Memantine, an antagonist of the NMDA glutamate receptor, affects cell proliferation, differentiation and the intracellular cycle and induces apoptosis in Trypanosoma cruzi

Chagas' disease is caused by the protozoan parasite Trypanosoma cruzi and affects approximately 10 million people in endemic areas of Mexico and Central and South America. Currently available chemotherapies are limited to two compounds: Nifurtimox and Benznidazole. Both drugs reduce the symptoms of the disease and mortality among infected individuals when used during the acute phase, but their efficacy during the chronic phase (during which the majority of cases are diagnosed) remains controversial. Moreover, these drugs have several side effects. The aim of this study was to evaluate the effect of Memantine, an antagonist of the glutamate receptor in the CNS of mammals, on the life cycle of T. cruzi. Memantine exhibited a trypanocidal effect, inhibiting the proliferation of epimastigotes (IC50 172.6 µM). Furthermore, this compound interfered with metacyclogenesis (approximately 30% reduction) and affected the energy metabolism of the parasite. In addition, Memantine triggered mechanisms that led to the apoptosis-like cell death of epimastigotes, with extracellular exposure of phosphatidylserine, increased production of reactive oxygen species, decreased ATP levels, increased intracellular Ca2+ and morphological changes. Moreover, Memantine interfered with the intracellular cycle of the parasite, specifically the amastigote stage (IC50 31 µM). Interestingly, the stages of the parasite life cycle that require more energy (epimastigote and amastigote) were more affected as were the processes of differentiation and cell invasion.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP grant #11/50631-1 to AMS)Instituto Nacional de Biologia Estrutural e Química Medicinal em Doenças Infecciosas (INBEQMeDI)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq grant #470272/201 to AMS

Protective effect of ions against cell death induced by acid stress in Saccharomyces

Saccharomyces boulardii is a probiotic used to prevent or treat antibiotic-induced gastrointestinal disorders and acute enteritis. For probiotics to be effective they must first be able to survive the harsh gastrointestinal environment. In this work, we show that S. boulardii displayed the greatest tolerance to simulated gastric environments compared with several Saccharomyces cerevisiae strains tested. Under these conditions, a pH 2.0 was the main factor responsible for decreased cell viability. Importantly, the addition of low concentrations of sodium chloride (NaCl) protected cells in acidic conditions more effectively than other salts. In the absence of S. boulardii mutants, the protective effects of Na 1 in yeast viability in acidic conditions was tested using S. cerevisiae Na 1 -ATPases (ena1-4), Na 1 /H 1 antiporter (nha1D) and Na 1 /H 1 antiporter prevacuolar (nhx1D) null mutants, respectively. Moreover, we provide evidence suggesting that this protection is determined by the plasma membrane potential, once altered by low pH and low NaCl concentrations. Additionally, the absence or low expression/activity of Ena proteins seems to be closely related to the basal membrane potential of the cells

Role of Δ1-Pyrroline-5-Carboxylate Dehydrogenase Supports Mitochondrial Metabolism and Host-Cell Invasion ofTrypanosoma cruzi

Proline is crucial for energizing critical events throughout the life cycle of Trypanosoma cruzi, the etiological agent of Chagas disease. The proline breakdown pathway consists of two oxidation steps, both of which producereducing equivalents as follows: the conversion of proline to Δ1-pyrroline-5-carboxylate (P5C), and the subsequent conversion of P5C to glutamate. We have identified and characterized the Δ1-pyrroline-5-carboxylate dehydrogenase from T. cruzi (TcP5CDH) and report here on how this enzyme contributes to a central metabolic pathway in this parasite. Size-exclusionchromatography, two-dimensional gel electrophoresis, and small angle x-ray scattering analysis of TcP5CDH revealed an oligomericstate composed of two subunits of six protomers. TcP5CDH was found to complement a yeast strain deficient in PUT2 activity,confirming the enzyme's functional role; and the biochemical parameters (Km, kcat, and kcat/Km) of the recombinant TcP5CDH were determined, exhibiting values comparable with those from T. cruzi lysates. In addition, TcP5CDH exhibited mitochondrial staining during the main stages of the T. cruzi life cycle. mRNA and enzymatic activity levels indicated the up-regulation (6-fold change) of TcP5CDH during the infectivestages of the parasite. The participation of P5C as an energy source was also demonstrated. Overall, we propose that thisenzymatic step is crucial for the viability of both replicative and infective forms of T. cruzi

Actions of a Proline Analogue, L-Thiazolidine-4-Carboxylic Acid (T4C), on Trypanosoma cruzi

It is well established that L-proline has several roles in the biology of trypanosomatids. In Trypanosoma cruzi, the etiological agent of Chagas' disease, this amino acid is involved in energy metabolism, differentiation processes and resistance to osmotic stress. In this study, we analyzed the effects of interfering with L-proline metabolism on the viability and on other aspects of the T. cruzi life cycle using the proline analogue L- thiazolidine-4-carboxylic acid (T4C). The growth of epimastigotes was evaluated using different concentrations of T4C in standard culture conditions and at high temperature or acidic pH. We also evaluated possible interactions of this analogue with stress conditions such as those produced by nutrient starvation and oxidative stress. T4C showed a dose-response effect on epimastigote growth (IC50 = 0.89±0.02 mM at 28°C), and the inhibitory effect of this analogue was synergistic (p<0.05) with temperature (0.54±0.01 mM at 37°C). T4C significantly diminished parasite survival (p<0.05) in combination with nutrient starvation and oxidative stress conditions. Pre-incubation of the parasites with L-proline resulted in a protective effect against oxidative stress, but this was not seen in the presence of the drug. Finally, the trypomastigote bursting from infected mammalian cells was evaluated and found to be inhibited by up to 56% when cells were treated with non-toxic concentrations of T4C (between 1 and 10 mM). All these data together suggest that T4C could be an interesting therapeutic drug if combined with others that affect, for example, oxidative stress. The data also support the participation of proline metabolism in the resistance to oxidative stress

Biochemical characterization of serine transport in Leishmania (Leishmania) amazonensis

In addition to its role as a protein component in Leishmania, serine is also a precursor for the synthesis of both phosphatidylserine, which is a membrane molecule involved in parasite invasion and inactivation of macrophages, and sphingolipids, which are necessary for Leishmania to differentiate into its infective forms. We have characterized serine uptake in both promastigote and amastigote forms of Leishmania (Leishmania) amazonensis. In promastigotes, kinetic data show a single, saturable transport system, with a Km of 0.253 +/- 0.01 mM and a maximum velocity of 0.246 +/- 0.04 nmol/min per 107 cells. Serine transport increased linearly with temperature in the range from 20 degrees C to 45 degrees C, allowing the calculation of an activation energy of 7.09 kJ/mol. Alanine, cysteine, glycine, threonine, valine and ethanolamine competed with the substrate at a ten-fold excess concentration. Serine uptake was dependent on pH, with an optimum activity at pH 7.5. The characterization of the serine transport process in amastigotes revealed a transport system with a similar Km, energy of activation and pH response to that found in promastigotes, suggesting that the same transport system is active in both insect vector and mammalian host Leishmania stages. This could constitute an evolutionary mechanism that guarantees the provision of such an essential molecule during host change events, such as differentiation into amastigotes and macrophage invasion, as well as to ensure that the parasite maintains the infection in the mammalian host. (C) 2008 Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq

Effect of Memantine on the intracellular cycle of <i>Trypanosoma cruzi</i>.

<p>Panel A: viability of CHO-K₁ cells treated with different concentrations of Memantine (range 50 µM to 1 mM). Viability was assessed by MTT assay. Panel B: effect on the infectivity of trypomastigotes treated only during the period of infection (50–400 µM). Panel C: effect of treatment after invasion of parasites in CHO-K₁ cells (5–300 µM). Panel D: effect of Memantine on intracellular stages. Cells were treated at different stages with 31 µM Memantine (IC₅₀ value): <b>T</b> (trypomastigote cell invasion), <b>A</b> (amastigote) and <b>IE</b> (intracellular epimastigote-like) stages. In all experiments, we evaluated the burst of trypomastigotes on the fifth day post-infection by counting parasites in a Neubauer chamber. Tukey test: *: p<0.05; **: p<0.01; ***: p<0.001.</p

Forward and side scattering values for epimastigotes treated or not with Memantine.

1<p>Geometrical mean of scatter values of epimastigotes treated or not with 172.6 µM Memantine.</p

Quantification of H₂O₂, Ca²⁺ and ATP levels in <i>T. cruzi</i>.

<p>Panel A: <b>H₂O₂ levels</b>, parasites treated with Memantine (172.6 µM) or not treated (control) for 24 hours. After this period, the parasites (1.0×10⁷) were incubated with 25 µM amplex red, and 0.05 U mL⁻¹ horseradish peroxidase and analyzed on a fluorometer (λ_ex 563 nm and λ_em 587 nm). Panel B: <b>Ca²⁺ levels</b>, parasites were treated for 4 days and incubated with Fluo-4 AM (5 µM) for 1 hour at 28°C, washed twice in HEPES-glucose and evaluated on a fluorometer (λ_ex 490 nm and λ_em 518 nm). Panel C: <b>ATP levels</b>, parasites were treated for 30 hours, and the levels of ATP were assessed using a bioluminescent assay kit (Sigma-Aldrich) and analyzed on a luminometer (λ 570 nm). <i>T</i> test: *: p<0.05; **: p<0.01; ***: p<0.001.</p

<i>Trypanosoma cruzi</i> epimastigote forms, 4^th day of growth.

<p>Panel A and B: untreated parasites. Panel C and D, parasites treated with Memantine (172.6 µM).</p

Growth curve of epimastigote forms of <i>Trypanosoma cruzi</i>.

<p><b>Left:</b> growth curves in the presence of different concentrations of compounds. Treatments with (A) Memantine (MM), (B) MK-801, (C) Amantadine (AMT); at 28°C and 7.4 pH: black square: 0 µM; black up-pointing triangle: 30 µM (MM or AMT), 100 µM (MK); black down-pointing triangle: 60 µM (MM), 90 µM (AMT), 200 µM (MK); black left-pointing triangle: 90 µM (MM), 150 µM (AMT), 300 µM (MK); black right-pointing triangle: 120 µM (MM), 250 µM (AMT), 400 µM (MK); black diamond: 150 µM (MM), 400 µM (AMT), 500 µM (MK); black pentagon: 180 µM (MM), 500 µM (AMT), 600 µM (MK); black hexagon: 210 µM (MM), 700 µM (AMT or MK); black star: 250 µM (MM), 1,000 µM (AMT), 800 µM (MK); inverse white circle: 300 µM (MM), 1 mM (MK); black circle: Inhibition control (0.5 µM antimycin and 60 µM rotenone). <b>Right:</b> dose-response curves.</p