Dramatic Increase in Glycerol Biosynthesis upon Oxidative Stress in the Anaerobic Protozoan Parasite <em>Entamoeba histolytica</em>

<div><p><em>Entamoeba histolytica</em>, a microaerophilic enteric protozoan parasite, causes amebic colitis and extra intestinal abscesses in millions of inhabitants of endemic areas. Trophozoites of <em>E. histolytica</em> are exposed to a variety of reactive oxygen and nitrogen species during infection. Since <em>E. histolytica</em> lacks key components of canonical eukaryotic anti-oxidative defense systems, such as catalase and glutathione system, alternative not-yet-identified anti-oxidative defense strategies have been postulated to be operating in <em>E. histolytica</em>. In the present study, we investigated global metabolic responses in <em>E. histolytica</em> in response to H₂O₂- and paraquat-mediated oxidative stress by measuring charged metabolites on capillary electrophoresis and time-of-flight mass spectrometry. We found that oxidative stress caused drastic modulation of metabolites involved in glycolysis, chitin biosynthesis, and nucleotide and amino acid metabolism. Oxidative stress resulted in the inhibition of glycolysis as a result of inactivation of several key enzymes, leading to the redirection of metabolic flux towards glycerol production, chitin biosynthesis, and the non-oxidative branch of the pentose phosphate pathway. As a result of the repression of glycolysis as evidenced by the accumulation of glycolytic intermediates upstream of pyruvate, and reduced ethanol production, the levels of nucleoside triphosphates were decreased. We also showed for the first time the presence of functional glycerol biosynthetic pathway in <em>E. histolytica</em> as demonstrated by the increased production of glycerol 3-phosphate and glycerol upon oxidative stress. We proposed the significance of the glycerol biosynthetic pathway as a metabolic anti-oxidative defense system in <em>E. histolytica</em>.</p> </div

Representation of major changes in central carbon metabolism upon oxidative stress.

<p>Shades in red and green indicate increase or decrease of metabolites, respectively. Enzymes catalyzing these reactions are also shown in bold letters. Enzymes in black or green are those whose activities were unchanged or downregulated, respectively, upon oxidative stress. Abbreviations are: HK, Hexokinase; HPI, Hexose phosphate isomerase; PFK, Phosphofructokinase; ALDO, Aldolase; TPI, Triose-phosphate isomerase; GAPDH, glyceraldehyde 3-P dehydrogenase; PGK, Phosphoglycerate kinase; ENO, Enolase; PPDK, Pyruvate phosphate dikinase; PK, Pyruvate kinase; MDH, Malate dehydrogenase; ME, malic enzyme; ADH, Alcohol dehydrogenase; GK, glycerol kinase; GPP, glycerol 3-phosphate phosphatase.</p

Oxidative stress causes energy depletion.

<p>(<b>A</b>) The average fold change ± SD (error bars) of the nucleotides at various time points during PQ- or H₂O₂-mediated oxidative stress. (<b>B</b>) Adenylate energy charge of the cell, which is calculated by the equation, [(ATP)+1/2(ADP)]/[(ATP)+(ADP)+(AMP)] during the course of oxidative stress.</p

PQ- or H₂O₂-mediated oxidative stress causes global metabolic changes.

<p>(<b>A–B</b>) Survival of <i>E. histolytica</i> trophozoites after 12 h of treatment with varying concentrations of PQ (<b>A</b>) or H₂O₂ (<b>B</b>). The average viability (%) ± standard deviation (SD, error bars) at various concentrations of PQ or H₂O₂ is shown. (<b>C</b>) Effect of 1 mM of PQ treatment for 1, 3, 6, or 12 h on the intracellular level of reactive oxygen species. Average level of 2′, 7′-DCF-DA fluorescence (arbitrary units) ±S.D. (error bars) in 5×10⁵ cells is shown (<b>D</b>) Heat map produced by hierarchical clustering of metabolite profiles obtained from CE-TOFMS analysis. Rows correspond to metabolites and columns correspond to the duration of treatment. Metabolite levels are expressed as fold change with respect to time 0 h. Shades in red and green indicate increase or decrease in the levels of metabolites, respectively, according to the scale bar shown at the bottom.</p

Oxidative stress inactivates glycolytic enzymes and redirects glycolytic flux towards glycerol synthesis.

<p>(<b>A</b>) Activities of enzymes involved in the central energy metabolism upon PQ (1 mM for 12 h) treatment. Enzymatic activities are expressed as percentage relative to untreated trophozoites. (<b>B–D</b>) The average fold change ± SD (error bars) of glycerol, ethanol, and acetate in untreated (control) or PQ-treated (1 mM for 12 h) trophozoites are shown. Abbreviations are: HK, Hexokinase; HPI, Hexose phosphate isomerase; PFK, Phosphofructokinase; ALDO, Aldolase; TPI, Triose-phosphate isomerase; GAPDH, glyceraldehyde 3-P dehydrogenase; PGK, Phosphoglycerate kinase; ENO, Enolase; PPDK, Pyruvate phosphate dikinase; PK, Pyruvate kinase; MDH, Malate dehydrogenase; ME, malic enzyme; ADH, Alcohol dehydrogenase.</p

The general reaction scheme for <i>N</i>-ethylmaleimide on biological thiols.

<p>The product formed is an <i>N</i>-ethylsuccinimido conjugate and the monoisotopic mass of the reactant is increased by 125.048. For example the glutathione (m/z 308.0911) conjugate is <i>N</i>-ethylsuccinimido-S-glutathione (m/z 433.1391).</p

Metabolites (attomol/cell) extracted from samples taken during respiratory oscillation (minimum dissolved oxygen; figure 4), detected by CE-MS and the methods used to extract them. nd – not detected.

1<p>-Q not quenched.</p>2<p>BB bead-beating.</p>3<p>All other samples were performed in triplicate, except this one.</p>4<p>NEM <i>N</i>-ethylmaleimide.</p>5<p>NEM added pre bead-beating.</p>6<p>NEM added post bead-beating.</p

A heatmap of the calibrated intracellular metabolite time-series during the respiratory oscillation (A) and time-series for ATP (B), glutathione (C), and aspartate (D) in <i>S. cerevisiae</i>.

<p>Cationic and anionic data are shown in red and blue (lines or shaded areas), respectively. The corresponding oscillation in dissolved oxygen (DO) is represented by a grey line.</p

Electrophoretograms of glutathione (GSH) and homocysteine (Hcys) and their NEM derivatives (ESG and ESHcys) in representative samples with (red line) and without (green line) the addition of 2 mM NEM.

<p>Electrophoretograms of glutathione (GSH) and homocysteine (Hcys) and their NEM derivatives (ESG and ESHcys) in representative samples with (red line) and without (green line) the addition of 2 mM NEM.</p

Time-resolved metabolomics reveals metabolic modulation in rice foliage-4

Pplied 3 CE-MS methods and a CE-DAD method to analyze 69 major metabolites. Dynamic changes in the metabolite levels were assessed at hourly intervals over a 24 h period. Averages of 2 samples (± SEM) are shown. The top bar (shown in only Ala) indicates light and dark conditions.<p><b>Copyright information:</b></p><p>Taken from "Time-resolved metabolomics reveals metabolic modulation in rice foliage"</p><p>http://www.biomedcentral.com/1752-0509/2/51</p><p>BMC Systems Biology 2008;2():51-51.</p><p>Published online 18 Jun 2008</p><p>PMCID:PMC2442833.</p><p></p