Charles Ebikeme

Increasing the mass accuracy of high-resolution LC-MS data using background ions - a case study on the LTQ-Orbitrap

With the advent of a new generation of high‐resolution mass spectrometers, the fields of proteomics and metabolomics have gained powerful new tools. In this paper, we demonstrate a novel computational method that improves the mass accuracy of the LTQ‐Orbitrap mass spectrometer from an initial ±1–2 ppm, obtained by the standard software, to an absolute median of 0.21 ppm (SD 0.21 ppm). With the increased mass accuracy it becomes much easier to match mass chromatograms in replicates and different sample types, even if compounds are detected at very low intensities. The proposed method exploits the ubiquitous presence of background ions in LC‐MS profiles for accurate alignment and internal mass calibration, making it applicable for all types of MS equipment. The accuracy of this approach will facilitate many downstream systems biology applications, including mass‐based molecule identification, ab initio metabolic network reconstruction, and untargeted metabolomics in general.

Cytosolic NADPH homeostasis in glucose-starved procyclic Trypanosoma brucei relies on malic enzyme and the pentose phosphate pathway fed by gluconeogenic flux.

Background: NADPH production is critical for growth and oxidative stress management. Results: Redundancy of the pentose phosphate pathway and the cytosolic malic enzyme for NADPH synthesis is carbon source-independent in procyclic trypanosomes. Conclusion: The parasite has gluconeogenic capacity from proline. Significance: This work illustrates the flexible carbon source-dependent flux changes for essential NADPH supply. All living organisms depend on NADPH production to feed essential biosyntheses and for oxidative stress defense. Protozoan parasites such as the sleeping sickness pathogen Trypanosoma brucei adapt to different host environments, carbon sources, and oxidative stresses during their infectious life cycle. The procyclic stage develops in the midgut of the tsetse insect vector, where they rely on proline as carbon source, although they prefer glucose when grown in rich media. Here, we investigate the flexible and carbon source-dependent use of NADPH synthesis pathways in the cytosol of the procyclic stage. The T. brucei genome encodes two cytosolic NADPH-producing pathways, the pentose phosphate pathway (PPP) and the NADP-dependent malic enzyme (MEc). Reverse genetic blocking of those pathways and a specific inhibitor (dehydroepiandrosterone) of glucose-6-phosphate dehydrogenase together established redundancy with respect to H2O2 stress management and parasite growth. Blocking both pathways resulted in ∼10-fold increase of susceptibility to H2O2 stress and cell death. Unexpectedly, the same pathway redundancy was observed in glucose-rich and glucose-depleted conditions, suggesting that gluconeogenesis can feed the PPP to provide NADPH. This was confirmed by (i) a lethal phenotype of RNAi-mediated depletion of glucose-6-phosphate isomerase (PGI) in the glucose-depleted Δmec/Δmec null background, (ii) an ∼10-fold increase of susceptibility to H2O2 stress observed for the Δmec/Δmec/RNAiPGI double mutant when compared with the single mutants, and (iii) the 13C enrichment of glycolytic and PPP intermediates from cells incubated with [U-13C]proline, in the absence of glucose. Gluconeogenesis-supported NADPH supply may also be important for nucleotide and glycoconjugate syntheses in the insect host.

Ablation of succinate production from glucose metabolism in the procyclic trypanosomes induces metabolic switches to the glycerol 3-phosphate/dihydroxyacetone phosphate shuttle and to proline metabolism.

Trypanosoma brucei is a parasitic protist that undergoes a complex life cycle during transmission from its mammalian host (bloodstream forms) to the midgut of its insect vector (procyclic form). In both parasitic forms, most glycolytic steps take place within specialized peroxisomes, called glycosomes. Here, we studied metabolic adaptations in procyclic trypanosome mutants affected in their maintenance of the glycosomal redox balance. T. brucei can theoretically use three strategies to maintain the glycosomal NAD+/NADH balance as follows: (i) the glycosomal succinic fermentation branch; (ii) the glycerol 3-phosphate (Gly-3-P)/dihydroxyacetone phosphate (DHAP) shuttle that transfers reducing equivalents to the mitochondrion; and (iii) the glycosomal glycerol production pathway. We showed a hierarchy in the use of these glycosomal NADH-consuming pathways by determining metabolic perturbations and adaptations in single and double mutant cell lines using a combination of NMR, ion chromatography-MS/MS, and HPLC approaches. Although functional, the Gly-3-P/DHAP shuttle is primarily used when the preferred succinate fermentation pathway is abolished in the Δpepck knock-out mutant cell line. In the absence of these two pathways (Δpepck/RNAiFAD-GPDH.i mutant), glycerol production is used but with a 16-fold reduced glycolytic flux. In addition, the Δpepck mutant cell line shows a 3.3-fold reduced glycolytic flux compensated by an increase of proline metabolism. The inability of the Δpepck mutant to maintain a high glycolytic flux demonstrates that the Gly-3-P/DHAP shuttle is not adapted to the procyclic trypanosome context. In contrast, this shuttle was shown earlier to be the only way used by the bloodstream forms of T. brucei to sustain their high glycolytic flux.

Cell-Penetrating Peptide TP10 Shows Broad-Spectrum Activity against both Plasmodium falciparum and Trypanosoma brucei brucei

ABSTRACT Malaria and trypanosomiasis are diseases which afflict millions and for which novel therapies are urgently required. We have tested two well-characterized cell-penetrating peptides (CPPs) for antiparasitic activity. One CPP, designated TP10, has broad-spectrum antiparasitic activity against Plasmodium falciparum, both blood and mosquito stages, and against blood-stage Trypanosoma brucei brucei.

The threonine degradation pathway of the Trypanosoma brucei procyclic form: the main carbon source for lipid biosynthesis is under metabolic control

The Trypanosoma brucei procyclic form resides within the digestive tract of its insect vector, where it exploits amino acids as carbon sources. Threonine is the amino acid most rapidly consumed by this parasite, however its role is poorly understood. Here, we show that the procyclic trypanosomes grown in rich medium only use glucose and threonine for lipid biosynthesis, with threonines contribution being ∼ 2.5 times higher than that of glucose. A combination of reverse genetics and NMR analysis of excreted end‐products from threonine and glucose metabolism, shows that acetate, which feeds lipid biosynthesis, is also produced primarily from threonine. Interestingly, the first enzymatic step of the threonine degradation pathway, threonine dehydrogenase (TDH, EC 1.1.1.103), is under metabolic control and plays a key role in the rate of catabolism. Indeed, a trypanosome mutant deleted for the phosphoenolpyruvate decarboxylase gene (PEPCK, EC 4.1.1.49) shows a 1.7‐fold and twofold decrease of TDH protein level and activity, respectively, associated with a 1.8‐fold reduction in threonine‐derived acetate production. We conclude that TDH expression is under control and can be downregulated in response to metabolic perturbations, such as in the PEPCK mutant in which the glycolytic metabolic flux was redirected towards acetate production.

ATP Synthesis-coupled and -uncoupled Acetate Production from Acetyl-CoA by Mitochondrial Acetate:Succinate CoA-transferase and Acetyl-CoA Thioesterase in Trypanosoma

Background: Mitochondrial acetate production is essential for viability of the procyclic trypanosomes and probably many other protists. Results: We identified an acetyl-CoA thioesterase (ACH) contributing to acetate production from acetyl-CoA. Conclusion: Acetate production by ASCT, but not by ACH, is involved in ATP production. Significance: In trypanosomes and probably other protists, ASCT/SCoAS cycle-derived ATP production can substitute for oxidative phosphorylation. Insect stage trypanosomes use an “acetate shuttle” to transfer mitochondrial acetyl-CoA to the cytosol for the essential fatty acid biosynthesis. The mitochondrial acetate sources are acetate:succinate CoA-transferase (ASCT) and an unknown enzymatic activity. We have identified a gene encoding acetyl-CoA thioesterase (ACH) activity, which is shown to be the second acetate source. First, RNAi-mediated repression of ASCT in the ACH null background abolishes acetate production from glucose, as opposed to both single ASCT and ACH mutants. Second, incorporation of radiolabeled glucose into fatty acids is also abolished in this ACH/ASCT double mutant. ASCT is involved in ATP production, whereas ACH is not, because the ASCT null mutant is ∼1000 times more sensitive to oligomycin, a specific inhibitor of the mitochondrial F0/F1-ATP synthase, than wild-type cells or the ACH null mutant. This was confirmed by RNAi repression of the F0/F1-ATP synthase F1β subunit, which is lethal when performed in the ASCT null background but not in the wild-type cells or the ACH null background. We concluded that acetate is produced from both ASCT and ACH; however, only ASCT is responsible, together with the F0/F1-ATP synthase, for ATP production in the mitochondrion.

Alanine aminotransferase of Trypanosoma brucei--a key role in proline metabolism in procyclic life forms.

African trypanosomes possess high levels of alanine aminotransferase (EC 2.6.1.2), although the function of their activity remains enigmatic, especially in slender bloodstream forms where the metabolism of ketoacids does not occur. Therefore, the gene for alanine aminotransferase enzyme in Trypanosoma brucei (TbAAT) was characterized and its function assessed using a combination of RNA interference and gene knockout approaches. Surprisingly, as much as 95% or more of the activity appears to be unnecessary for growth of either bloodstream or procyclic forms respiring on glucose. A combination of RNA interference and NMR spectroscopy revealed an important role for the activity in procyclic forms respiring on proline. Under these conditions, the major end product of proline metabolism is alanine, and a reduction in TbAAT activity led to a proportionate decrease in the amount of alanine excreted along with an increase in the doubling time of the cells. These results provide evidence of a role for alanine aminotransferase in the metabolism of proline in African trypanosomes by linking glutamate produced by the initial oxidative steps of the pathway with pyruvate produced by the final oxidative step of the pathway. This step appears to be essential when proline is the primary carbon source, which is likely to be the physiological situation in the tsetse fly vector.

TrypanoCyc : a community-led biochemical pathways database for Trypanosoma brucei

The metabolic network of a cell represents the catabolic and anabolic reactions that interconvert small molecules (metabolites) through the activity of enzymes, transporters and non-catalyzed chemical reactions. Our understanding of individual metabolic networks is increasing as we learn more about the enzymes that are active in particular cells under particular conditions and as technologies advance to allow detailed measurements of the cellular metabolome. Metabolic network databases are of increasing importance in allowing us to contextualise data sets emerging from transcriptomic, proteomic and metabolomic experiments. Here we present a dynamic database, TrypanoCyc (http://www.metexplore.fr/trypanocyc/), which describes the generic and condition-specific metabolic network of Trypanosoma brucei, a parasitic protozoan responsible for human and animal African trypanosomiasis. In addition to enabling navigation through the BioCyc-based TrypanoCyc interface, we have also implemented a network-based representation of the information through MetExplore, yielding a novel environment in which to visualise the metabolism of this important parasite.

Transketolase in Trypanosoma brucei

A single copy gene, encoding a protein highly similar to transketolase from other systems, was identified in the Trypanosoma brucei genome. The gene was expressed in E. coli and the purified protein demonstrated transketolase activity with K(m) values of 0.2mM and 0.8mM respectively for xylulose 5-phosphate and ribose 5-phosphate. A peroxisomal targeting signal (PTS-1) present at the C-terminus of the protein suggested a glycosomal localisation. However, subcellular localisation experiments revealed that while the protein was present in glycosomes it was found mainly within the cytosol and thus has a dual localisation. Transketolase activity was absent from the long slender bloodstream form of the parasite and the protein was not detectable in this life cycle stage, with the RNA present only at low abundance, indicating a strong differential regulation, being present predominantly in the procyclic form. The gene was knocked out from procyclic T. brucei and transketolase activity was lost but no growth phenotype was evident in the null mutants. Metabolite profiling to compare wild type and TKT null mutants revealed substantial increases in transketolase substrate metabolites coupled to loss of sedoheptulose 7-phosphate, a principal product of the transketolase reaction.

Acetate and succinate production in amoebae, helminths, diplomonads, trichomonads and trypanosomatids: common and diverse metabolic strategies used by parasitic lower eukaryotes.

Parasites that often grow anaerobically in their hosts have adopted a fermentative strategy relying on the production of partially oxidized end products, including lactate, glycerol, ethanol, succinate and acetate. This review focuses on recent progress in understanding acetate production in protist parasites, such as amoebae, diplomonads, trichomonads, trypanosomatids and in the metazoan parasites helminths, as well as the succinate production pathway(s) present in some of them. We also describe the unconventional organisation of the tricarboxylic acid cycle associated with the fermentative strategy adopted by the procyclic trypanosomes, which may resemble the probable structure of the primordial TCA cycle in prokaryotes.