A Pyrophosphatase Regulating Polyphosphate Metabolism in Acidocalcisomes Is Essential for Trypanosoma brucei Virulence in Mice
2004; Elsevier BV; Volume: 279; Issue: 5 Linguagem: Inglês
10.1074/jbc.m309974200
ISSN1083-351X
AutoresGuillaume Lemercier, Benoît Espiau, Félix A. Ruiz, Maurício Vieira, Shuhong Luo, Théo Baltz, Roberto Docampo, Norbert Bakalara,
Tópico(s)Lysosomal Storage Disorders Research
ResumoWe report the functional characterization of a soluble pyrophosphatase (TbVSP1), which localizes to acidocalcisomes, a vesicular acidic compartment of Trypanosoma brucei. Depending on the pH and the cofactors Mg2+ or Zn2+, both present in the compartment, the enzyme hydrolyzes either inorganic pyrophosphate (PPi) (kcat = 385 s–1) or tripolyP (polyP3) and polyphosphate (polyP) of 28 residues (polyP28) with kcat values of 52 and 3.5 s–1, respectively. An unusual N-terminal domain of 160 amino acids, containing a putative calcium EF-hand-binding domain, is involved in protein oligomerization. Using double-stranded RNA interference methodology, we produced an inducible bloodstream form (BF) deficient in the TbVSP1 protein (BFiVSP1). The long-chain polyP levels of these mutants were reduced by 60%. Their phenotypes revealed a deficient polyP metabolism, as indicated by their defective response to phosphate starvation and hyposmotic stress. BFiVSP1 did not cause acute virulent infection in mice, demonstrating that TbVSP1 is essential for growth of bloodstream forms in the mammalian host. We report the functional characterization of a soluble pyrophosphatase (TbVSP1), which localizes to acidocalcisomes, a vesicular acidic compartment of Trypanosoma brucei. Depending on the pH and the cofactors Mg2+ or Zn2+, both present in the compartment, the enzyme hydrolyzes either inorganic pyrophosphate (PPi) (kcat = 385 s–1) or tripolyP (polyP3) and polyphosphate (polyP) of 28 residues (polyP28) with kcat values of 52 and 3.5 s–1, respectively. An unusual N-terminal domain of 160 amino acids, containing a putative calcium EF-hand-binding domain, is involved in protein oligomerization. Using double-stranded RNA interference methodology, we produced an inducible bloodstream form (BF) deficient in the TbVSP1 protein (BFiVSP1). The long-chain polyP levels of these mutants were reduced by 60%. Their phenotypes revealed a deficient polyP metabolism, as indicated by their defective response to phosphate starvation and hyposmotic stress. BFiVSP1 did not cause acute virulent infection in mice, demonstrating that TbVSP1 is essential for growth of bloodstream forms in the mammalian host. Inorganic polyphosphate (polyP) 1The abbreviations used are: polyPpolyphosphatepolyP3tripolyPpolyP28polyP of 28 residuesPPasepyrophosphatasePFprocyclic formsBFbloodstream formsPBSphosphate-buffered salineMES4-morpholineethanesulfonic acidV-H+-ATPasevacuolar H+-ATPaseH+-PPasevacuolar H+-PPase.1The abbreviations used are: polyPpolyphosphatepolyP3tripolyPpolyP28polyP of 28 residuesPPasepyrophosphatasePFprocyclic formsBFbloodstream formsPBSphosphate-buffered salineMES4-morpholineethanesulfonic acidV-H+-ATPasevacuolar H+-ATPaseH+-PPasevacuolar H+-PPase. is a ubiquitous molecule formed by phosphate (Pi) residues linked by high energy phosphoanhydride bonds. Although polyP had been dismissed as a molecular fossil (1Kornberg A. J. Bacteriol. 1995; 177: 491-496Google Scholar), the recent introduction of novel quantitative enzymatic analytical methods (2Kornberg A. Rao N.N. Ault-Riche D. Annu. Rev. Biochem. 1999; 68: 89-125Google Scholar) has permitted reexamination of its biological functions. A study of the compartmentalization of polyP performed on various eukaryotic microorganisms has shown that each compartment contains its own type of polyP (3Vagabov V.M. Trilisenko L.V. Kulaev I.S. Biochemistry (Mosc.). 2000; 65: 349-354Google Scholar), and a correlation has been established between the basic biological processes occurring in each compartment and the structure and metabolism of polyP (3Vagabov V.M. Trilisenko L.V. Kulaev I.S. Biochemistry (Mosc.). 2000; 65: 349-354Google Scholar). Among the enzymes involved in the synthesis and utilization of polyP, exopolyphosphatases were considered as the central regulatory enzymes of its metabolism (4Kulaev I.S. Vagabov V.M. Kulakovskaya T.V. Lichko L.P. Andreeva N.A. Trilisenko L.V. Biochemistry (Mosc.). 2000; 65: 271-278Google Scholar). In yeast, polyphosphatases differ from each other in their molecular masses, substrate specificity, and requirement for divalent cations (4Kulaev I.S. Vagabov V.M. Kulakovskaya T.V. Lichko L.P. Andreeva N.A. Trilisenko L.V. Biochemistry (Mosc.). 2000; 65: 271-278Google Scholar). However, only one gene encoding an exopolyphosphatase has been identified so far (5Wurst H. Shiba T. Kornberg A. J. Bacteriol. 1995; 177: 898-906Google Scholar). In eukaryotic organisms, genetic approaches have uncovered key roles for an endopolyphosphatase and an exopolyphophatase in polyP metabolism (6Sethuraman A. Rao N.N. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8542-8547Google Scholar). polyphosphate tripolyP polyP of 28 residues pyrophosphatase procyclic forms bloodstream forms phosphate-buffered saline 4-morpholineethanesulfonic acid vacuolar H+-ATPase vacuolar H+-PPase. polyphosphate tripolyP polyP of 28 residues pyrophosphatase procyclic forms bloodstream forms phosphate-buffered saline 4-morpholineethanesulfonic acid vacuolar H+-ATPase vacuolar H+-PPase. In trypanosomatids and many other protozoa causing human disease, an acidic electron-dense compartment has been shown to contain the major part of the cellular polyP. This compartment, initially named the acidocalcisome in T. brucei, was shown to be implicated in storage of cations and phosphate (7Docampo R. Moreno S.N. Parasitol. Today. 1999; 15: 443-448Google Scholar), regulation of intracellular pH (8LeFurgey A. Ingram P. Blum J.J. Mol. Biochem. Parasitol. 1990; 40: 77-86Google Scholar, 9Lemercier G. Dutoya S. Luo S. Ruiz F.A. Rodrigues C.O. Baltz T. Docampo R. Bakalara N. J. Biol. Chem. 2002; 277: 37369-37376Google Scholar), and osmoregulation (10LeFurgey A. Ingram P. Blum J.J. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 2001; 128: 385-394Google Scholar, 11Rohloff P. Rodrigues C.O. Docampo R. Mol. Biochem. Parasitol. 2003; 126: 219-230Google Scholar). To better understand the relation between polyP metabolism and its cellular role, studies were performed on the polyP composition and on the characterization of enzymes involved in the metabolism of these compounds. Short- and long-chain polyP and PPi were shown to be present in the acidocalcisomes of T. brucei bloodstream forms (BF) (12Moreno B. Urbina J.A. Oldfield E. Bailey B.N. Rodrigues C.O. Docampo R. J. Biol. Chem. 2000; 275: 28356-28362Google Scholar, 13Moreno B. Rodrigues C.O. Bailey B.N. Urbina J.A. Moreno S.N. Docampo R. Oldfield E. FEBS Lett. 2002; 523: 207-212Google Scholar). The turnover of the acidocalcisome PPi pool is low (14Urbina J.A. Moreno B. Vierkotter S. Oldfield E. Payares G. Sanoja C. Bailey B.N. Yan W. Scott D.A. Moreno S.N. Docampo R. J. Biol. Chem. 1999; 274: 33609-33615Google Scholar), suggesting that PPi could be a product of polyP metabolism. In this work, we report the identification in acidocalcisomes of a novel type I soluble pyrophosphatase (TbVSP1). According to the divalent metal cofactor used, Mg2+ or Zn2+, TbVSP1 specifically hydrolyzes either PPi or short-chain polyP. TbVSP1 was also shown to play a central role in the regulation of polyP metabolism in acidocalcisomes and to be essential for osmoregulation and virulence in mice. Strains Used—T. brucei brucei bloodstream form host cell line 90-13, co-expressing the T7 RNA polymerase and the Tet repressor, was a gift from G. A. M. Cross (15Wirtz E. Leal S. Ochatt C. Cross G.A. Mol. Biochem. Parasitol. 1999; 99: 89-101Google Scholar). Cells were cultured in vitro in modified essential medium supplemented with 10% fetal calf serum (16Baltz T. Baltz D. Giroud C. Crockett J. EMBO J. 1985; 4: 1273-1277Google Scholar). T. brucei strain 427 procyclic forms (PF) were cultured in SDM-79 medium (17Brun R. Jenni L. Tanner M. Schonenberger M. Schell K.F. Acta Trop. 1979; 36: 387-390Google Scholar) supplemented with 10% fetal calf serum. Cloning of TbVSP1—To screen for genes encoding soluble pyrophosphatases (PPases) in T. brucei, the amino acid sequence of the yeast PPase (accession number 2781300) was used to search all available sequence databases using TBLASTN. This search yielded a 0.535-kb gene fragment (accession number AL496002), which closely resembled the region encompassed by nucleotides encoding amino acid residues 42–215 of the yeast PPase. In silico chromosome walking revealed two overlapping sequences (AQ951034 and AZ216908) encoding the N-terminal extremity of the T. brucei PPase. A genomic DNA fragment, PCR-amplified with the following set of primers, PPasePF1Nter (5′-GGGCGGCATATGAACAACACACACGGT-3′) and PPasePF1Cter (5′-GGCCGGCTCGAGCTTCTCAAATGT-3′), was used as a probe to screen a T. brucei cosmid library (18Sambrook J. Fritsh E. Maniatis T. Ford N. Nolan C. Ferguson M. Molecular Cloning: A Laboratory Manual. 2nd Ed. vols. 3. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York1989Google Scholar), generated into the c2X75 cosmid vector (19Campbell D.A. Nucleic Acids Res. 1989; 17: 458Google Scholar) as described previously (20Bates P. Swift R. Gene (Amst.). 1983; 26: 137-146Google Scholar). Hinc2 fragments of the isolated cosmids were subcloned into the pUC18 vector from Appligene and screened with the genomic DNA fragment PPasePF1Nter/PPase-PF1Cter. An Hinc2 fragment of 4000 bp was isolated and sequenced using the AmpliTaq DNA polymerase, as described by the manufacturer (ABI PRISM®, PerkinElmer Life Sciences). The complete gene sequence of the T. brucei PPase was obtained. Cloning, Expression, and Purification of the N-terminal Domain of TbVSP1 (N-TbVSP1)—A 405-bp fragment comprising the N-terminal domain was generated by PCR. The 5′ primer PPasePF1Nter contained an 18-nucleotide linker with a NdeI restriction site to facilitate subcloning and 5′ adjacent N-terminal residues. The 3′ primer, PPasePF1Cter included an 18-nucleotide linker with a XhoI site for cloning. The PCR product was inserted into the XhoI/NdeI sites of the pET23a plasmid (Novagen). The resulting recombinant protein was expressed in Escherichia coli BL21 (DE3) from Novagen according to the manufacturer's instructions. Cells were lysed, and the recombinant protein was purified as described previously (21Bakalara N. Santarelli X. Davis C. Baltz T. J. Biol. Chem. 2000; 275: 8863-8871Google Scholar). Cloning, Expression, and Purification of the rec-TbVSP1 and ΔN-TbVSP1—Genomic DNA fragments were PCR-amplified with the following set of primers: PPasePF1Nter (5′-GGGCGGCATATGAACAACACACACGGT-3′) and PPasePF2Nter (5′-GGCCGGCATATGCCGTATCACCAGCAG-3′) as the 5′ primer for the rec-TbVSP1 and ΔN-TbVSP1, respectively, and the PPasePF2Cter (5′-CCGGCCCTCGAGCAGTTCTCCCATCTTGTT-3′) as the 3′ primer for both products. The fragments were cloned into the XhoI/NdeI sites of the pET23a plasmid (Novagen). Purification of the Recombinant N-TbVSP1, rec-TbVSP1 and ΔN-Tb-VSP1, Immobilized Metal Affinity Chromatography—The recombinant proteins expressed in E. coli were purified from the His·Bind® column from Amersham Biosciences used according to the manufacturer's instructions (Novagen). Before performing the PPase assays, the enzyme was desalted in TrisDES buffer (50 mm Tris-HCl, pH 7.5, and 1 mm MgCl2) on a PD10 column (Amersham Biosciences). The exclusion chromatography of the rec-TbVSP1 and ΔN-TbVSP1 proteins was performed in a buffer containing 50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 5 mm MgCl2 using a Superdex 200 HR column from Amersham Biosciences. Determination of Protein Concentration—Protein concentration was estimated by the method of Bradford (22Bradford M.M. Anal. Biochem. 1976; 72: 248-254Google Scholar) and by Coomassie Blue staining after SDS-PAGE. Bovine serum albumin was used as standard. Production and Western Blot Analysis of Immune Serum against the Recombinant N-TbVSP1—Rabbits were first injected with 80 μg of the N-TbVSP1 in complete Freund's adjuvant and with another 80 μg in incomplete Freund's adjuvant 15 and 30 days later. Blood was collected before the first injection (preimmune serum) and 10, 13, and 15 days following the last injection. Western blotting was performed as described (23Dutoya S. Gibert S. Lemercier G. Santarelli X. Baltz D. Baltz T. Bakalara N. J. Biol. Chem. 2001; 276: 49117-49124Google Scholar), with serum diluted 1200 and incubated overnight at 4 °C. The secondary antibody, a 15000 dilution of goat anti-mouse IgG conjugated to horseradish peroxidase (Sigma), was incubated for 2 h in PBS-Tween-milk. Immunoreactive bands were revealed by washing in 50 mm Tris-HCl, pH 7.5, 20 mm NaCl, and revealed either by 0.5 mg/ml 3,3′-diaminobenzidine or by the ECL™ revelation kit (Amersham Biosciences). PPase and Exopolyphosphatase Activity—0.020–2 μg of desalted recombinant PPase was incubated for 1–5 min at 25 °C in a buffer containing 50 mm Tris-HCl, pH 7.5, 1 mm MgCl2, or 1 mm ZnCl2 and the indicated concentration of sodium PPi, polyP3, or polyP28. The reaction was stopped, and released Pi was quantified as described by Ref. 24Shatton J.B. Ward C. Williams A. Weinhouse S. Anal. Biochem. 1983; 130: 114-119Google Scholar. Acetate buffer was used from pH 4.5 to 5.0, MES buffer from 5.5 to 6.5, Tris-HCl from 7.0 to 8.8, and glycine NaOH at pH 9.5. Before use, polyP28 was purified on G25 Sephadex. TbVSP1 and Ca2+ kinetic constants were determined by preincubation of the enzyme with 100 μm CaCl2. The enzyme was then diluted 100 times in the reaction buffer. Because polyP3 hydrolysis produces Pi and PPi, which could be hydrolyzed to Pi, we used the equation developed for an enzyme with two substrates, as published by Zyryanov et al. (25Zyryanov A.B. Shestakov A.S. Lahti R. Baykov A.A. Biochem. J. 2002; 367: 901-906Google Scholar), to calculate the Michaelis constants. Expression of the Proteolipidic Subunit of the Vacuolar H+-ATPase (V-H+-ATPase) in T. brucei PF—The gene was amplified from AnTat genomic DNA with the two following primers: SC2 (5′-CTATATTTCGAAATGCTAAGTGACGATACCTGTCAAC-3′) and SC1 (5′-CTCCGTGGATCCTTAGTGATGGTGATGGTGATGAGAGCAACCACCCGTATAGGA-3′) designed from the T. brucei V-H+-ATPase proteolipidic subunit and cloned in the BamH1/HindIII restriction sites of the pLEW100 vector. Cells were then transfected with that construction and selected as described previously (23Dutoya S. Gibert S. Lemercier G. Santarelli X. Baltz D. Baltz T. Bakalara N. J. Biol. Chem. 2001; 276: 49117-49124Google Scholar). Electron Microscopy—For immunocytochemistry, procyclic trypomastigotes were washed with Dulbecco's PBS, fixed for 1 h in a solution containing 0.1% grade I glutaraldehyde, 4% freshly prepared formaldehyde, 0.8% picric acid, in 0.1 m cacodylate buffer, pH 7.2. Fixed parasites were washed with Dulbecco's PBS and dehydrated by successive incubations of 6 min with increasing concentrations of ethanol (10, 25, 50, 75, 95, and 100%) at –20 °C. Samples were embedded in Unicryl at 4 °C by incubation with 1:1 ethanol/Unicryl for 1 h and 100% Unicryl for 1, 16, and 8 h. Embedded samples were polymerized under UV irradiation at –20 °C for 48 h. Thin sections were collected on 300 mesh nickel grids and blocked for 60 min with PBS containing 0.1% Tween 20 and 0.5% cold fish gelatin (PBS-TW-FG). Grids were incubated for 3 h with a mix of a rabbit polyclonal antibody against TbVSP1 (1:50) and a mouse anti-His monoclonal antibody (1:50), diluted in PBS-TW-FG. After washing in PBS-TW-FG, grids were incubated for 1 h with a mixture of a 10-nm gold-conjugated goat anti-rabbit antibody (1:75) and a 20-nm gold-conjugated goat anti-mouse antibody (1:20), diluted in PBS-TW-FG. After incubations, grids were washed with 5 ml of PBS and with 5 ml of distilled water before being stained with uranyl acetate and lead citrate. Routine and immunocytochemistry samples were observed in a Hitachi H 600 electron microscope. Double-stranded RNA Expression and Trypanosome Transfection— The inducible T7 RNA polymerase-based protein expression system developed by Wirtz et al. (15Wirtz E. Leal S. Ochatt C. Cross G.A. Mol. Biochem. Parasitol. 1999; 99: 89-101Google Scholar) (kindly provided by G. A. M. Cross) and the p2T7 vector (kindly provided by D. LaCount) (26LaCount D.J. Bruse S. Hill K.L. Donelson J.E. Mol. Biochem. Parasitol. 2000; 111: 67-76Google Scholar) were used in this study. DNA fragments corresponding to the coding regions of TbVSP1 from nucleotides 1–462 were PCR-amplified. The 462-bp fragment was amplified using the following set of primers: DB/PP1 (5′-CGGGGCAAGCTTATGAACAACACACACGG-3′) and DB/PP2 (5′-GCCCCGGAATTCCATCGAGAAACATTTCG-3′). After precloning in the pGEMt vector (Promega), the resulting fragment was cloned between the HindIII/SacII restriction sites of p2T7 generating the p2T7PPase construction. For stable transfection of bloodstream forms, cells were harvested from a log phase culture, washed once in ZPFMG (ZPFM (Zimmerman Post Fusion Medium) and 55 mm glucose), and resuspended in ZPMG (containing 2 mm ATP and 5 mm glutathione) at a density of 2 × 107 cells/ml (27Wirtz E. Hoek M. Cross G.A. Nucleic Acids Res. 1998; 26: 4626-4634Google Scholar, 28van den Hoff M.J. Moorman A.F. Lamers W.H. Nucleic Acids Res. 1992; 20: 2902Google Scholar). 2 × 107 cells were electroporated with 10 μg of DNA at 1600 V, infinite resistance, 40 microfarads using the Eurogentech cellject machine. Surviving cells were then seeded in a microtiter plate as described by Wirtz et al. (27Wirtz E. Hoek M. Cross G.A. Nucleic Acids Res. 1998; 26: 4626-4634Google Scholar), and selection was applied the following day by adding phleomycin to a final concentration of 0.5 μg/ml. The genetically modified strain was named BFiTbVSP1 and induced for 4 days with 1 μg/ml of tetracycline. Several clones with identical phenotypes were obtained. Analysis of PolyP—PolyP levels were determined as described by Ruiz et al. (29Ruiz F.A. Rodriguez C.O. Docampo R. J. Biol. Chem. 2001; 22: 22Google Scholar) using the recombinant exopolyphosphatase (rPPX1) from Saccharomyces cerevisiae. BFiVSP1 bloodstream forms were induced or not with 1 μg/ml tetracycline during 4 days. Then, 2 × 107cells were washed twice with Dulbecco's PBS, and long- and short-chain polyP were quantified. Gene Cloning, Classification, and Sequence Analysis of T. brucei VSP1—The nucleotide sequence revealed an open reading frame of 1245 bp, which encodes a protein of 415 amino acids with a relative molecular mass of 47.3 kDa and a calculated pI of 5.79 (Fig. 1). A Southern blot analysis indicated the presence of a muticopy gene family (data not shown). While this manuscript was in preparation, a search in the T. brucei genome project databases (www.genedb.org) using the TbVSP1 coding sequence indicated the existence on chromosome X and XI of at least three copies of the TbVSP1. The sequence on chromosome X encodes for the pyrophosphatase we worked with. Sequences on chromosomes XI are isoforms. One sequence diverged on 11 amino acids (Fig. 1) (V52F,L55S; H56L,K362E; N373S,R378H; M380L; K381R; S434A; L442R, and M447R. Positions are given according to Fig. 1), and another one diverged on 6 of these 11 amino acids (K362E; N373S; R378H; M380L; K381R; and M447R). Sequence comparison analysis (www.ncbi.nlm.nih.gov/BLAST) revealed that the TbVSP1 belongs to the family I PPase. The 17 polar amino acids present in the active site of yeast PPase (30Sivula T. Salminen A. Parfenyev A.N. Pohjanjoki P. Goldman A. Cooperman B.S. Baykov A.A. Lahti R. FEBS Lett. 1999; 454: 75-80Google Scholar) are conserved in the TbVSP1 protein (Fig. 1). With respect to size and primary structure, type I PPases have been divided into three families (30Sivula T. Salminen A. Parfenyev A.N. Pohjanjoki P. Goldman A. Cooperman B.S. Baykov A.A. Lahti R. FEBS Lett. 1999; 454: 75-80Google Scholar). The best identity was obtained with the animal/fungal (Ic) subfamily (Fig. 1). In particular, the TbVSP1 contains three of the four insertions characteristic of the animal/fungal subfamily (Fig. 1). In addition, the TbVSP1 has a large N-terminal extension domain of 160 amino acids (Fig. 1). A search of conserved domains performed by INTERPRO (www.ebi.ac.uk/interpro/scan.html) revealed the presence of a calcium-binding type II EF-hand domain (IPR002048) in this N-terminal region. This analysis suggested that the TbVSP1 might possess specific functions or a particular location. Therefore, in a first step toward the characterization of the TbVSP1, we analyzed its expression and its subcellular localization. Expression and Localization of TbVSP1—Immunoblotting with a specific rabbit antiserum directed against the N-terminal domain (amino acids 1–160) of TbVSP1 indicated that expression levels by the mammalian host bloodstream and the insect procyclic forms were similar (Fig. 2A). To determine the localization of the TbVSP1, immunoelectron microscopy was performed on thin sections of PF parasites using the rabbit anti-N-TbVSP1 antiserum. The results obtained (Fig. 2B) indicated that TbVSP1 was located in large vesicles containing electron-dense material, a characteristic feature of acidocalcisomes (7Docampo R. Moreno S.N. Parasitol. Today. 1999; 15: 443-448Google Scholar). Co-localization studies were performed on procyclic forms expressing a His tag subunit c of the vacuolar H+-ATPase using an anti-His monoclonal antibody (Fig. 2B). This co-localization confirmed the presence of TbVSP1 protein in acidocalcisomes (31Vercesi A.E. Moreno S.N. Docampo R. Biochem. J. 1994; 304: 227-233Google Scholar, 32Rodrigues C.O. Scott D.A. Docampo R. Mol. Cell. Biol. 1999; 19: 7712-7723Google Scholar). No reaction was detected when the primary antibodies were omitted (data not shown). To further analyze the subcellular localization of TbVSP1, we prepared acidocalcisomes from procyclic forms from a high density Percoll fraction of trypanosomes as described previously (9Lemercier G. Dutoya S. Luo S. Ruiz F.A. Rodrigues C.O. Baltz T. Docampo R. Bakalara N. J. Biol. Chem. 2002; 277: 37369-37376Google Scholar). TbVSP1 was detected by Western blots in this high density fraction (Fig. 2C), which was shown to contain the acidocalcisomal protein marker V-H+-PPase (9Lemercier G. Dutoya S. Luo S. Ruiz F.A. Rodrigues C.O. Baltz T. Docampo R. Bakalara N. J. Biol. Chem. 2002; 277: 37369-37376Google Scholar). We estimated at around 10-fold the enrichment of the acidocalcisome fraction in TbVSP1. Although some membrane-associated PPases have been shown to localize in mitochondria (33Lundin M. Baltscheffsky H. Ronne H. J. Biol. Chem. 1991; 266: 12168-12172Google Scholar) and chloroplast thylakoid (34Jiang S.S. Fan L.L. Yang S.J. Kuo S.Y. Pan R.L. Arch. Biochem. Biophys. 1997; 346: 105-112Google Scholar) previously, none has been localized to a vacuolar compartment before. Substrate Specificity of TbVSP1—In the presence of Mg2+, PPases display nearly absolute PPi specificity. However, this specificity is lost when transition metal ions such as Zn2+, Mn2+, or Co2+ are used as cofactors (25Zyryanov A.B. Shestakov A.S. Lahti R. Baykov A.A. Biochem. J. 2002; 367: 901-906Google Scholar). Because acidocalcisomes contain high levels of Mg2+, Zn2, and Ca2+ (35Docampo R. Moreno S.N. Mol. Biochem. Parasitol. 2001; 114: 151-159Google Scholar), we determined the Michaelis-Menten constants of the TbVSP1 for the three substrates polyP28, polyP3, and PPi, in the presence of Mg2+, Zn2+, and Ca2+. With 1 mm Mg2+, TbVSP1 specifically hydolyzed PPi with an optimum pH of 7.5 and a kcat value of 385 s–1 (Fig. 3A). The replacement of Mg2+ for Zn2+ in the reaction buffer resulted in the hydrolysis of polyP3 and polyP28, in addition to PPi. However, the optimum pH for hydrolysis shifted to a more acidic pH of 6.5 with kcat values of 52, 3.5, and 56 s–1, respectively (Fig. 3, B and C). This effect of specificity change was discussed previously by Zyryanov et al. (25Zyryanov A.B. Shestakov A.S. Lahti R. Baykov A.A. Biochem. J. 2002; 367: 901-906Google Scholar) and attributed to the fact that the Zn2+ transition metal ion allowed a more favorable positioning of the terminal phosphate group for catalysis (25Zyryanov A.B. Shestakov A.S. Lahti R. Baykov A.A. Biochem. J. 2002; 367: 901-906Google Scholar). Ca2+-PPi, conversely, is an efficient competitive inhibitor of Mg2+-PPi hydrolysis. TbVSP1 was inhibited by Ca2+ with an inhibition constant value (Ki) of 14 μm, calculated for PPi hydrolysis in the presence of 1 mm MgCl2. In contrast, even when used at 200 μm, Ca2+ did not inhibit polyP3 and polyP28 hydrolysis. In conclusion, TbVSP1 substrate specificity in acidocalcisomes is regulated in a complex manner by the pH and Ca2+, Zn2+, and Mg2+ content of the compartment. Role of the N-terminal Extension in the Oligomerization and Enzymatic Activity of TbVSP1—The sequence that directs proteins to acidocalcisomes has not been identified. To analyze the potential implication of the N-terminal domain for acidocalcisome protein targeting, we constructed a chimeric protein containing this N-terminal domain and the green fluorescent protein. The resulting fusion protein expressed in T. brucei procyclic forms was not addressed to acidocalcisomes (data not shown). However, the main roles attributed to the EF-hand domain are the regulation of cellular activity, buffering/transporting Ca2+, or anchoring protein complexes (36Davis J.P. Rall J.A. Reiser P.J. Smillie L.B. Tikunova S.B. J. Biol. Chem. 2002; 277: 49716-49726Google Scholar, 37Lewit-Bentley A. Rety S. Curr. Opin. Struct. Biol. 2000; 10: 637-643Google Scholar). Therefore, to further investigate the role of the N-terminal extension, we deleted it from a mutant protein (ΔN-TbVSP1). The resulting kinetic constants of ΔN-TbVSP1 for PPi hydrolysis and the Ca2+ inhibition constant did not vary (13 μm), suggesting that the EF-hand domain of this N-terminal region was not implicated in PPi hydrolysis or Ca2+ regulation. Nevertheless, whereas size analysis performed by exclusion chromatography indicated that ΔN-TbVSP1 principally existed as a dimer (Fig. 4), TbVSP1, in the presence of 5 mm MgCl2, was shown to mainly exist as an hexamer. Because of the type II Ca2+ EF-hand domain, we tested the effect of Ca2+ on the enzyme oligomerization state. Exclusion chromatography showed that addition of 100 μm CaCl2 favored the formation of large complexes of around 600,000 Da (Fig. 4.). Large complexes did not show different kinetic constants for PPi (kcat = 324 s–1), polyP3 (kcat = 33.5 s–1), and polyP28 (kcat = 3.7 s–1). In conclusion, Ca2+ could regulate the oligomerization state of the enzyme by its binding to the N-terminal region. This oligomerization could be a mechanism for association of TbVSP1 with the membrane compartment (38Godsel L.M. Engman D.M. EMBO J. 1999; 18: 2057-2065Google Scholar) in which polyP is present (7Docampo R. Moreno S.N. Parasitol. Today. 1999; 15: 443-448Google Scholar). Implication of the TbVSP1 in the Regulation of PolyP Metabolism in Acidocalcisomes—We used RNA interference to produce bloodstream TbVSP1 mutants (BFiVSP1). After 4 days of double-stranded RNA expression, TbVSP1 protein completely disappeared (Fig. 5A). Concomitantly, the level of short- and long-chain polyP greatly decreased. In particular, the steady state level of long-chain polyP underwent a 60% reduction (Fig. 5B). Moreno et al. (12Moreno B. Urbina J.A. Oldfield E. Bailey B.N. Rodrigues C.O. Docampo R. J. Biol. Chem. 2000; 275: 28356-28362Google Scholar) have reported that short-chain polyP of an average chain length of 3.3 phosphates and PPi are the most abundant phosphorus-containing compounds in acidocalcisomes. The short-chain polyP concentration in T. brucei PF has been previously reported to be around 50 mm (9Lemercier G. Dutoya S. Luo S. Ruiz F.A. Rodrigues C.O. Baltz T. Docampo R. Bakalara N. J. Biol. Chem. 2002; 277: 37369-37376Google Scholar). Fig. 5B shows that the concentration in BF is around 600 μm. The respective Km values for PPi and polyP3 hydrolysis in the presence of Mg2+ and Zn2+ were estimated as 19 and 373 μm (Fig. 3). Interestingly, the Km for polyP28, which is present in a much lower concentration in acidocalcisomes, was estimated as 14 μm (Fig. 3). Moreover, Km values for the same substrates and cofactors calculated for the hexameric and larger complexes were similar. Therefore, according to the Km values of TbVSP1, it appears that the TbVSP1 would work at Vmax to produce Pi from PPi and short-chain polyP. However, before it could hydrolyze PPi, TbVSP1 would require a pH increase and a calcium concentration decrease in acidocalcisomes. This conclusion suggests a central role for the enzyme in the regulation of polyP metabolism. In S. cerevisiae, polyP is hydrolyzed through the combined action of an endopolyphosphatase and an exopolyphosphatase (39Kumble K.D. Kornberg A. J. Biol. Chem. 1995; 270: 5818-5822Google Scholar), which hydrolyze long- and short-chain polyP to polyP3 and PPi. In Leishmania major (40Rodrigues C.O. Ruiz F.A. Vieira M. Hill J.E. Docampo R. J. Biol. Chem. 2002; 277: 50899-50906Google Scholar) and in T. brucei (41Lemercier G. Bakalara N. Santarelli X. J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci. 2003; 786: 305-309Google Scholar), genes encoding for exopolyphosphatases have been sequenced, but in T. brucei, we do not have evidence for its location in acidocalcisomes. This exopolyphosphatase is active at pH 7.5 and is inhibited by CaCl2 (40Rodrigues C.O. Ruiz F.A. Vieira M. Hill J.E. Docampo R. J. Biol. Chem. 2002; 277: 50899-50906Google Scholar, 41Lemercier G. Bakalara N. Santarelli X. J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci. 2003; 786: 305-309Google Scholar). To avoid polyP3 accumulation in the cell compartment where polyP hydrolysis is occurring and to produce Pi, polyP3 should be further hydrolyzed. We hypothesize that polyP3 at acidic pH in the presence of Zn2+ is hydrolyzed by the TbVSP1 and that, following compartment alkalinization and the release of H+ and Ca2+, the exopolyphosphatase and TbVSP1 could drive the reaction by producing Pi directly from polyP3 and PPi within acidocalcisomes. The produced Pi could be either exported or further utilized for polyP synthesis. Acidocalcisomes have been shown to be involved in several biological processes after partial alkalinization (9Lemercier G. Dutoya S. Luo S. Ruiz F.A. Rodrigues C.O. Baltz T. Docampo R. Bakalara N. J. Biol. Chem. 2002; 277: 37369-37376Google Scholar, 29Ruiz F.A. Rodriguez C.O. Docampo R. J. Biol. Chem. 2001; 22: 22Google Scholar). Alkalinization creates environmental conditions that activate TbVSP1 and the previously reported exopolyphosphatase activity (9Lemercier G. Dutoya S. Luo S. Ruiz F.A. Rodrigues C.O. Baltz T. Docampo R. Bakalara N. J. Biol. Chem. 2002; 277: 37369-37376Google Scholar, 29Ruiz F.A. Rodriguez C.O. Docampo R. J. Biol. Chem. 2001; 22: 22Google Scholar, 32Rodrigues C.O. Scott D.A. Docampo R. Mol. Cell. Biol. 1999; 19: 7712-7723Google Scholar, 40Rodrigues C.O. Ruiz F.A. Vieira M. Hill J.E. Docampo R. J. Biol. Chem. 2002; 277: 50899-50906Google Scholar). Accordingly, TbVSP1 might be essential for polyP hydrolysis to occur during these processes. We therefore tested the BF mutant cells under different physiological conditions that have been shown to require polyP hydrolysis. Phenotypic Analysis of TbVSP1 BF Mutants (BFiVSP1), pH and Ca2+Homeostasis and Survival in a Deprived Phosphate Medium and under Osmotic Stress—In contrast to what we reported previously for V-H+-PPase mutated parasites (9Lemercier G. Dutoya S. Luo S. Ruiz F.A. Rodrigues C.O. Baltz T. Docampo R. Bakalara N. J. Biol. Chem. 2002; 277: 37369-37376Google Scholar), BFiVSP1 recovered as well as wild type cells from pH stresses (acidic or basic, data not shown). Moreover, Ca2+ release induced by nigericin, NH4Cl, or ionomycin was not altered in the induced BFiVSP1 (data not shown), whereas that amount was significantly reduced in the V-H+-PPase mutants (9Lemercier G. Dutoya S. Luo S. Ruiz F.A. Rodrigues C.O. Baltz T. Docampo R. Bakalara N. J. Biol. Chem. 2002; 277: 37369-37376Google Scholar). Because the polyP content was reduced by 90% in V-H+-PPase mutants, we suggest that the remaining polyP in the BFiVSP1 mutants was sufficient for these homeostatic mechanisms to take place. It has been established that polyP serves as a Pi reservoir for cellular metabolism and growth. We showed that neither induced nor non-induced BFiVSP1 mutant cells failed to overcome the effects of an incubation in a phosphate-depleted medium (data not shown). However, after 45 min of incubation in low PO4-containing medium, the percent of cell survival is about 30% for non-induced BFiVSP1 parasites and 1% for those induced (Fig. 6A). This observation indicated that induced parasites were more sensitive to a low PO4-containing medium. Ruiz et al. (29Ruiz F.A. Rodriguez C.O. Docampo R. J. Biol. Chem. 2001; 22: 22Google Scholar) have demonstrated that polyP hydrolysis was induced by hyposmotic stress. LeFurgey et al. (10LeFurgey A. Ingram P. Blum J.J. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 2001; 128: 385-394Google Scholar) also confirmed that acidocalcisomes play a physiological role in osmoregulation. Induced BFiVSP1 forms were therefore tested for growth under hyposmotic growth conditions. Induced mutated cells did not develop under hyposmotic stress, whereas non-induced cells started to develop normally after 30 h (Fig. 6B). As a control, we found that the addition of NaCl to a final concentration of 120 mm (isoosmotic conditions) was required for normal growth of induced BFiVSP1. This cellular response is not immediate since we noted a 20-h latent period before growth of the non-induced BFiVSP1. In conclusion, these results suggested that the absence of TbVSP1 largely reduced the ability of acidocalcisomes to hydrolyze polyP and recover from hyposmotic stress. TbVSP1 Is Essential for T. brucei Virulence in Mice—First, we examined whether induced BFiVSP1 grew normally in isoosmotic modified essential medium. Induced BFiVSP1 started to grow at about 120 h and then declined in numbers to an undetectable level for at least 10 days (Fig. 6C). In the absence of either tetracycline induction or doxycline treatment, BFiVSP1 parasites killed the mice after 100 h of infection. After 100 h, there were 40-fold fewer induced BFiVSP1 parasites in the blood than in the controls (non-induced and wild type) (Fig. 6C). Taken together, these results indicate that the absence of TbVSP1 decreases acute virulence of T. brucei in mice such that animals survive an otherwise lethal infection with this strain. These data confirmed that TbVSP1 plays a key role in polyP metabolism and might be an alternative to exopolyphosphatase activity in eukaryotic cells. Because TbVSP1 was essential for osmoregulation and the establishment of full virulence in mice, it could be considered as a potential pharmacological target. We also have identified a gene encoding this enzyme in Leishmania amazonensis (accession number AL354096). Moreover, bisphosphonates, which are known as good trypanocides (42Montalvetti A. Bailey B.N. Martin M.B. Severin G.W. Oldfield E. Docampo R. J. Biol. Chem. 2001; 276: 33930-33937Google Scholar, 43Martin M.B. Grimley J.S. Lewis J.C. Heath 3rd, H.T. Bailey B.N. Kendrick H. Yardley V. Caldera A. Lira R. Urbina J.A. Moreno S.N. Docampo R. Croft S.L. Oldfield E. J. Med. Chem. 2001; 44: 909-916Google Scholar), are potent inhibitors of pyrophosphatase activities (44Hyytia T. Halonen P. Salminen A. Goldman A. Lahti R. Cooperman B.S. Biochemistry. 2001; 40: 4645-4653Google Scholar). Since these molecules accumulate on polyphosphate (45Schroder H.C. Kurz L. Muller W.E. Lorenz B. Biochemistry (Mosc.). 2000; 65: 296-303Google Scholar), it would be of great interest to evaluate bisphosphonate accumulation in this compartment and to test them for inhibitory effects on TbVSP1 enzymatic activity. We thank Arthur Kornberg for E. coli CA38 pTrcPPX1, Frédéric Bringaud for the pyrophosphatase sequence, and Charles Davis for critical reading of the manuscript.
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