Mapping the metabolism of five amino acids in bloodstream form <i>Trypanosoma brucei</i> using U-13C-labelled substrates and LC–MS

Johnston, Katharina; Kim, Dong-Hyun; Kerkhoven, Eduard J.; Burchmore, Richard; Barrett, Michael P.; Achcar, Fiona

doi:10.1042/bsr20181601

Cited by 20 publications

(55 citation statements)

References 61 publications

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“…In a recent report, it was shown that in T brucei bloodstream forms phosphoarginine is exclusively generated by the action of arginine kinases. 73 Although arginine kinase knock-out parasites were viable in culture, 73 it is believed that phosphoarginine plays a central role in regenerating ATP from ADP in situations of high (short term) energy demand. 75 Remarkably, while the analysis of the proteome during CL depletion revealed a pronounced decrease in the levels of several subunits of the inner mitochondrial membrane F o F 1 -ATPase (Table S1; see also above), one protein annotated as putative ATP synthase subunit (Tb927.11.9420, 1.4-fold upregulated) was found to be increased (Table S2).…”

Section: Discussionmentioning

confidence: 99%

Depletion of cardiolipin induces major changes in energy metabolism in Trypanosoma brucei bloodstream forms

et al. 2020

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The mitochondrial inner membrane glycerophospholipid cardiolipin (CL) associates with mitochondrial proteins to regulate their activities and facilitate protein complex and supercomplex formation. Loss of CL leads to destabilized respiratory complexes and mitochondrial dysfunction. The role of CL in an organism lacking a conventional electron transport chain (ETC) has not been elucidated. Trypanosoma brucei bloodstream forms use an unconventional ETC composed of glycerol-3-phosphate dehydrogenase and alternative oxidase (AOX), while the mitochondrial membrane potential (ΔΨm) is generated by the hydrolytic action of the F o F 1 -ATP synthase (aka F o F 1 -ATPase). We now report that the inducible depletion of cardiolipin synthase (TbCls) is essential for survival of T brucei bloodstream forms. Loss of CL caused a rapid drop in ATP levels and a decline in the ΔΨm. Unbiased proteomic analyses revealed a reduction in the levels of many mitochondrial proteins, most notably of F o F 1 -ATPase subunits and AOX, resulting in a strong decline of glycerol-3-phosphate-stimulated oxygen consumption.The changes in cellular respiration preceded the observed decrease in F o F 1 -ATPase stability, suggesting that the AOX-mediated ETC is the first pathway responding to the decline in CL. Select proteins and pathways involved in glucose and amino acid metabolism were upregulated to counteract the CL depletion-induced drop in cellular ATP.

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Section: Discussionmentioning

confidence: 99%

Depletion of cardiolipin induces major changes in energy metabolism in Trypanosoma brucei bloodstream forms

et al. 2020

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“…It has recently been described as an epigenetic modifier that governs T cell differentiation and plays a role in cancer initiation and progression [28,46]. This metabolite was also detected in the metabolome of BSF trypanosomes, being derived from the metabolism of glutamate [30]. Here we showed that 2-hydroxyglutarate represents 37% of the total end-products excreted by PCF from catabolism of α-ketoglutarate, with an excretion rate of 2-hydroxyglutarate 3.4 times higher than that of end-products from glucose metabolism (6.77 versus 2.02 µmole/h/mg of protein).…”

Section: Discussionmentioning

confidence: 94%

“…The ∆kdh-e2 null mutant excretes only 2-hydroxyglutarate from metabolism of α-ketoglutarate, suggesting that a single reduction step, catalyzed by an as yet unknown enzyme, produces 2hydroxyglutarate from α-ketoglutarate [28,29]. It has recently been proposed that 2hydroxyglutarate detected in the T. brucei BSF metabolome results from the promiscuous action of the NADH-dependent malate dehydrogenase on α-ketoglutarate [30]. 2-hydroxyglutarate is also produced from malate and succinate by the ∆kdh-e2 null mutant ( Figs 3B and 5A).…”

Section: Metabolism Of α-Ketoglutarate In the Presence Of Prolinementioning

confidence: 99%

Fly stage trypanosomes recycle glucose catabolites and TCA cycle intermediates to stimulate growth in near physiological conditions

Villafraz

Biran

Pineda

et al. 2020

Preprint

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Trypanosoma brucei, a protist responsible for human African trypanosomiasis (sleeping sickness), is transmitted by the tsetse fly, where the procyclic forms of the parasite develop in the proline-rich (1-2 mM) and glucose-depleted digestive tract. Proline is essential for the midgut colonization of the parasite in the insect vector, however other carbon sources could be available and used to feed its central metabolism. Here we show that procyclic trypanosomes can consume and metabolize metabolic intermediates, including those excreted from glucose catabolism (succinate, alanine and pyruvate), with the exception of acetate, which is the ultimate end-product excreted by the parasite. Among the tested metabolites, tricarboxylic acid (TCA) cycle intermediates (succinate, malate and α-ketoglutarate) stimulated growth of the parasite in the presence of 2 mM proline. The pathways used for their metabolism were mapped by proton-NMR metabolic profiling and phenotypic analyses of a dozen RNAi and/or null mutants affecting central carbon metabolism. We showed that (i) malate is converted to succinate by both the reducing and oxidative branches of the TCA cycle, which demonstrates that procyclic trypanosomes can use the full TCA cycle, (ii) the enormous rate of α-ketoglutarate consumption (15-times higher than glucose) is possible thanks to the balanced production and consumption of NADH at the substrate level and (iii) α-ketoglutarate is toxic for trypanosomes if not appropriately metabolized as observed for an α-ketoglutarate dehydrogenase null mutant. In addition, epimastigotes produced from procyclics upon overexpression of RBP6, showed a growth defect in the presence of 2 mM proline, which is rescued by α-ketoglutarate, suggesting that physiological amounts of proline are not sufficient per se for the development of trypanosomes in the fly. In conclusion, these data show that trypanosomes can metabolize multiple metabolites, in addition to proline, which allows them to confront challenging environments in the fly.Author SummaryIn the midgut of its insect vector, trypanosomes rely on proline to feed their energy metabolism. However, the availability of other potential carbon sources that can be used by the parasite is currently unknown. Here we show that tricarboxylic acid (TCA) cycle intermediates, i.e. succinate, malate and α-ketoglutarate, stimulate growth of procyclic trypanosomes incubated in medium containing 2 mM proline, which is in the range of the amounts measured in the midgut of the fly. Some of these additional carbon sources are needed for the development of epimastigotes, which differentiate from procyclics in the midgut of the fly, since their growth defect observed in the presence of 2 mM proline is rescued by addition of α-ketoglutarate. In addition, we have implemented new approaches to study a poorly explored branch of the TCA cycle converting malate to α-ketoglutarate, which was previously described as non-functional in the parasite, regardless of the glucose levels available. The discovery of this branch reveals that a full TCA cycle can operate in procyclic trypanosomes. Our data broaden the metabolic potential of trypanosomes and pave the way for a better understanding of the parasite’s metabolism in various organ systems of the tsetse fly, where it evolves.

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“…It is well established that trypanosomatid parasites scavenge amino acids, key nutrients for survival, from their hosts [85, 86]. Therefore, comparative analyses of T. congolense and T. brucei amino acid metabolism were undertaken.…”

Section: Resultsmentioning

confidence: 99%

“…BSF T. brucei utilizes exogenous L-glutamine as the primary source of intracellular glutamate and 2-oxoglutarate and produce significant levels of glutamine-derived succinate [42, 85] (Fig 6I). Given the high levels of succinate excreted by T. congolense , stable isotope labelling was used to determine the contribution of L-glutamine to this pool.…”

Section: Resultsmentioning

confidence: 99%

Divergent metabolism betweenTrypanosoma congolenseandTrypanosoma bruceiresults in differential drug sensitivity

Steketee

Dickie

Iremonger

et al. 2021

Preprint

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Animal African Trypanosomiasis (AAT) is a debilitating livestock disease prevalent across sub-Saharan Africa, a main cause of which is the protozoan parasite Trypanosoma congolense. In comparison to the well-studied T. brucei, there is a major paucity of knowledge regarding the biology of T. congolense. Here, we use a combination of omics technologies and novel genetic tools to characterise core metabolism in T. congolense mammalian-infective bloodstream-form parasites, and test whether metabolic differences compared to T. brucei impact upon drug sensitivity. Like T. brucei, glycolysis plays a major part in T. congolense energy metabolism. However, the rate of glucose uptake is significantly reduced in T. congolense, with cells remaining viable when cultured in concentrations as low as 2 mM. Instead of pyruvate, the primary glycolytic endpoints are succinate, malate and acetate. Comparative transcriptomics analysis showed higher levels of activity associated with the mitochondrial pyruvate dehydrogenase complex, acetate generation and the succinate shunt in T. congolense. However, based on omics analysis and chemical inhibition, there does not appear to be significant levels of oxidative phosphorylation. Stable-isotope labelling of glucose enabled the comparison of carbon usage between T. brucei and T. congolense, highlighting differences in nucleotide and fatty acid metabolism. To validate the metabolic similarities and differences, both species were treated with pharmacological inhibitors, confirming a lack of essential electron transport chain activity in T. congolense, but increased sensitivity to inhibition of mitochondrial pyruvate import. Strikingly, T. congolense exhibited significant resistance to inhibitors of fatty acid synthesis, including a 780-fold greater EC50 against the lipase and fatty acid synthase inhibitor Orlistat, compared to T. brucei. These data highlight that bloodstream form T. congolense diverges from T. brucei in key areas of metabolism, with several features that are intermediate between bloodstream- and insect-stage T. brucei. These results have implications for drug development, mechanisms of drug resistance and host-pathogen interactions.

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Mapping the metabolism of five amino acids in bloodstream form Trypanosoma brucei using U-13C-labelled substrates and LC–MS

Cited by 20 publications

References 61 publications

Depletion of cardiolipin induces major changes in energy metabolism in Trypanosoma brucei bloodstream forms

Depletion of cardiolipin induces major changes in energy metabolism in Trypanosoma brucei bloodstream forms

Fly stage trypanosomes recycle glucose catabolites and TCA cycle intermediates to stimulate growth in near physiological conditions

Divergent metabolism betweenTrypanosoma congolenseandTrypanosoma bruceiresults in differential drug sensitivity

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