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

Abstract

We report the cloning, expression, purification, and characterization of the Trypanosoma cruzi exopolyphosphatase (TcPPX). The product of this gene (TcPPX), has 383 amino acids and a molecular mass of 43.1 kDa. TcPPX differs from most exopolyphosphatases in its preference for short-chain polyphosphate (poly P). Heterologous expression of TcPPX in Escherichia coli produced a functional enzyme that had a neutral optimum pH and was dramatically inhibited by low concentrations of Zn 2؉ , high concentrations of basic amino acids (lysine and arginine), and heparin. TcPPX is a processive enzyme and does not hydrolyze ATP, pyrophosphate, or p-nitrophenyl phosphate, although it hydrolyzes guanosine 5-tetraphosphate very efficiently. Overexpression of TcPPX resulted in a dramatic decrease in total short-chain poly P and partial decrease in longchain poly P. This was accompanied by a delayed regulatory volume decrease after hyposmotic stress. These results support the role of poly P in T. cruzi osmoregulation. Polyphosphate (poly P)8 is a ubiquitous polymer of a few to several hundred orthophosphate residues linked by high-energy phosphoanhydride bonds. Poly P is found in the environment and in all life forms, where it is involved in a number of functions (1, 2). Poly P is essential for bacterial responses to stresses and starvation, as well as for survival and virulence (1, 2). Similar functions in adaptation to stress have been attributed to poly P in eukaryotic cells such as yeast (3, 4), fungi (5), and algae (6 -8). Poly P is also involved in blood clotting (9), eukaryotic cell proliferation (10, 11), and induction of apoptosis in plasma and myeloma cells (12).In Trypanosoma cruzi, the etiologic agent of Chagas disease, most poly P is accumulated in acidocalcisomes. Acidocalcisomes are acidic, calcium-containing organelles that have been demonstrated to be involved in osmoregulation (13-16). Rapid hydrolysis of acidocalcisome poly P occurs when epimastigotes of T. cruzi are exposed to hyposmotic stress (16) resulting in increased osmolarity and swelling of the organelle (15). In addition, a microtubule and cAMP-mediated fusion of acidocalcisomes to the contractile vacuole complex with translocation of an aquaporin results in water movement and a regulatory volume decrease (15), indicating a link between acidocalcisomes and osmotic homeostasis.In eukaryotic cells the hydrolysis of poly P is performed by the action of endopolyphosphatases and exopolyphosphatases (1, 2). Genes encoding for exopolyphosphatases from Saccharomyces cerevisiae (17), Trypanosoma brucei (18), and Leishmania major (19) have been cloned and expressed in Escherichia coli, whereas a gene encoding an endopolyphosphatase from S. cerevisiae (20) is the only one that has been cloned and expressed. The L. major exopolyphosphatase (LmPPx) has been characterized recently and demonstrated to be located in acidocalcisomes and in the cytosol (19). An exopolyphosphatase activity has also been detected in acidocalcisomes of T. cruzi (16). The crystal structures...

Vtc5, a Novel Subunit of the Vacuolar Transporter Chaperone Complex, Regulates Polyphosphate Synthesis and Phosphate Homeostasis in Yeast

“…This is relevant because polyP synthesis is not the only determinant of cytosolic P i homeostasis, where the SPX domains and inositol pyrophosphates play a role. In amino acid and metal homeostasis, polyP also constitutes an important polymeric counter-ion to basic amino acids and divalent metal ions (45,46). Basic amino acids can be stored in vacuoles and acidocalcisomes in concentrations of several hundred millimolars.…”

Evolution of acidocalcisomes and their role in polyphosphate storage and osmoregulation in eukaryotic microbes

“…The T. cruzi PPX is similar to the LmPPX although its localization has not been reported. Interestingly, overexpression of TcPPX led to a significant decrease in short-chain poly P and in the staining of acidocalcisomes with DAPI, suggesting that it is also localized to acidocalcisomes (Fang et al 2007b).…”

“…Acidocalcisomes are enriched in basic amino acids that probably complex with the negatively charged poly P (Rohloff et al 2003). Only a few soluble enzymes have been detected in the acidocalcisomal matrix, including an exopolyphosphatase (PPX) in Leishmania major and Trypanosoma cruzi (Fang et al 2007b), a soluble inorganic pyrophosphatase in Trypanosoma brucei (Lemercier et al 2004), and a metacaspase in Leishmania donovani (Lee et al 2007). Acid phosphatase activity has also been detected using cytochemical methods (Gomes et al 2006).…”

One inositol ring to rule thrombosis

“…It is possible to compare these outcomes with representative historical data sets including the Medical Research Council (MRC) study where the overall survival (OS) in the nontransplanted group for melphalan-prednisone vs cyclophosphamide-thalidomidedexamethasone was 30.6 vs 33.2 months, respectively, and for patients in the transplanted group .64 years of age was 53 months. 2 In the Intergroup Français du Myélome (IFM) 99-06 study comparing melphalan-prednisone with melphalanthalidomide-prednisone and with autologous stem cell transplant (ASCT) with 100 mg/m 2 of melphalan, the OS in each group was 33.2, 51.6, and 38.3 months, respectively. 3 Thus, the 5-year OS of 63% in the current study is good, using this novel sequential approach…”

“…which is secreted upon platelet activation. 2 In 2006, we showed that polyphosphate of the size secreted by activated platelets is a potent prohemostatic agent, capable of accelerating plasma clotting and substantially delaying fibrinolysis. 3 In the ensuing years, our laboratory and others have further delineated the contributions of polyphosphate to hemostasis and thrombosis, showing in particular that long-chain polyphosphate (of the size that accumulates in microorganisms) is an extremely potent trigger of the contact pathway of clotting, while platelet-sized polyphosphate greatly accelerates downstream clotting reactions, enhances fibrin clot structure, and promotes the feedback activation of factor XI by thrombin.…”