pyrophosphate and Chagas-Disease

Trypanosoma cruzi, the causative agent of Chagas' disease, belongs to the Trypanosomatidae family. The parasite undergoes multiple morphological and metabolic changes during its life cycle, in which it can use both glucose and amino acids as carbon and energy sources. The glycolytic pathway is peculiar in that its first six or seven steps are compartmentalized in glycosomes, and has a two-branched auxiliary glycosomal system functioning beyond the intermediate phosphoenolpyruvate (PEP) that is also used in the cytosol as substrate by pyruvate kinase. The pyruvate phosphate dikinase (PPDK) is the first enzyme of one branch, converting PEP, PPi, and AMP into pyruvate, Pi, and ATP. Here we present a kinetic study of PPDK from T. cruzi that reveals its hysteretic behavior. The length of the lag phase, and therefore the time for reaching higher specific activity values is affected by the concentration of the enzyme, the presence of hydrogen ions and the concentrations of the enzyme's substrates. Additionally, the formation of a more active PPDK with more complex structure is promoted by it substrates and the cation ammonium, indicating that this enzyme equilibrates between the monomeric (less active) and a more complex (more active) form depending on the medium. These results confirm the hysteretic behavior of PPDK and are suggestive for its functioning as a regulatory mechanism of this auxiliary pathway. Such a regulation could serve to distribute the glycolytic flux over the two auxiliary branches as a response to the different environments that the parasite encounters during its life cycle.

Topics: Adenosine Monophosphate; Chagas Disease; Diphosphates; Glucose; Glycolysis; Hydrogen-Ion Concentration; Kinetics; Microbodies; Phosphoenolpyruvate; Pyruvate, Orthophosphate Dikinase; Pyruvates; Recombinant Proteins; Trypanosoma cruzi

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.

Topics: Animals; Cells, Cultured; Chagas Disease; Chlorocebus aethiops; Cloning, Molecular; Diphosphates; Disease Models, Animal; Humans; Mice; Mice, Inbred C57BL; Mice, Knockout; Polyphosphates; Protozoan Proteins; Pyrophosphatases; Trypanosoma cruzi; Vacuoles; Vero Cells; Virulence Factors

Trypanosoma cruzi, the etiologic agent of Chagas disease, has an unusual ATP-dependent hexokinase (TcHK) that is not affected by D-glucose 6-phosphate, but is non-competitively inhibited by inorganic pyrophosphate (PP(i)), suggesting a heterotropic modulator effect. In a previous study we identified a novel family of bisphosphonates, metabolically stable analogs of PP(i), which are potent and selective inhibitors of TcHK as well as the proliferation of the clinically relevant intracellular amastigote form of the parasite in vitro (Hudock, M. P., Sanz-Rodriguez, C. E., Song, Y., Chan, J. M., Zhang, Y., Odeh, S., Kosztowski, T., Leon-Rossell, A., Concepcion, J. L., Yardley, V., Croft, S. L., Urbina, J. A., and Oldfield, E. (2006) J. Med. Chem. 49, 215-223). In this work, we report a detailed kinetic analysis of the effects of three of these bisphosphonates on homogeneous TcHK, as well as on the enzyme in purified intact glycosomes, peroxisome-like organelles that contain most of the glycolytic pathway enzymes in this organism. We also investigated the effects of the same compounds on glucose consumption by intact and digitonin-permeabilized T. cruzi epimastigotes, and on the growth of such cells in liver-infusion tryptose medium. The bisphosphonates investigated were several orders of magnitude more active than PP(i) as non-competitive or mixed inhibitors of TcHK and blocked the use of glucose by the epimastigotes, inducing a metabolic shift toward the use of amino acids as carbon and energy sources. Furthermore, there was a significant correlation between the IC(50) values for TcHK inhibition and those for epimastigote growth inhibition for the 12 most potent compounds of this series. Finally, these bisphosphonates did not affect the sterol composition of the treated cells, indicating that they do not act as inhibitors of farnesyl diphosphate synthase. Taken together, our results suggest that these novel bisphosphonates act primarily as specific inhibitors of TcHK and may represent a novel class of selective anti-T. cruzi agents.

Topics: Animals; Chagas Disease; Diphosphates; Diphosphonates; Enzyme Inhibitors; Geranyltranstransferase; Glucose-6-Phosphate; Hexokinase; Humans; Kinetics; Organelles; Protozoan Proteins; Sterols; Trypanosoma cruzi

Chagas' disease is a serious protozoan infection affecting up to 20% of populations in some endemic areas. Myocarditis and cardiomyopathy occur in 50% of patients who go on to develop chronic Chagas' disease. We have studied a patient with no overt cardiac symptoms who revealed intense myocardial uptake of Tc-99m pyrophosphate. The significance of this finding in relation to early detection and progress of therapy is explored.

Topics: Chagas Cardiomyopathy; Chagas Disease; Diphosphates; Humans; Male; Middle Aged; Radionuclide Imaging; Technetium; Time Factors