31P NMR Spectroscopy of Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major

Abstract

High resolution31 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.Infections caused by trypanosomatid parasites continue to be among the most widespread diseases in developing nations, and the increasing fraction of immunocompromised individuals in the global population further contributes to morbidity and economic loss. New chemotherapeutic leads based on unique aspects of parasite biology are therefore of great interest, particularly in the case of emerging resistance to traditional treatments.One approach to the discovery of new drug targets is to identify metabolic pathways in parasites that are essential for parasite survival but do not have an equivalent in the host. Then, one can pursue specific inhibitors of such metabolic activities as possible means of controlling the parasites without damaging the host. NMR spectroscopy has already been used by several groups to investigate the carbohydrate and/or phosphate metabolism of protozoa, including Crithidia fasciculata (1), Entamoeba histolytica (2), Trypanosoma cruzi (3, 4), Trypanosoma brucei (5, 6), Leishmania pifanoi (7), Babesia microti (8), and the free-living ciliate Tetrahymena pyriformis (9). In Tetrahymena, large levels of pyrophosphate were found, and more recently we have found similarly large levels of pyrophosphate in T. cruzi (10). These observations are of interest since a number of protozoa are already known to utilize pyrophosphate in addition to ATP in their high energy phosphate metabolic processes. For example, E. histolytica possesses a pyrophosphatedependent phosphofructokinase (11), a pyruvate-phosphate dikinase (12), a phosphoenolpyruvate carboxytransphosphorylase (13), and a pyrophosphate-acetate kinase (14). Several of these pyrophosphate-utilizing enzymes have also been discovered in other parasitic protists (see, e.g., the discussion in Ref. 15), and more recently a pyruvate-phosphate dikinase (16) and a plantlike vacuolar proton-translocating pyrophosphatase (17-19) were discovered in many trypanosomatids as well.An unusual character...

Phospholipid and glycolipid composition of acidocalcisomes of Trypanosoma cruzi

“…Poly P is a linear chain of a few to many hundreds of phosphate (P i ) residues linked by high-energy phosphoanhydride bonds [8]. T. cruzi is especially rich in short chain poly P such as poly P 3 , poly P 4 , and poly P 5 [9]. 31 P NMR spectra of purified acidocalcisomes of T. cruzi indicated that the poly P has an average chain length of 3.25 phosphates [9].…”

“…T. cruzi is especially rich in short chain poly P such as poly P 3 , poly P 4 , and poly P 5 [9]. 31 P NMR spectra of purified acidocalcisomes of T. cruzi indicated that the poly P has an average chain length of 3.25 phosphates [9]. Based on the total concentration of poly Ps in different stages of T. cruzi, the relative volume of the acidocalcisomes in these cells (0.86%, 2.3%, and 0.26% of the total cell volume of epimastigotes, amastigotes and trypomastigotes, respectively; ref.…”

A Pyrophosphatase Regulating Polyphosphate Metabolism in Acidocalcisomes Is Essential for Trypanosoma brucei Virulence in Mice

“…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 PP i were shown to be present in the acidocalcisomes of T. brucei bloodstream forms (BF) (12,13). The turnover of the acidocalcisome PP i pool is low (14), suggesting that PP i could be a product of polyP metabolism.…”

“…Moreno et al (12) have reported that short-chain polyP of an average chain length of 3.3 phosphates and PP i 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 (9).…”

Dynamics of polymorphism of acidocalcisomes in Leishmania parasites

“…Two exceptions are the bloodstream forms of T. cruzi (Correa et al 2002) and the culture forms of Phytomonas françai (Miranda et al 2004) that have been shown to contain iron-rich acidocalcisomes. 31 P NMR and biochemical analysis have shown that the phosphorus present in these organelles is primarily in the form of pyrophosphate, and short and long chain polyphosphate (Urbina et al 1999, Moreno et al 2000, Ruiz et al 2001b.…”