TAG species analysis and uptake of labeled oleate.

<p>(A) Dominant TAG species in procyclic <i>T. brucei</i> cells identified by ESI/MS/MS after oleate feeding for three days (black columns) or in the control (white columns). For a complete list of TAG species detected see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114628#pone.0114628.s001" target="_blank">S1 Figure</a>. The nomenclature 54:X indicates the total carbon number of all three acyl chains and the sum of all unsaturated double bonds within the acyl chains. (B) Uptake kinetics upon growth in the presence of radiolabeled oleate for up to 8 h. The incorporation of ¹⁴C oleate into lipid species was quantified by HPTLC and a Storm 860 phosphorimager. PPL, phospholipids; TAG, triacylglycerol; SE, Steryl-esters; DAG, diacylglycerol.</p

NADPH-dependent 3-hydroxyacyl-CoA dehydrogenase activity in WT and Δ<i>tfeα1</i>/Δ<i>tfeα1</i> cells.

1<p>WCE, whole cell exctract.</p>2<p>glyco, partially purified glycosome fraction.</p>3<p>Mean ± SEM of n experiments (mU/mg of protein).</p>4<p>+gluc: cells cultured in SDM79 containing 10 mM glucose.</p>5<p>−gluc: cells cultured in glucose-depleted SDM79GluFree.</p><p>NADPH-dependent 3-hydroxyacyl-CoA dehydrogenase activity in WT and Δ<i>tfeα1</i>/Δ<i>tfeα1</i> cells.</p

Phenotypic analysis of Δ<i>tfeα1</i>/Δ<i>tfeα1</i> cell.

<p>(A) growth curve of WT and Δ<i>tfeα1</i>/Δ<i>tfeα1</i> cell knock cells in glucose-rich (SDM79 with 10 mM glucose) or glucose-free (SDM79GluFree) conditions. (B) Global protein abundance in the partially purified glycosome fraction of WT (x-axis) and Δ<i>tfeα1</i>/Δ<i>tfeα1</i> cell knock cells (y-axis). Each protein identification is presented by a point at log₁₀ of normalized peptide count values taken from the proteome data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114628#pone.0114628.s004" target="_blank">S4 Figure</a>. Proteins on the dashed grey line have identical normalized peptide counts in both samples; the grey lines represent a 2-fold abundance in one condition.</p

Oleate feeding stimulates lipid droplet formation in procyclic <i>T. brucei</i> cells.

<p>Staining of lipid droplets with nile red (A) or BODIPY 493/503 (B) was as detailed in experimental procedures. Myriocin treatment (0.5 µM for 24 h) was included for comparison to a previous report <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114628#pone.0114628-Bird1" target="_blank">[36]</a>. An example of several experiments is shown.</p

Expression and immunolocalization of enzymes involved in acetate metabolism.

<p>In Panel A, expression of AceCS, ASCT, ACH, PDH-E2 and TDH was analyzed by western blotting with specific immune sera on total protein lysates (5×10⁶ cells per lane) of wild-type 427 monomorphic bloodstream forms (LS-BSF) and procyclic cells (PF). The anti-hps60 antibodies were used as loading controls. The positions of the molecular weight markers are indicated in kDa on the right margin of each panel. The PDH and TDH activities (milliunits/mg of protein) were determined in both forms of the parasite. The “±” signs indicate SD of at least 3 independent experiments. In panel B, bloodstream cells were stained with mouse anti-AceCS (Alexa 488 channel), mouse anti-PDH-E1α (Alexa 488 channel) or rabbit anti-TDH (Alexa 488 channel) with MitoTracker as mitochondrial control. Differential interference contrast (DIC) of cells is shown to the left of each panel. Scale bar, 1 µm.</p

LD and TAG turnover in WT and Δ<i>tfeα1</i>/Δ<i>tfeα1</i> cells.

<p>Cells were fed with oleate in glucose-rich SDM79 medium for three days, and after oleate withdrawal samples were taken at the time points indicated. (A) WT cells stained with BODIPY and analyzed by flow cytometry (left y-axis). Error bars represent the SEM of independent replicates (n = 3). The growth curve is given as dashed line (right y-axis). (B) Growth curve and sampling time points (arrows) for the experiments in panels (C) and (D). Total TAG content was determined in triplicate by HPTLC and densitometry in WT (C) and Δ<i>tfeα1</i>/Δ<i>tfeα1</i> (D) cells. Error bars represent the SEM of independent replicates (n = 3). The calculated values (filled symbols) account for dilution of LDs or TAG content by cell division, based on the matched growth data.</p

Quantification of the oleate-induced lipid droplet formation.

<p>(A) BODIPY 493/503 stained LDs were counted in stacks of confocal laser scanning microscopy (CLSM) images; the average number of LDs per cell is given after oleate feeding (black column) or in the control (white column). (B) Distribution of LD numbers per cells in the population after oleate feeding (black columns) or in the control (white columns). (C) Quantification of BODIPY-stained LDs by flow cytometry after oleate feeding (black column) or in the control (white column). BODIPY 493/503 preferentially stains nonpolar lipids. Error bars give the SEM (n = 3) of values normalized to the control. (D) Quantification of TAG content by HPTLC and densitometry after oleate feeding (black columns) or in the control (white columns). Values are normalized to the control.</p

Schematic representation of acetate production from glucose and threonine in BSF.

<p>Black arrows indicate enzymatic steps of glucose and threonine metabolism and the double line arrow represents <i>de novo</i> biosynthesis of fatty acids. Excreted end-products of metabolism of glucose and threonine are boxed. The thick arrows illustrate the high glycolytic flux leading to production of pyruvate, which accounts for 85.1% of the excreted end-products from glucose metabolism. Abbreviations: AcCoA, acetyl-CoA; AOB, amino oxobutyrate; DHAP, dihydroxyacetone phosphate; G3P, glyceraldehyde 3-phosphate; MAL, malate; PEP, phosphoenolpyruvate; PYR, pyruvate; SCoA, succinyl-CoA. Indicated enzymes are: 1, pyruvate dehydrogenase complex (PDH); 2, acetate∶succinate CoA-transferase (ASCT); 3, acetyl-CoA thioesterase (ACH); 4, succinyl-CoA synthetase (SCoAS); 5, threonine 3-dehydrogenase (TDH); 6, 2-amino-3-ketobutyrate coenzyme A ligase (AKCT); 7, acetyl-CoA synthetase (AMP-dependent enzyme, AceCS).</p

Analysis of mutant cell lines.

<p>Panels A and C–E show growth curve of the parental 427 90-13 BSF cells (WT) and mutant cell lines incubated in the IMDM medium. The mutant cell lines are <i>^RNAi</i>PDH (A and D), Δ<i>tdh</i> (C) and Δ<i>tdh</i>/<i>^RNAi</i>PDH (E) incubated in the presence (.i, +tet) or in the absence (.ni) of tetracycline. In panel D, the <i>^RNAi</i>PDH.i cell line was incubated in threonine-depleted medium supplemented with 0 to 150 µM threonine. The cells were maintained in the exponential growth phase (between 2×10⁵ and 2×10⁶ cells/mL) and cumulative cell numbers reflect normalization for dilution during cultivation. The insets in panels A and C–E show western blot analyses of the parental (WT) and mutant cell lines with the immune sera indicated in the right margin. The lower inset in panel C shows the TDH activity (milliunits/mg of protein) measured in the WT and Δ<i>tdh</i> cell lines (ND stands for not detectable). Panel B shows a PCR analysis of genomic DNA isolated from the parental WT and Δ<i>tdh</i> cell lines. Amplifications were performed with primers based on sequences that flank the 5′UTR and 3′UTR fragments used to target the <i>TDH</i> gene depletion (black boxes) and internal sequences of the puromycin (<i>PAC</i>, PCR products 3 and 5) or blasticidin (<i>BSD</i>, PCR products 4 and 6) resistance genes. As controls, we also used primers corresponding to the 5′UTR (PCR product 1), the <i>TDH</i>, <i>PAC</i> and <i>BLE</i> genes (PCR products 2, 7 and 8, respectively). As expected, PCR amplification of the 5′UTR was observed for both cell lines (lane 1), while the <i>TDH</i> gene was PCR-amplified only in the WT cells (lane 2) and PCR products with marker-derived primers were observed only in the Δ<i>tdh</i> cell line (lanes 3–8).</p

Excreted end-products of glucose and threonine metabolism by <i>T. brucei</i> long-slender bloodstream form (BSF) and procyclic form (PF) cell lines.

<p>The extracellular PBS medium of trypanosomes incubated with 4 mM [U-¹³C]-glucose and 4 mM threonine was analyzed by ¹H-NMR spectrometry to detect and quantify excreted end-products.</p>a<p>.i: RNAi cell lines tetracycline-induced for 5 to 10 days depending on the cell line and the experiments; .ni: uninduced RNAi cell lines.</p>b<p>PF and BSF were incubated at the 2×10⁷ cells/mL (0.1 mg of proteins/mL) and 10⁷ cells/mL (0.1 mg of proteins/mL), respectively.</p>c<p>Number of experiments.</p>d<p>Mean ± SD of 3 or 9 experiments of cells incubated in PBS containing 4 mM [U-¹³C]-glucose and 4 mM threonine (nmol/h/10⁸ cells) are presented.</p>e<p>Values into brackets represent percent of each excreted molecules from [U-¹³C]-glucose metabolism.</p>f<p>Ratio between glucose-derived and threonine-derived [¹³C]-acetate excreted.</p>g<p>Not detectable.</p