81 research outputs found
Relative levels of different amino acids measured in WT and Δtkt cells and their growth media.
<p>(A) Selected amino acids in fresh and spent media of WT and Δtkt cultures over four days of cultivation as detected by LC-MS analysis, FM—fresh medium, n = 4. (B) Intracellular levels of amino acids in WT, Δtkt, and Δtkt + TKT cultures detected by LC-MS. Intensity detected in WT is considered reference and the other values indicate relative abundance, n = 4. All the amino acids were identified based on matches with respective standards.</p
Pentose phosphate pathway.
<p>Glucose is phosphorylated into glucose 6-phosphate (G6P) by hexokinase (HK) and utilized further in glycolysis, shaded in blue, or channelled into the pentose phosphate pathway (PPP). In the oxidative branch of the PPP, shaded in green, G6P is converted via three subsequent steps comprising glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconolactonase (6-PGL), and 6-phosphogluocnate dehydrogenase (6-PGDH), resulting in the production of ribulose 5-phosphate, reduced NADPH and CO<sub>2</sub>. In the non-oxidative branch, shaded in lilac, ribulose 5-phosphate is converted by ribulose-5-phosphate epimerase (RuPE) to xylulose 5-phosphate or by ribose-5-phosphate isomerase (RPI) to ribose 5-phosphate. These and other sugar phosphates can be utilized by transketolase (TKT) and transaldolase (TAL) in reactions shuffling two or three carbons, respectively. Adapted from [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006953#ppat.1006953.ref035" target="_blank">35</a>].</p
Scheme of mannogen biosynthesis.
<p>Blue and red colours indicate enzymes and their relative mRNA abundance in Δtkt when compared to WT. GlcNAc, N-acetylglucosamine; GlcN, glucosamine; Glc, glucose; GlcNAc6P, N-acetylglucosamine 6-phosphate; GlcN6P, glucosamine 6-phosphate; G6P, glucose 6-phosphate; F6P, fructose 6-phosphate; Man6P, mannose 6-phosphate; UDP-GlcNAc, uridine diphosphate N-acetylglucosamine; GDP-Man, guanosine diphosphate mannose; HK, hexokinase; PGI, glucose-6-phosphate isomerase; NAGD, N-acetylglucosamine-6-phosphate deacetylase; GND, glucosamine-6-phosphate deaminase; GNAT, glucosamine-6-phosphate acetylase; GFAT, glutamine:Fru6P aminotransferase; PMM, phosphomannomutase; MPGT, mannose-1-phosphate guanylyltransferase; dashed arrows indicate multiple enzymatic steps; based om Naderer, et al. [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006953#ppat.1006953.ref036" target="_blank">36</a>] and Garami, et al. [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006953#ppat.1006953.ref070" target="_blank">70</a>].</p
Immunofluorescence microscopy of cells transfected with all variants of altered GFP-TKT constructs.
<p>Shown from the longest at the top to the shortest at the bottom. Antibody against triosephosphate isomerase (TIM) was used as a glycosomal marker. The GFP signal corresponds to GFP-TKT complexes as verified by a Western blot analysis (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006953#ppat.1006953.s005" target="_blank">S5C Fig</a>).</p
Relative shift in mRNA levels detected for the respective enzymes in glycolysis and the PPP by RNAseq analysis.
<p>Relative values detected in Δtkt when compared to WT are indicated. mRNA increased more than two fold is indicated in red, and decreases of more than two fold in blue, respectively. Red and blue arrows accompanying metabolites represent results of the GC-MS analysis, as depicted in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006953#ppat.1006953.g004" target="_blank">Fig 4B</a>. * p < 0.001.</p
Sensitivity to oxidative stress inducing agents and other drugs.
<p>Sensitivity to oxidative stress inducing agents and other drugs.</p
Central carbon flux is decreased in Δtkt.
<p>(A) Glucose consumption by WT and Δtkt cell lines. The graph indicates the concentration of glucose detected in spent media of the respective cultures, starting in Homem medium containing 1 mM glucose, n = 4. (B) Metabolic end products as detected in spent media of WT and Δtkt cultures by LC-MS, all three metabolites were identified based on matches with the respective standards. Growth rates were not significantly different. FM, fresh medium.</p
Scheme of the flow of carbon atoms from glucose through glycolysis and the PPP.
<p>If 1,2-<sup>13</sup>C<sub>2</sub>-glucose is used, products from glycolysis contain two or no carbons labelled, whereas in the PPP one carbon is cleaved as CO<sub>2</sub>, hence the three carbon products contain one or no carbons labelled. Based on Lee et al. [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006953#ppat.1006953.ref028" target="_blank">28</a>]. Glyceraldehyde 3-phosphate (GA3P) used for the quantification is highlighted.</p
Metabolomic analysis of AAC DKO cells.
The long slender bloodstream form Trypanosoma brucei maintains its essential mitochondrial membrane potential (ΔΨm) through the proton-pumping activity of the FoF1-ATP synthase operating in the reverse mode. The ATP that drives this hydrolytic reaction has long been thought to be generated by glycolysis and imported from the cytosol via an ATP/ADP carrier (AAC). Indeed, we demonstrate that AAC is the only carrier that can import ATP into the mitochondrial matrix to power the hydrolytic activity of the FoF1-ATP synthase. However, contrary to expectations, the deletion of AAC has no effect on parasite growth, virulence or levels of ΔΨm. This suggests that ATP is produced by substrate-level phosphorylation pathways in the mitochondrion. Therefore, we knocked out the succinyl-CoA synthetase (SCS) gene, a key mitochondrial enzyme that produces ATP through substrate-level phosphorylation in this parasite. Its absence resulted in changes to the metabolic landscape of the parasite, lowered virulence, and reduced mitochondrial ATP content. Strikingly, these SCS mutant parasites become more dependent on AAC as demonstrated by a 25-fold increase in their sensitivity to the AAC inhibitor, carboxyatractyloside. Since the parasites were able to adapt to the loss of SCS in culture, we also analyzed the more immediate phenotypes that manifest when SCS expression is rapidly suppressed by RNAi. Importantly, when performed under nutrient-limited conditions mimicking various host environments, SCS depletion strongly affected parasite growth and levels of ΔΨm. In totality, the data establish that the long slender bloodstream form mitochondrion is capable of generating ATP via substrate-level phosphorylation pathways.</div
Mannogen is decreased in Δtkt.
<p>(A) Intermediates of glycolysis and the PPP as detected by LC-MS, when WT and Δtkt cells were grown in Homem medium containing either glucose (Glu), or fructose (Fru) as the main carbon source. Values in the first column (WT in glucose) are considered reference and the following values indicate relative change in intensity detected, n = 4. <sup>#</sup>—metabolites identified based on matches with respective standards, all the other annotations are predicted. * p < 0.05. (B) Levels of sugar oligomers detected in WT and Δtkt cells grown in the presence of glucose or fructose, n = 4. Identity of these metabolites is predicted based on mass and retention time, however, the mass is very specific for sugar oligomers. (C) Mannogen as detected by HPTLC. Shown is one of three replicates.</p
- …