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

Abstract

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.

Divergent metabolism betweenTrypanosoma congolenseandTrypanosoma bruceiresults in differential drug sensitivity

“…Interestingly, we observed a reproducible reduction in pyruvate levels in T. congolense supernatants over time, before abundance of this metabolite returned to levels similar to those observed in negative controls. A recent study in PCF T. brucei demonstrated that these parasites can re-metabolize glycolytic end products such as pyruvate and succinate [37]. Stable isotope labelling patterns in catabolic products derived from glucose do not support cyclical TCA activity, nor re-uptake of excreted metabolites in BSF T. congolense .…”

“…Rather than oxidative phosphorylation, we propose it is likelier that considerable ATP production occurs in the acetate:succinate CoA transferase – succinyl-CoA synthetase (SCS) cycle, which would explain the high levels of acetate generated by T. congolense , in addition to increased sensitivity to inhibition of mitochondrial uptake of pyruvate, the key metabolic precursor. Given that 2-oxoglutarate dehydrogenase complex expression appears to be less than, or equal to, that in T. brucei (under in vitro culturing conditions, Fig 9), it is likely that SCS activity occurs in the acetate-generating pathway rather than in the TCA cycle, which is not thought to be fully functional in BSF African trypanosomes [35], although recent data have challenged this paradigm in PCF T. brucei [37]. The mechanisms proposed here bear some similarities to the scheme proposed by Dewar and colleagues for stumpy-form T. brucei metabolism, which also exhibit increased mitochondrial metabolism compared to BSF T. brucei [107].…”

“…Until recently, it was thought that PCF T. brucei did not exhibit active TCA metabolism, although recent data have shown that TCA intermediates such as succinate and 2-oxoglutarate can stimulate PCF T. brucei growth. [35–37].…”