The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Roberto Docampo - One of the best experts on this subject based on the ideXlab platform.
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Magic-angle spinning 31 P NMR spectroscopy of condensed phosphates in parasitic protozoa: visualizing the invisible
2020Co-Authors: Benjamin Moreno, Roberto Docampo, Silvia N. J. Moreno, Claudia O Rodrigues, Brian N Bailey, Julio A Urbina, Eric Old¢eldAbstract:Abstract We report the results of a solid-state 31 P nuclear magnetic resonance (NMR) spectroscopic investigation of the Acidocalcisome organelles from Trypanosoma brucei (bloodstream form), Trypanosoma cruzi and Leishmania major (insect forms). The spectra are characterized by a broad envelope of spinning sidebands having isotropic chemical shifts at V V0, 3 37 and 3 321 ppm. These resonances are assigned to orthophosphate, terminal (K K) phosphates of polyphosphates and bridging (L L) phosphates of polyphosphates, respectively. The average polyphosphate chain length is V V3.3 phosphates. Similar results were obtained with whole L. major promastigotes. 31 P NMR spectra of living L. major promastigotes recorded under conventional solution NMR conditions had spectral intensities reduced with respect to solution-state NMR spectra of acid extracts, consistent with the invisibility of the solid-state phosphates. These results show that all three parasites contain large stores of condensed phosphates which can be visualized by using magic-angle spinning NMR techniques. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies
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the Acidocalcisome inositol 1 4 5 trisphosphate receptor of trypanosoma brucei is stimulated by luminal polyphosphate hydrolysis products
Journal of Biological Chemistry, 2019Co-Authors: Evgeniy Potapenko, Nuria Waddington Negrao, Guozhong Huang, Roberto DocampoAbstract:Acidocalcisomes are acidic calcium stores rich in polyphosphate (polyP) and are present in trypanosomes and also in a diverse range of other organisms. Ca2+ is released from these organelles through a channel, inositol 1,4,5-trisphosphate receptor (TbIP3R), which is essential for growth and infectivity of the parasite Trypanosoma brucei However, the mechanism by which TbIP3R controls Ca2+ release is unclear. In this work, we expressed TbIP3R in a chicken B lymphocyte cell line in which the genes for all three vertebrate IP3Rs were stably ablated (DT40-3KO). We show that IP3-mediated Ca2+ release depends on Ca2+ but not on ATP concentration and is inhibited by heparin, caffeine, and 2-aminomethoxydiphenyl borate (2-APB). Excised patch clamp recordings from nuclear membranes of DT40 cells expressing only TbIP3R disclosed that luminal inorganic orthophosphate (Pi) or pyrophosphate (PPi), and neutral or alkaline pH can stimulate IP3-generated currents. In contrast, polyP or acidic pH did not induce these currents, and nuclear membranes obtained from cells expressing rat IP3R were unresponsive to polyP or its hydrolysis products. Our results are consistent with the notion that polyP hydrolysis products within Acidocalcisomes or alkalinization of their luminal pH activate TbIP3R and Ca2+ release. We conclude that TbIP3R is well-adapted to its role as the major Ca2+ release channel of Acidocalcisomes in T. brucei.
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Pyrophosphate Stimulates the Phosphate-Sodium Symporter of Trypanosoma brucei Acidocalcisomes and Saccharomyces cerevisiae Vacuoles
'American Society for Microbiology', 2019Co-Authors: Evgeniy Potapenko, Guozhong Huang, Ciro D. Cordeiro, Roberto DocampoAbstract: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.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
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Acidocalcisome-Mitochondrion Membrane Contact Sites in Trypanosoma brucei.
Pathogens (Basel Switzerland), 2018Co-Authors: S. Ramakrishnan, Beejan Asady, Roberto DocampoAbstract:Membrane contact sites are regions of close apposition between two organelles, typically less than 30 nanometers apart, that facilitate transfer of biomolecules. The presence of contact sites has been demonstrated in yeast, plants, and mammalian cells. Here, we investigated the presence of such contact sites in Trypanosoma brucei. In mammalian cells, endoplasmic reticulum-mitochondria contact sites facilitate mitochondrial uptake of Ca2+ released by the ER-located inositol 1,4,5-trisphosphate receptor (InsP3R). However, the InsP3R in trypanosomes localizes to Acidocalcisomes, which serve as major Ca2+ stores in these parasites. In this work, we have used super-resolution structured illumination microscopy and electron microscopy to identify membrane contact sites that exist between Acidocalcisomes and mitochondria. Furthermore, we have confirmed the close association of these organelles using proximity ligation assays. Characterization of these contact sites may be a necessary starting point towards unraveling the role of Ca2+ in regulating trypanosome bioenergetics.
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crispr cas9 mediated endogenous c terminal tagging of trypanosoma cruzi genes reveals the Acidocalcisome localization of the inositol 1 4 5 trisphosphate receptor
Journal of Biological Chemistry, 2016Co-Authors: Noelia Lander, Roberto Docampo, Miguel Angel Chiurillo, Melissa Storey, Anibal E VercesiAbstract:Methods for genetic manipulation of Trypanosoma cruzi, the etiologic agent of Chagas disease, have been highly inefficient, and no endogenous tagging of genes has been reported to date. We report here the use of the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated gene 9) system for endogenously tagging genes in this parasite. The utility of the method was established by tagging genes encoding proteins of known localization such as TcFCaBP (flagellar calcium binding protein) and TcVP1 (vacuolar proton pyrophosphatase), and two proteins of undefined or disputed localization, the TcMCU (mitochondrial calcium uniporter) and TcIP3R (inositol 1,4,5-trisphosphate receptor). We confirmed the flagellar and Acidocalcisome localization of TcFCaBP and TcVP1 by co-localization with antibodies to the flagellum and Acidocalcisomes, respectively. As expected, TcMCU was co-localized with the voltage-dependent anion channel to the mitochondria. However, in contrast to previous reports and our own results using overexpressed TcIP3R, endogenously tagged TcIP3R showed co-localization with antibodies against VP1 to Acidocalcisomes. These results are also in agreement with our previous reports on the localization of this channel to Acidocalcisomes of Trypanosoma brucei and suggest that caution should be exercised when overexpression of tagged genes is done to localize proteins in T. cruzi.
Silvia N. J. Moreno - One of the best experts on this subject based on the ideXlab platform.
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Magic-angle spinning 31 P NMR spectroscopy of condensed phosphates in parasitic protozoa: visualizing the invisible
2020Co-Authors: Benjamin Moreno, Roberto Docampo, Silvia N. J. Moreno, Claudia O Rodrigues, Brian N Bailey, Julio A Urbina, Eric Old¢eldAbstract:Abstract We report the results of a solid-state 31 P nuclear magnetic resonance (NMR) spectroscopic investigation of the Acidocalcisome organelles from Trypanosoma brucei (bloodstream form), Trypanosoma cruzi and Leishmania major (insect forms). The spectra are characterized by a broad envelope of spinning sidebands having isotropic chemical shifts at V V0, 3 37 and 3 321 ppm. These resonances are assigned to orthophosphate, terminal (K K) phosphates of polyphosphates and bridging (L L) phosphates of polyphosphates, respectively. The average polyphosphate chain length is V V3.3 phosphates. Similar results were obtained with whole L. major promastigotes. 31 P NMR spectra of living L. major promastigotes recorded under conventional solution NMR conditions had spectral intensities reduced with respect to solution-state NMR spectra of acid extracts, consistent with the invisibility of the solid-state phosphates. These results show that all three parasites contain large stores of condensed phosphates which can be visualized by using magic-angle spinning NMR techniques. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies
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proteomic analysis of the Acidocalcisome an organelle conserved from bacteria to human cells
PLOS Pathogens, 2014Co-Authors: Guozhong Huang, Silvia N. J. Moreno, Melissa Storey, Paul N Ulrich, Darryl Johnson, Julie Tischer, Javier A Tovar, Ron Orlando, Roberto DocampoAbstract:Acidocalcisomes are acidic organelles present in a diverse range of organisms from bacteria to human cells. In this study Acidocalcisomes were purified from the model organism Trypanosoma brucei, and their protein composition was determined by mass spectrometry. The results, along with those that we previously reported, show that Acidocalcisomes are rich in pumps and transporters, involved in phosphate and cation homeostasis, and calcium signaling. We validated the Acidocalcisome localization of seven new, putative, Acidocalcisome proteins (phosphate transporter, vacuolar H+-ATPase subunits a and d, vacuolar iron transporter, zinc transporter, polyamine transporter, and acid phosphatase), confirmed the presence of six previously characterized Acidocalcisome proteins, and validated the localization of five novel proteins to different subcellular compartments by expressing them fused to epitope tags in their endogenous loci or by immunofluorescence microscopy with specific antibodies. Knockdown of several newly identified Acidocalcisome proteins by RNA interference (RNAi) revealed that they are essential for the survival of the parasites. These results provide a comprehensive insight into the unique composition of Acidocalcisomes of T. brucei, an important eukaryotic pathogen, and direct evidence that Acidocalcisomes are especially adapted for the accumulation of polyphosphate.
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a vacuolar h pyrophosphatase tgvp1 is required for microneme secretion host cell invasion and extracellular survival of toxoplasma gondii
Molecular Microbiology, 2014Co-Authors: Jing Liu, Douglas A Pace, Zhicheng Dou, Thayer P King, Daniel Guidot, Vern B Carruthers, Silvia N. J. MorenoAbstract:The vacuolar proton pyrophosphatase (H(+) -PPase) of Toxoplasma gondii (TgVP1), a membrane proton pump, localizes to Acidocalcisomes and a novel lysosome-like compartment termed plant-like vacuole (PLV) or vacuolar compartment (VAC). We report the characterization of a T. gondii null mutant for the TgVP1 gene. Propagation of these mutants decreased significantly because of deficient attachment and invasion of host cells, which correlated with deficient microneme secretion. Processing of cathepsin L (CPL) in these mutants was deficient only when the parasites were incubated in the presence of low concentrations of the vacuolar H(+) -ATPase (V-H(+) -ATPase) inhibitor bafilomycin A1 , suggesting that either TgVP1 or the T. gondii V-H(+) -ATPase (TgVATPase) are sufficient to support CPL processing. The lack of TgVP1 did not affect processing of micronemal proteins, indicating that it does not contribute to proMIC maturations. The TgVP1 null mutants were more sensitive to extracellular conditions and were less virulent in mice. We demonstrate that T. gondii tachyzoites possess regulatory volume decrease capability during hypo-osmotic stress and this ability is impaired in TgVP1 null mutants implicating TgVP1 in osmoregulation. We hypothesize that osmoregulation is needed for host cell invasion and that TgVP1 plays a role during the normal lytic cycle of T. gondii.
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Proteomic Analysis of the Acidocalcisome, an Organelle Conserved from Bacteria to Human Cells
2014Co-Authors: Guozhong Huang, Silvia N. J. Moreno, Melissa Storey, Paul N Ulrich, Darryl Johnson, Julie Tischer, Javier A Tovar, Ron Orl, Roberto DocampoAbstract:Acidocalcisomes are acidic organelles present in a diverse range of organisms from bacteria to human cells. In this study Acidocalcisomes were purified from the model organism Trypanosoma brucei, and their protein composition was determined by mass spectrometry. The results, along with those that we previously reported, show that Acidocalcisomes are rich in pumps and transporters, involved in phosphate and cation homeostasis, and calcium signaling. We validated the Acidocalcisome localization of seven new, putative, Acidocalcisome proteins (phosphate transporter, vacuolar H+-ATPase subunits a and d, vacuolar iron transporter, zinc transporter, polyamine transporter, and acid phosphatase), confirmed the presence of six previously characterized Acidocalcisome proteins, and validated the localization of five novel proteins to different subcellular compartments by expressing them fused to epitope tags in their endogenous loci or by immunofluorescence microscopy with specific antibodies. Knockdown of several newly identified Acidocalcisome proteins by RNA interference (RNAi) revealed that they are essential for the survival of the parasites. These results provide a comprehensive insight into the unique composition of Acidocalcisomes of T. brucei, an important eukaryotic pathogen, an
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Purification of Acidocalcisomes on iodixanol step gradients.
2014Co-Authors: Guozhong Huang, Silvia N. J. Moreno, Melissa Storey, Paul N Ulrich, Darryl Johnson, Julie Tischer, Javier A Tovar, Ron Orlando, Roberto DocampoAbstract:Yield values are percentages relative to the 15,000×g pellet fraction and represent averages from number of preparations in parentheses.*Pyrophosphatase activities in the 15,000 g pellet, and the 1st and 2nd gradient Acidocalcisome preparations were 0.22±0.09, 15.6±3.2, and 22.6±2.1 µmol min−1 mg−1 protein, respectively (mean ± SD).Purification of Acidocalcisomes on iodixanol step gradients.
Wanderley De Souza - One of the best experts on this subject based on the ideXlab platform.
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Structural organization of the tachyzoite of Toxoplasma gondii [Abstract in English]
2010Co-Authors: Wanderley De Souza, Leandro Lemgruber, Márcia Attias, Erica S. Martins-duarte, Rossiane C. VommaroAbstract:AIMS: To review basic aspects on the ultrastructure of the tachyzoite of Toxoplasma gondii , the causative agent of toxoplasmosis. SOURCE OF DATA: The data presented are based on recent publications by the most distinguished research groups in the area dedicated to the study of Toxoplasma gondii , including studies from the present authors. SUMMARY OF FINDINGS: The tachyzoites are responsible for the acute phase of the infection by actively penetrating, through the parasites' apical complex, the host cells where they multiply. Both ultrastructural and molecular particularities of the pellicle, the cytoskeleton, secretory (rhoptries, micronemas and dense granules) and non secretory (apicoplast) organelles, specific to Apicomplexa phylum, besides peculiar features of the nucleus, mitochondrion, Acidocalcisomes, endoplasmic reticulum and Golgi complex of these intracellular parasites. CONCLUSIONS: These characteristics confirm that the success in the process of adhesion, invasion and multiplication of Toxoplasma gondii is clearly correlated to its morphology.
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Calcium- and polyphosphate-containing acidic granules of sea urchin eggs are similar to Acidocalcisomes, but are not the targets for NAADP
2010Co-Authors: Ramos, Isabela Barbosa, Wanderley De Souza, Kildare Rocha De ,miranda, Pace, Douglas A., Verbist, Katherine C., Lin Fu-yang, Zhang Yonghui, Oldfield Eric, Machado, Ednildo De Alcântara, Roberto Do CampoAbstract:11 p. : il.Acidocalcisomes are acidic calcium-storage compartments described from bacteria to humans and characterized by their high content in poly P (polyphosphate), a linear polymer ofmany tens to hundreds of Pi residues linked by high-energy phosphoanhydride bonds. In the present paper we report that millimolar levels of short-chain poly P (in terms of Pi residues) and inorganic PPi are present in sea urchin extracts as detected using 31P-NMR, enzymatic determinations and agarose gel electrophoresis. Poly P was localized to granules randomly distributed in the sea urchin eggs, as shown by labelling with the poly-P-binding domain of Escherichia coli exopolyphosphatase. These granules were enriched using iodixanol centrifugation and shown to be acidic and to contain poly P, as determined by Acridine Orange and DAPI (4 ,6 -diamidino-2-phenylindole) staining respectively. These granules also contained large amounts of calcium, sodium, magnesium, potassium and zinc, as detected by X-ray microanalysis, and bafilomycin A1-sensitive ATPase, pyrophosphatase and exopolyphosphatase activities, as well as Ca2+/H+ and Na+/H+ exchange activities, being therefore similar to Acidocalcisomes described in other organisms. Calcium release from these granules induced by nigericin was associated with poly P hydrolysis. Although NAADP (nicotinic acid–adenine dinucleotide phosphate) released calcium from the granule fraction, this activity was not significantly enriched as compared with the NAADP-stimulated calcium release from homogenates and was not accompanied by poly P hydrolysis. GPN (glycyl-Lphenylalanine- naphthylamide) released calcium when added to sea urchin homogenates, but was unable to release calcium from Acidocalcisome-enriched fractions, suggesting that these acidic stores are not the targets for NAADP
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organizacao estrutural do taquizoito de toxoplasma gondii structural organization of the tachyzoite of toxoplasma gondii
2010Co-Authors: Wanderley De Souza, Leandro Lemgruber, Márcia Attias, Erica S Martinsduarte, Rossiane C. VommaroAbstract:Aims: To review basic aspects on the ultrastructure of the tachyzoite of gondii, the causative agent of toxoplasmosis. Source of data: The data presented are based on recent publications by the most distinguished research groups in the area dedicated to the study of Toxoplasma gondii, including studies from the present authors. Summary of findings: The tachyzoites are responsible for the acute phase of the infection by actively penetrating, through the parasites’ apical complex, the host cells where they multiply. Both ultrastructural and molecular particularities of the pellicle, the cytoskeleton, secretory (rhoptries, micronemas and dense granules) and non secretory (apicoplast) organelles, specific to Apicomplexa phylum, besides peculiar features of the nucleus, mitochondrion, Acidocalcisomes, endoplasmic reticulum and Golgi complex of these intracellular parasites. Conclusions: These characteristics confirm that the success in the process of adhesion, invasion and multiplication of this parasite is clearly correlated to its morphology.
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proton pyrophosphatase and polyphosphate in Acidocalcisome like vesicles from oocytes and eggs of periplaneta americana
Insect Biochemistry and Molecular Biology, 2009Co-Authors: Roberto Docampo, Wanderley De Souza, Kildare Miranda, Isabela Ramos, Lucimar S Motta, Fabio M Gomes, Donald E Champagne, Marcelo F Santiago, Ednildo A MachadoAbstract:Abstract Acidocalcisomes are acidic organelles containing large amounts of polyphosphate (poly P), a number of cations, and a variety of cation pumps in their limiting membrane. The vacuolar proton-pyrophosphatase (V-H+-PPase), a unique electrogenic proton-pump that couples pyrophosphate (PPi) hydrolysis to the active transport of protons across membranes, is commonly present in membranes of Acidocalcisomes. In the course of insect oogenesis, a large amount of yolk protein is incorporated by the oocytes and stored in organelles called yolk granules (YGs). During embryogenesis, the content of these granules is degraded by acid hydrolases. These enzymes are activated by the acidification of the YG by a mechanism that is mediated by proton-pumps present in their membranes. In this work, we describe an H+-PPase activity in membrane fractions of oocytes and eggs of the domestic cockroach Periplaneta americana. The enzyme activity was optimum at pH around 7.0, and was dependent on Mg2+ and inhibited by NaF, as well as by IDP and Ca2+. Immunolocalization of the yolk preparation using antibodies against a conserved sequence of V-H+-PPases showed labeling of small vesicles, which also showed the presence of high concentrations of phosphorus, calcium and other elements, as revealed by electron probe X-ray microanalysis. In addition, poly P content was detected in ovaries and eggs and localized inside the yolk granules and the small vesicles. Altogether, our results provide evidence that numerous small vesicles of the eggs of P. americana present Acidocalcisome-like characteristics. In addition, the possible role of these organelles during embryogenesis of this insect is discussed.
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Structural organization of Trypanosoma cruzi.
Memórias do Instituto Oswaldo Cruz, 2009Co-Authors: Wanderley De SouzaAbstract:Since the initial description of Trypanosoma cruzi by Carlos Chagas in 1909, several research groups have used different microscopic techniques to obtain detailed information about the various developmental stages found in the life cycle of this intracellular parasite. This review describes the present knowledge on the organization of the most important structures and organelles found in the protozoan, such as the cell surface, flagellum, cytoskeleton, kinetoplast-mitochondrion complex, glycosome, Acidocalcisome, contractile vacuole, lipid inclusions, the secretory pathway, endocytic pathway and the nucleus.
Kildare Miranda - One of the best experts on this subject based on the ideXlab platform.
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the Acidocalcisome vacuolar transporter chaperone 4 catalyzes the synthesis of polyphosphate in insect stages of trypanosoma brucei and t cruzi
Journal of Eukaryotic Microbiology, 2014Co-Authors: Noelia Lander, Paul N Ulrich, Kildare Miranda, Samarchith P Kurup, Laura Reiss, Jessica Brewer, Lia Carolina Soares Medeiros, Roberto DocampoAbstract:Polyphosphate is a polymer of inorganic phosphate found in both prokaryotes and eukaryotes. Polyphosphate typically accumulates in acidic, calcium-rich organelles known as Acidocalcisomes, and recent research demonstrated that vacuolar transporter chaperone 4 catalyzes its synthesis in yeast. The human pathogens Trypanosoma brucei and T. cruzi possess vacuolar transporter chaperone 4 homologs. We demonstrate that T. cruzi vacuolar transporter chaperone 4 localizes to Acidocalcisomes of epimastigotes by immunofluorescence and immuno-electron microscopy and that the recombinant catalytic region of the T. cruzi enzyme is a polyphosphate kinase. RNA interference of the T. brucei enzyme in procyclic form parasites reduced short chain polyphosphate levels and resulted in accumulation of pyrophosphate. These results suggest that this trypanosome enzyme is an important component of a polyphosphate synthase complex that utilizes ATP to synthesize and translocate polyphosphate to Acidocalcisomes in insect stages of these parasites.
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evidence for the role of vacuolar soluble pyrophosphatase and inorganic polyphosphate in trypanosoma cruzi persistence
Molecular Microbiology, 2013Co-Authors: Melina Galizzi, Jianmin Fang, Kildare Miranda, Lia Carolina Soares Medeiros, Juan M Bustamante, Rick L Tarleton, Roberto DocampoAbstract:Summary Trypanosoma cruzi infection leads to development of a chronic disease but the mechanisms that the parasite utilizes to establish a persistent infection despite activation of a potent immune response by the host are currently unknown. Unusual characteristics of T. cruzi are that it possesses cellular levels of pyrophosphate (PPi) at least 10 times higher than those of ATP and molar levels of inorganic polyphosphate (polyP) within Acidocalcisomes. We characterized an inorganic soluble EF-hand containing pyrophosphatase from T. cruzi (TcVSP) that, depending on the pH and cofactors, can hydrolyse either pyrophosphate (PPi) or polyphosphate (polyP). The enzyme is localized to both Acidocalcisomes and cytosol. Overexpression of TcVSP (TcVSP-OE) resulted in a significant decrease in cytosolic PPi, and short and long-chain polyP levels. Additionally, the TcVSP-OE parasites showed a significant growth defect in fibroblasts, less responsiveness to hyperosmotic stress, and reduced persistence in tissues of mice, suggesting that PPi and polyP are essential for the parasite to resist the stressful conditions in the host and to maintain a persistent infection.
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target of rapamycin tor like 1 kinase is involved in the control of polyphosphate levels and Acidocalcisome maintenance in trypanosoma brucei
Journal of Biological Chemistry, 2010Co-Authors: Teresa Cristina Leandro De Jesus, Paul N Ulrich, Kildare Miranda, Renata R Tonelli, Sheila Cristina Nardelli, Leonardo Da Silva Augusto, Maria Cristina M Motta, Wendell Girarddias, Veronica Jimenez, Antonio BarquillaAbstract:Abstract Target of rapamycin (TOR) kinases are highly conserved protein kinases that integrate signals from nutrients and growth factors to coordinate cell growth and cell cycle progression. It has been previously described that two TOR kinases control cell growth in the protozoan parasite Trypanosoma brucei, the causative agent of African trypanosomiasis. Here we studied an unusual TOR-like protein named TbTOR-like 1, containing a PDZ domain and found exclusively in kinetoplastids. TbTOR-like 1 localizes to unique cytosolic granules. After hyperosmotic stress the localization of the protein shifts to the cell periphery, differently from other organelle markers. Ablation of TbTOR-like 1 causes a progressive inhibition of cell proliferation, producing parasites accumulating in S/G2 phase of the cell cycle. TbTOR-like 1 knocked down cells have an increased area occupied by acidic vacuoles, known as Acidocalcisomes, and are enriched in polyphosphate and pyrophosphate. These results suggest that TbTOR-like 1 might be involved in the control of Acidocalcisome and polyphosphate metabolism in T. brucei.
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target of rapamycin tor like 1 kinase is involved in the control of polyphosphate levels and Acidocalcisome maintenance in trypanosoma brucei
Journal of Biological Chemistry, 2010Co-Authors: Paul N Ulrich, Kildare Miranda, Teresa Cristina Leandro De Jesus, Renata R Tonelli, Sheila Cristina Nardelli, Maria Cristina M Motta, Wendell Girarddias, Leonardo Augusto, Veronica JimenezAbstract:Target of rapamycin (TOR) kinases are highly conserved protein kinases that integrate signals from nutrients and growth factors to coordinate cell growth and cell cycle progression. It has been previously described that two TOR kinases control cell growth in the protozoan parasite Trypanosoma brucei, the causative agent of African trypanosomiasis. Here we studied an unusual TOR-like protein named TbTOR-like 1 containing a PDZ domain and found exclusively in kinetoplastids. TbTOR-like 1 localizes to unique cytosolic granules. After hyperosmotic stress, the localization of the protein shifts to the cell periphery, different from other organelle markers. Ablation of TbTOR-like 1 causes a progressive inhibition of cell proliferation, producing parasites accumulating in the S/G2 phase of the cell cycle. TbTOR-like 1 knocked down cells have an increased area occupied by acidic vacuoles, known as Acidocalcisomes, and are enriched in polyphosphate and pyrophosphate. These results suggest that TbTOR-like 1 might be involved in the control of Acidocalcisome and polyphosphate metabolism in T. brucei.
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proton pyrophosphatase and polyphosphate in Acidocalcisome like vesicles from oocytes and eggs of periplaneta americana
Insect Biochemistry and Molecular Biology, 2009Co-Authors: Roberto Docampo, Wanderley De Souza, Kildare Miranda, Isabela Ramos, Lucimar S Motta, Fabio M Gomes, Donald E Champagne, Marcelo F Santiago, Ednildo A MachadoAbstract:Abstract Acidocalcisomes are acidic organelles containing large amounts of polyphosphate (poly P), a number of cations, and a variety of cation pumps in their limiting membrane. The vacuolar proton-pyrophosphatase (V-H+-PPase), a unique electrogenic proton-pump that couples pyrophosphate (PPi) hydrolysis to the active transport of protons across membranes, is commonly present in membranes of Acidocalcisomes. In the course of insect oogenesis, a large amount of yolk protein is incorporated by the oocytes and stored in organelles called yolk granules (YGs). During embryogenesis, the content of these granules is degraded by acid hydrolases. These enzymes are activated by the acidification of the YG by a mechanism that is mediated by proton-pumps present in their membranes. In this work, we describe an H+-PPase activity in membrane fractions of oocytes and eggs of the domestic cockroach Periplaneta americana. The enzyme activity was optimum at pH around 7.0, and was dependent on Mg2+ and inhibited by NaF, as well as by IDP and Ca2+. Immunolocalization of the yolk preparation using antibodies against a conserved sequence of V-H+-PPases showed labeling of small vesicles, which also showed the presence of high concentrations of phosphorus, calcium and other elements, as revealed by electron probe X-ray microanalysis. In addition, poly P content was detected in ovaries and eggs and localized inside the yolk granules and the small vesicles. Altogether, our results provide evidence that numerous small vesicles of the eggs of P. americana present Acidocalcisome-like characteristics. In addition, the possible role of these organelles during embryogenesis of this insect is discussed.
Guozhong Huang - One of the best experts on this subject based on the ideXlab platform.
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the Acidocalcisome inositol 1 4 5 trisphosphate receptor of trypanosoma brucei is stimulated by luminal polyphosphate hydrolysis products
Journal of Biological Chemistry, 2019Co-Authors: Evgeniy Potapenko, Nuria Waddington Negrao, Guozhong Huang, Roberto DocampoAbstract:Acidocalcisomes are acidic calcium stores rich in polyphosphate (polyP) and are present in trypanosomes and also in a diverse range of other organisms. Ca2+ is released from these organelles through a channel, inositol 1,4,5-trisphosphate receptor (TbIP3R), which is essential for growth and infectivity of the parasite Trypanosoma brucei However, the mechanism by which TbIP3R controls Ca2+ release is unclear. In this work, we expressed TbIP3R in a chicken B lymphocyte cell line in which the genes for all three vertebrate IP3Rs were stably ablated (DT40-3KO). We show that IP3-mediated Ca2+ release depends on Ca2+ but not on ATP concentration and is inhibited by heparin, caffeine, and 2-aminomethoxydiphenyl borate (2-APB). Excised patch clamp recordings from nuclear membranes of DT40 cells expressing only TbIP3R disclosed that luminal inorganic orthophosphate (Pi) or pyrophosphate (PPi), and neutral or alkaline pH can stimulate IP3-generated currents. In contrast, polyP or acidic pH did not induce these currents, and nuclear membranes obtained from cells expressing rat IP3R were unresponsive to polyP or its hydrolysis products. Our results are consistent with the notion that polyP hydrolysis products within Acidocalcisomes or alkalinization of their luminal pH activate TbIP3R and Ca2+ release. We conclude that TbIP3R is well-adapted to its role as the major Ca2+ release channel of Acidocalcisomes in T. brucei.
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Pyrophosphate Stimulates the Phosphate-Sodium Symporter of Trypanosoma brucei Acidocalcisomes and Saccharomyces cerevisiae Vacuoles
'American Society for Microbiology', 2019Co-Authors: Evgeniy Potapenko, Guozhong Huang, Ciro D. Cordeiro, Roberto DocampoAbstract: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.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
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proteomic analysis of Acidocalcisomes of trypanosoma brucei uncovers their role in phosphate metabolism cation homeostasis and calcium signaling
Communicative & Integrative Biology, 2015Co-Authors: Guozhong Huang, Roberto DocampoAbstract:Trypanosoma brucei, the causative agent of African trypanosomiasis, is a unicellular parasite that possesses lysosome-related organelles known as Acidocalcisomes. These organelles have been found from bacteria to human cells, and are characterized by their acidic nature and high calcium and polyphosphate (polyP) content. Our proteomic analysis of Acidocalcisomes of T. brucei procyclic stages, together with in situ epitope-tagging and immunofluorescence assays with specific antibodies against selected proteins, established the presence of 2 H+ pumps, a vacuolar H+-ATPase and a vacuolar H+-pyrophosphatase, that acidify the organelles as well as of a number of transporters and channels involved in phosphate metabolism, cation uptake and calcium signaling. Together with recent work in other organisms, these results provide direct evidence that Acidocalcisomes are especially adapted to accumulate polyP bound to cations and for calcium signaling.
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proteomic analysis of the Acidocalcisome an organelle conserved from bacteria to human cells
PLOS Pathogens, 2014Co-Authors: Guozhong Huang, Silvia N. J. Moreno, Melissa Storey, Paul N Ulrich, Darryl Johnson, Julie Tischer, Javier A Tovar, Ron Orlando, Roberto DocampoAbstract:Acidocalcisomes are acidic organelles present in a diverse range of organisms from bacteria to human cells. In this study Acidocalcisomes were purified from the model organism Trypanosoma brucei, and their protein composition was determined by mass spectrometry. The results, along with those that we previously reported, show that Acidocalcisomes are rich in pumps and transporters, involved in phosphate and cation homeostasis, and calcium signaling. We validated the Acidocalcisome localization of seven new, putative, Acidocalcisome proteins (phosphate transporter, vacuolar H+-ATPase subunits a and d, vacuolar iron transporter, zinc transporter, polyamine transporter, and acid phosphatase), confirmed the presence of six previously characterized Acidocalcisome proteins, and validated the localization of five novel proteins to different subcellular compartments by expressing them fused to epitope tags in their endogenous loci or by immunofluorescence microscopy with specific antibodies. Knockdown of several newly identified Acidocalcisome proteins by RNA interference (RNAi) revealed that they are essential for the survival of the parasites. These results provide a comprehensive insight into the unique composition of Acidocalcisomes of T. brucei, an important eukaryotic pathogen, and direct evidence that Acidocalcisomes are especially adapted for the accumulation of polyphosphate.
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Proteomic Analysis of the Acidocalcisome, an Organelle Conserved from Bacteria to Human Cells
2014Co-Authors: Guozhong Huang, Silvia N. J. Moreno, Melissa Storey, Paul N Ulrich, Darryl Johnson, Julie Tischer, Javier A Tovar, Ron Orl, Roberto DocampoAbstract:Acidocalcisomes are acidic organelles present in a diverse range of organisms from bacteria to human cells. In this study Acidocalcisomes were purified from the model organism Trypanosoma brucei, and their protein composition was determined by mass spectrometry. The results, along with those that we previously reported, show that Acidocalcisomes are rich in pumps and transporters, involved in phosphate and cation homeostasis, and calcium signaling. We validated the Acidocalcisome localization of seven new, putative, Acidocalcisome proteins (phosphate transporter, vacuolar H+-ATPase subunits a and d, vacuolar iron transporter, zinc transporter, polyamine transporter, and acid phosphatase), confirmed the presence of six previously characterized Acidocalcisome proteins, and validated the localization of five novel proteins to different subcellular compartments by expressing them fused to epitope tags in their endogenous loci or by immunofluorescence microscopy with specific antibodies. Knockdown of several newly identified Acidocalcisome proteins by RNA interference (RNAi) revealed that they are essential for the survival of the parasites. These results provide a comprehensive insight into the unique composition of Acidocalcisomes of T. brucei, an important eukaryotic pathogen, an
