Date Published: December 19, 2013
Publisher: Public Library of Science
Author(s): Muriel Mazet, Pauline Morand, Marc Biran, Guillaume Bouyssou, Pierrette Courtois, Sylvie Daulouède, Yoann Millerioux, Jean-Michel Franconi, Philippe Vincendeau, Patrick Moreau, Frédéric Bringaud, Reza Salavati. http://doi.org/10.1371/journal.pntd.0002587
Abstract: BackgroundThe bloodstream forms of Trypanosoma brucei, the causative agent of sleeping sickness, rely solely on glycolysis for ATP production. It is generally accepted that pyruvate is the major end-product excreted from glucose metabolism by the proliferative long-slender bloodstream forms of the parasite, with virtually no production of succinate and acetate, the main end-products excreted from glycolysis by all the other trypanosomatid adaptative forms, including the procyclic insect form of T. brucei.Methodology/Principal FindingsA comparative NMR analysis showed that the bloodstream long-slender and procyclic trypanosomes excreted equivalent amounts of acetate and succinate from glucose metabolism. Key enzymes of acetate production from glucose-derived pyruvate and threonine are expressed in the mitochondrion of the long-slender forms, which produces 1.4-times more acetate from glucose than from threonine in the presence of an equal amount of both carbon sources. By using a combination of reverse genetics and NMR analyses, we showed that mitochondrial production of acetate is essential for the long-slender forms, since blocking of acetate biosynthesis from both carbon sources induces cell death. This was confirmed in the absence of threonine by the lethal phenotype of RNAi-mediated depletion of the pyruvate dehydrogenase, which is involved in glucose-derived acetate production. In addition, we showed that de novo fatty acid biosynthesis from acetate is essential for this parasite, as demonstrated by a lethal phenotype and metabolic analyses of RNAi-mediated depletion of acetyl-CoA synthetase, catalyzing the first cytosolic step of this pathway.Conclusions/SignificanceAcetate produced in the mitochondrion from glucose and threonine is synthetically essential for the long-slender mammalian forms of T. brucei to feed the essential fatty acid biosynthesis through the “acetate shuttle” that was recently described in the procyclic insect form of the parasite. Consequently, key enzymatic steps of this pathway, particularly acetyl-CoA synthetase, constitute new attractive drug targets against trypanosomiasis.
Partial Text: Trypanosoma brucei is a unicellular eukaryote, belonging to the protozoan order Kinetoplastida that causes sleeping sickness in humans and economically important livestock diseases . This parasite undergoes a complex life cycle during transmission from the bloodstream of a mammalian host (bloodstream forms of the parasite – BSF) to the alimentary tract (procyclic form – PF) and salivary glands (epimastigote and metacyclic forms) of a blood feeding insect vector, the tsetse fly. In the bloodstream of the mammalian host, the pleomorphic BSF strains proliferate as “long-slender” BSF (LS-BSF) and differentiate into the non-proliferative “short-stumpy” trypanosomes (SS-BSF), which are preadapted for differentiation into PF in the insect midgut . The environmental changes encountered by the parasite require significant morphological and metabolic adaptations, as exemplified by important qualitative and quantitative differences in glucose metabolism between BSF and PF , .
LS-BSF trypanosomes are well known for their glucose-dependency to satisfy ATP requirements . Indeed, net production of all cellular ATP is fulfilled by the last glycolytic step catalyzed by pyruvate kinase, which produces pyruvate, the excreted glycolytic end-product. Excretion of significant amounts of other partially oxidized end-products of glycolysis, such as glycerol, succinate and alanine, has been previously reported , , , . However, in the late seventies emerged a general dogma whereby pyruvate was considered the exclusive glycolytic end-product excreted from LS-BSF under aerobic conditions , , because the minor end-products were assigned to either non-growing conditions or contamination with non-dividing SS-BSF , . Here, we used as an experimental model the 427 BSF strain, which has lost the ability to differentiate into SS-BSF, in order to focus our analysis of glucose metabolism on LS-BSF trypanosomes. This laboratory-adapted monomorphic strain is insensitive to the stumpy inductor factor, but, it successfully differentiates in vitro into bona fide SS-BSF, for instance when expression of the protein kinase target of rapamycin (TOR4) is inhibited . This suggests that the 427 strain can be considered as a slender-like BSF that has lost the ability to respond to the stumpy inductor factor, and as such is the relevant model to study the metabolism of proliferative BSF. Our metabolic analyses showed that LS-BSF can produce almost as much succinate and acetate from glucose as PF incubated in the same conditions. This suggests that most, if not all, enzymes involved in the “succinate and acetate branches” previously characterized in PF are also expressed in LS-BSF. To produce acetate, PDH (step 1 in Fig. 1) converts pyruvate into acetyl-CoA, which is the substrate of ASCT (step 2) and ACH (step 3) for acetate production. ASCT expression is low in BSF (Fig. 2 and ), while ACH is relatively abundant (Fig. 2) with an ACH activity ∼2-fold higher than PF (data not shown). Three of the four PDH subunits have been investigated so far and are expressed in BSF (PDH-E1α and PDH-E2, see Fig. 2; PDH-E3, ), with a PDH enzymatic activity only 4-fold lower than in PF (Fig. 2). This relatively high PDH activity is in agreement with a recent comparative SILAC proteomics analysis showing that PDH-E1α, PDH-E1ß, PDH-E2 and PDH-E3 are 5.3-, 7.6-, 5.2- and 8.1-fold more abundant in PF than LS-BSF, respectively . The same proteomics analysis in LS-BSF also detected most, if not all, of the enzymes involved in succinate production from phosphoenolpyruvate, although at a lower level of expression than in PF (between 3- and 20-fold). Altogether, this clearly demonstrates that LS-BSF have maintained the capacity to produce and excrete acetate and succinate from glycolysis. The relatively high rate of acetate and succinate production (only ∼2-fold higher in PF), while the enzymes involved in the corresponding metabolic pathways are 5- to 20-times more abundant in PF, may be due to the considerably higher glycolytic flux in BSF. We determined that the excretion rate of glycolytic end-products is 9.5-fold higher in BSF than in PF (9115 versus 968 nmol of excreted end-products/h/108 cells), which is consistent with the previously measured 5- to 10-fold difference in glycolytic flux . A recent analysis of the glycolytic flux in the same BSF strain incubated in growing conditions (IMDM containing 20 mM glucose) showed a higher rate of pyruvate production compared to our analysis performed in PBS containing 4 mM glucose and threonine (19.2 versus 12.0 µmol/h/108 cells) . The reduced glycolytic flux observed in PBS conditions certainly reflects the difference between non-growing conditions (PBS) and exponential growth (IMDM) with a doubling time in the range of 5 h . Also, the 35% reduction in total end-product fluxes for the wild type cells depending on the available substrates (PBS/glucose versus PBS/glucose/threonine) shows that metabolic fluxes are dependent on the exact context of substrates present (Table 1).