(+)/H(+) exchanger. 3,5-Dibutyl-4-hydroxytoluene, in the concentration range of 0-20 microM, inhibited the Na(+)/H(+) exchanger, and strongly stimulated the activity of the vacuolar H(+)-ATPase responsible for vacuolar acidification. As occurs with Na(+), the cationic anti-leishmanial drugs pentamidine, WR-6026, and chloroquine promoted a fast and extensive alkalization of the L. donovani acidocalcisomes.

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.