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Sampling of T. brucei Metabolome
Comprehensive reviews of sampling methods applied to trypanosomatids have been published a couple of years ago 25 and only the main aspects are discussed below. For cells cultivated in vitro, the exo-metabolome simply refers to the metabolites detected in the cultivation medium and resulting from cellular activity. The culture medium can be sampled by centrifugation or fast filtration. It is usually analysed by NMR, which allows measurement of both substrate consumption and production of metabolic end-products. The media used for cultivation of T. brucei often contain buffers that can be incompatible with analytical methods, such as HEPES or MOPS that can impair the chromatographic separation of metabolites in LC-MS. The buffers in general should not be an issue for the acquisition of NMR spectra; however, their signals can potentially overlap with the signals of analytes of interest. Significant information about the metabolism of T. brucei has been collected by isotopic profiling of the exo-metabolome, as is illustrated later in this chapter.
Sampling of intracellular components requires separation of media and cells, which can be achieved by centrifugation or fast filtration. Fast filtration has been successfully applied to T. brucei PCFs 8,26,27. In this method, the cultivation broth is rapidly filtered and cells on the filter are rapidly washed with cold and diluted medium to maintain the salt and nutrient balances during the sampling process. The presence of the carbon source in the washing solution was also shown to prevent substrate limitation during sampling 28. The metabolites can be extracted immediately after filtration, though for practical reasons in most cases they are quenched in liquid nitrogen or other cold solvents before metabolite extraction is performed. Fast filtration takes 4-10 sec, which is well adapted for maintaining the pools of fast-turnover compounds such as central metabolites (e.g. intermediates of glycolysis, TCA cycle, amino acid metabolism and anabolic pathways). However, metabolite leakage was reported to occur in T. brucei BSF during fast filtration or aqueous methanol quenching 25. An alternative method has been applied to T. brucei and Leishmania 25,29,30 in which cells are separated from medium by centrifugation. In this approach, cells are rapidly chilling in a bath of dry ice and ethanol at 4°C, centrifuged (5-10 min) at 4°C and subsequently the cell pellet is extracted in methanol: chloroform: water (1:3:1). This protocol is well adapted for the analysis of metabolites with slow turnover, however cells might undergo undefined conditions (substrate or oxygen limitation) during centrifugation 31. So, as for all other organisms, there is currently no absolute method for sampling trypanosomatid metabolome, but different protocols that are adapted to specific objectives. Fast filtration is well adapted to the investigation of central and energy metabolism, meanwhile centrifugation-based protocols appear better suited for analysing biosynthetic processes. Finally, various methods have been applied to extract trypanosomatid metabolome. Extraction of polar metabolites, organic acids and phosphorylated compounds in T. brucei PCF has been performed with boiling water 26,27. Methanol: chloroform has been used for the simultaneous extraction of both polar and non-polar metabolites 32. Extraction of whole T. brucei PCF culture medium with boiling ethanol has been used for untargeted metabolome 33. Non-polar lipids were reported to be extracted by chloroform: methanol (2:1) 34–37. The broad range of methods applied to trypanosomatids is a direct illustration of the current necessity to adapt metabolite extraction according to the investigated metabolism/metabolites.
Analytical Platforms in Metabolomics
This section provides a brief description of the two main analytical platforms used in metabolomics, NMR and MS. For more details the reader should refer to comprehensive reviews that have been published in recent years on analytical methods in metabolomics 38, MS 39,40 and NMR 41. The choice of the analytical technique(s) depends on instrument availability and on the particular objectives of the investigations.
NMR allows the detection of any atom nucleus with non-zero magnetic moment (1H, 13C, 31P, 14N, etc.), which are called spins, by applying a radiofrequency pulse to samples placed in a high magnetic field. The nuclei absorb the energy, excite to high-energy state and subsequently emit detectable energy in form of an electromagnetic radiation emitted at a specific frequency. This resonance frequency is altered by electrons surrounding the nuclei in the molecule, hence depends on the chemical environment of the nucleus. For instance, the resonance of a CH3 group will be different from that of a CH2 group. This chemical dependence, called chemical shift, is scaled according to known chemical references (e.g. trimethylsilyl, TMS, for 1H- and 13C-NMR) and can be used for identification. Additional structural information about the molecules is obtained from the so-called ‘scalar coupling’. This phenomenon is due to the interaction between two or more neighbouring spins.
It results in the splitting of the NMR resonances into two or more lines, according to the nature and number of spins in interaction. For instance, the resonance of a proton will be a singlet (one line) if it has no proton neighbour, a doublet (two lines) with one proton neighbour, a triplet (three lines) with two proton neighbours, etc. NMR provides highly detailed chemical information from which the structure of the molecules can be resolved. Furthermore, multi-dimensional NMR methods can be applied to resolve chemically-complex samples – such as those considered in metabolomics – by spreading the signals over 2 (2D-NMR) or 3 (3D-NMR) spectral dimensions. This ‘magnetic separation’ of chemical groups makes possible the resolution of metabolome samples without the need for physically (chromatographically) separating the compounds. Therefore, very often little sample preparation is needed in NMR, especially for the analysis of bio fluids. In principle all compounds occurring in the sample can be detected by NMR independently of their chemical nature and in a quantitative manner. All these features make NMR very attractive for untargeted (or without a priori) and quantitative approaches in metabolomics, such as metabolic fingerprinting or metabolic profiling, respectively. In practice, NMR has a rather low sensitivity (nanomole range) and allows the detection of predominant metabolites (around one hundred species at a maximum). With current developments in NMR technology, however, the sensitivity of instruments is increasing, which provides better metabolome coverage. Since it requires minimal sample preparation, and is very robust, NMR is well suited for high-throughput metabolomics. Finally, NMR is also a valuable method for the detection of stable isotopes, and is extensively used for isotopic studies of metabolism as developed further in this chapter.
Application of Metabolomics to T. brucei – Selected Illustrations.
In this section, selected applications of metabolomics to trypanosomatids are given to illustrate the interest of these approaches for getting comprehensive understanding of the unique metabolic traits of these organisms. The examples are taken from T. brucei, for which significant metabolic knowledge has been recently gathered using metabolomics. More specifically, considerable progress in the identification of metabolic pathways in T. brucei was achieved by applying 13C isotopic profiling strategies to mutants impaired for specific enzymes by means of gene silencing (RNAi mutants) or deletion (KO mutants). Table 1 provides a list of these investigations, though probably not exhaustive. To help the reader in following the metabolic processes discussed below, a schematic representation of the current view of T. brucei central carbon metabolism is given in Fig. 2. For simplicity and consistency reasons, the examples are taken from the investigation of T. brucei PCFs.
Exo-metabolomics and Succinate Fermentation
The analysis of the exo-metabolome provides detailed information about the compounds that are either consumed or produced by cells. It gives the global result of cellular metabolic activity since it can be measured which compounds are used by the cells and into which products they are converted in. T. brucei PCFs convert glucose into succinate and acetate as major end-products while alanine, lactate and malate as minor ones 27,35. Succinate is an intermediate of TCA cycle and all enzymes of this pathway can be detected in PCFs 48. Together with the fact that PCFs have a functional ATP synthase coupled to aerobic respiration, this suggested that pyruvate was oxidized in the mitochondrion to fulfil the cells’ energetic needs by oxidative phosphorylation. It also suggested a mitochondrial origin of succinate, which is produced from succinyl-CoA in the TCA cycle. However, succinate could also be produced from fumarate via a controversial NADH-dependent fumarate reductase (FRD) activity, described in T. brucei PCFs 49,50. In 2002, Besteiro et al. investigated the metabolic origin of succinate in PCFs by applying isotopic profiling of the exo-metabolome to the study of relevant mutants 51. First, these authors established the glycosomal localization of FRD (accordingly called FRDg), and generated mutants in which the activity of the enzyme was switched off by using an RNA interference (RNAi) strategy. Then, they incubated PCFs with [1-13C]-labeled glucose, and analysed the labeling patterns of metabolic end-products in the medium using 13C-NMR. In FRDg-depleted parasites, succinate production was decreased by 60% compared to wild-type parasites, whereas a significant increase in both fumarate and malate were observed, indicating that most of succinate was produced via FRDg.
Moreover, detailed analysis of the labeling patterns of the metabolic end-products measured by 13C-NMR provided evidence that the TCA cycle could not be responsible for the production of the remaining succinate. Briefly, the glycolytic degradation of [1-13C]-glucose generates [3-13C]-pyruvate, which can be further converted into [2-13C]-acetyl-CoA to feed the TCA cycle. The singly-labeled acetyl-CoA entering the TCA cycle produces doubly – or more – labeled species after several turns of the cycle. Given the labeling pattern measured for acetyl-CoA, it was calculated that 15% of succinate molecules should be doubly labeled at both C2 and C3 ([2, 3-13C2]-succinate) if a complete operation of the TCA cycle was assumed. As mentioned above, NMR allows unambiguous discrimination of singly and doubly labeled species. But Besteiro et al. could not observe doubly-labeled succinate, but only singly labeled forms, in the exo-metabolome of PCFs incubated with [1-13C]-glucose, showing that the TCA cycle was unlikely to be operating as a complete cycle in T. brucei PCFs 51. One year later, van Weldeen et al. confirmed this hypothesis by demonstrating that deletion of the aconitase gene, encoding a TCA cycle enzyme, does not affect central metabolism of the parasite 52, although several branches of the cycle can be used in upon utilization of glucose or proline 53,54.
Acyl Acceptor in Central Metabolism
CoA functions as essential cofactor in many enzyme catalysed reactions. It is required by pyruvate dehydrogenase complex (PDH), which catalyses dehydrogenation and decarboxylation of pyruvate into acetyl-CoA. This enzyme uses CoA as acceptor of two carbon residues from decarboxylated pyruvate. Acetyl-CoA produced from decarboxylation of pyruvate and other sources i.e. β-oxidation of lipids or breakdown of proteins, is completely oxidized into CO2 and H2O in TCA cycle. The α-ketoglutarate dehydrogenase complex (AKGDH) operates in similar manner as PDH. AKGDH catalyses conversion of α-ketoglutarate into succinyl-CoA and CO2. The enzyme is metabolically important, because it also reduces NAD+ to NADH. In trypanosomes, AKGDH participates on catabolism of amino acids L-glutamine and L-proline. In this reaction, NAD+ functions as electron acceptor and CoA carries succinyl group. Succinyl-CoA then undergoes reversible reaction by succinyl-CoA thiokinase (succinyl-CoA synthetase, SCS or SCoAS) to form succinate and free CoA. The thioester bond in succinyl-CoA is energy-rich, its hydrolysis leads to energy release which is subsequently stored in form of phosphate bond of ATP.
Fatty Acid and Polyketide Biosynthesis
One of the intermediates in CoA biosynthesis, 4’-phosphopantetheine, is required for activation of acyl carrier protein (ACP) for fatty acid and polyketide biosynthesis103. ACP are autonomous domains or proteins of 80-100AA and they function as stabilization of acyl groups in fatty acid and polyketide biosynthesis. This protein link assures covalent catalysis in multistep processes of sequential condensation reactions 104,105 because it carries intermediates of fatty acid biosynthesis. Acyl groups are attached to the sulfhydryl terminus of 4’-phosphopantetheine prosthetic group 106. Inactive apo-form of ACP is activated by attack of β-OH-sidechain of serine residue onto pyrophosphate bond in CoA, which releases the 4’-phosphopantetheinyl moiety. Released moiety is transferred by 4′-phosphopantetheine transferase onto serine residue of ACP and free thiol may be esterified by substrate, i.e. acetyl-CoA or malonyl-CoA 107. In higher organisms, this synthetic function is taken over by fatty acid synthetase complex (FAS) where the carrier domain is also called ACP.
Degradation of Lipids and Branched Amino Acids
Another metabolically important process which requires participation of Coenzyme A, are catabolic pathways – such as β-odixation of fatty acids which is source of energy or catabolism of amino acids (proteins). Conjugation of fatty acid with CoA (activation) is a pre-requisite needed for their participation in catabolism. This reaction is catalysed by ATP-dependent acyl-CoA synthetases108. CoA conjugated fatty acids also play role in cell signalling and metabolic control109. In process of β-oxidation, two-carbon residues are cyclically cleaved from the fatty acyl chain which is catalysed by acyl-CoA synthetases. Eukaryotic organisms possess acyl-CoA synthetases of various chain length specificity, while E. coli contains only one synthetase (fadD).
Degradation of branched amino acids proceeds through acyl-CoA thioesters, in mammals branched amino acids feed the pool of propionyl-CoA which is then converted to succinyl-CoA. Mitochondrial enzyme responsible for conversion of propionyl-CoA and succinyl-CoA (methylmalonyl-CoA mutase) seems to be absent in T. brucei. Furthermore, L-threonine is converted into acetyl-CoA and glycine by activity of threonine dehydrogenase (TDH) because threonine dehydratase (threonine ammonia lyase) is missing – it would convert threonine into 2-oxobutyrate and ammonia110.
Table of contents :
1 Investigation of Acyl-CoA Metabolism in T. brucei
1.1 Methods to Investigate Metabolic Systems in Trypanosoma
1.2 Trypanosomes, Unconventional Organisms
1.4 Sample Preparation in Metabolomics
1.4.1 General Considerations
1.4.2 Sampling of T. brucei Metabolome
1.5 Analytical Platforms in Metabolomics
1.6 Isotopes and Isotopic Profiling
1.7 Application of Metabolomics to T. brucei – Selected Illustrations.
1.7.1 Exo-metabolomics and Succinate Fermentation
1.8 Endo-metabolomics, Redox Balances and Gluconeogenesis
1.9 Concluding Remarks
1.10 CoA Thioesters Metabolism in Trypanosomes
1.11 General Considerations
1.12 Acyl-CoA structure
1.13 CoA Biosynthesis
1.14 Functions of CoA and Acyl-CoAs in Metabolism
1.14.1 Acyl Acceptor in Central Metabolism
1.14.2 Fatty Acid and Polyketide Biosynthesis
1.14.3 Degradation of Lipids and Branched Amino Acids
1.14.4 Global Control of Metabolism
1.14.5 Metabolism of Acyl-CoAs in T. brucei
1.15 Objectives of the Thesis
2 Application of Method for Analysis of Acyl-CoA Thioesters
2.1 Introduction to Acyl-CoA Analytical Methodology
2.1.1 Introduction to Acyl-CoA Extraction Methodology
2.2 Application of Separation and Detection of Acyl-CoA thioesters
2.2.1 HPLC Separation of Acyl-CoAs
2.2.2 MS-based Detection of Acyl-CoAs
2.3 Validation of Analytical method
2.3.1 Parameters of Analytical Method
2.3.2 Isotope Dilution Mass Spectrometry (IDMS)
2.3.3 Applied Procedure
2.4 Results of Method Validation
2.4.1 Carry Over
2.4.2 Mass Accuracy
2.4.3 Repeatability and Reproducibility
2.4.4 Calibration Curves and Linearity
2.4.6 Limits of Detection and Quantitation
2.5 Extracting Acyl-CoA thioesters
2.5.1 Acyl-CoA Extraction in E. coli
2.5.2 Acyl-CoA Profile in E. coli
2.6 Analysis of Acyl-CoA Isotopic Profile
2.6.1 Isotopic Profile
3 Investigation of Acyl-CoA Network in E. coli
3.1 Functional Studies of Metabolic Systems by Instationary 13C-Metabolic Flux Analysis
3.1.1 General Principle of 13C-Metabolic Flux Analysis
3.1.2 Stationary and Instationary 13C-Metabolic Flux Analysis
3.2.1 Workflow for Functional Analysis of Acyl-CoAs Metabolism in E. coli
3.2.2 Quantitative Metabolomics and Isotopic Analyses of Acyl-CoA Thioesters in E. coli
3.2.3 Labeling Kinetics of Acyl-CoAs and Central Metabolites
3.2.4 Metabolic Model Linking Central Metabolism to Acyl-CoAs Metabolism
3.2.5 Metabolic Fluxes
3.2.6 Propionyl-CoA is Produced by The BCAAs Biosynthetic Pathway
3.2.7 Propionyl-CoA is Produced by The Pyruvate Formate Lyase PflB Under Aerobic Conditions
4 Acyl-CoA Thioesters in Trypanosoma brucei
4.1 Adaptation of Fast-Filtration method for CoA analysis in T. brucei
4.1.1 Sampling and Quenching
4.1.2 Extraction of Acyl-CoAs
4.2 Development of a New Sampling Method for Metabolomics and Isotopic Profiling Analyses of Acyl-CoA Thioesters
5 Discussion and outlook
5.1 Perspectives from Analytical Point of View
5.2 Perspectives in Metabolic Flux Analysis of E. coli
5.3 Perspectives in Study of T. brucei Metabolism
6 Materials and Methods
6.1 E. coli cultivation
6.1.1 Pre-cultivation E. coli
6.1.2 Cultivation E. coli
6.1.3 Acyl-CoA Quantification in E. coli
6.1.4 Isotopic Profiling
6.1.5 Quantification of Amino Acids and Central Metabolites
6.1.6 Calculation of Metabolite Intracellular Concentration in E. coli
6.2 T. brucei PCF Cultivation
6.2.1 T. brucei sample Collection
6.3 Isotopically Enriched Standards
6.4 IDMS E.coli
6.5 Preparation of Solvents
6.6 HPLC-HRMS Analysis of Acyl-Coenzyme A Thioesters
6.7 HPLC-HRMS Analysis of Amino Acids
6.8 IC-MS analysis of Organic Acids and Phosphorylated Compounds
6.9 Analysis of Extracellular Metabolites
6.10 Data Treatment Software
6.11 Calculation of metabolic fluxes