Gluconeogenesis using glycerol as a substrate in bloodstream-form Trypanosoma brucei

Abstract

Bloodstream form African trypanosomes are thought to rely exclusively upon glycolysis, using glucose as a substrate, for ATP production. Indeed, the pathway has long been considered a potential therapeutic target to tackle the devastating and neglected tropical diseases caused by these parasites. However, plasma membrane glucose and glycerol transporters are both expressed by trypanosomes and these parasites can infiltrate tissues that contain glycerol. Here, we show that bloodstream form trypanosomes can use glycerol for gluconeogenesis and for ATP production, particularly when deprived of glucose following hexose transporter depletion. We demonstrate that Trypanosoma brucei hexose transporters 1 and 2 (THT1 and THT2) are localized to the plasma membrane and that knockdown of THT1 expression leads to a growth defect that is more severe when THT2 is also knocked down. These data are consistent with THT1 and THT2 being the primary routes of glucose supply for the production of ATP by glycolysis. However, supplementation of the growth medium with glycerol substantially rescued the growth defect caused by THT1 and THT2 knockdown. Metabolomic analyses with heavy-isotope labelled glycerol demonstrated that trypanosomes take up glycerol and use it to synthesize intermediates of gluconeogenesis, including fructose 1,6-bisphosphate and hexose 6-phosphates, which feed the pentose phosphate pathway and variant surface glycoprotein biosynthesis. We used Cas9-mediated gene knockout to demonstrate a gluconeogenesis-specific, but fructose-1,6-bisphosphatase (Tb927.9.8720)-independent activity, converting fructose 1,6-bisphosphate into fructose 6-phosphate. In addition, we observed increased flux through the tricarboxylic acid cycle and the succinate shunt. Thus, contrary to prior thinking, gluconeogenesis can operate in bloodstream form T. brucei. This pathway, using glycerol as a physiological substrate, may be required in mammalian host tissues.

Sterol 14-α-demethylase is vital for mitochondrial functions and stress tolerance in Leishmania major

“…While glycerol could be converted into glucose, it is unlikely to account for the rescue as the energy required for gluconeogenesis exceeds that generated from glycolysis. Instead, it is possible that Leishmania parasites metabolize glycerol into glycerol-3-phosphate (by glycerol kinase) and then dihydroxyacetone phosphate (by the mitochondrial FAD-dependent glycerol-3-phosphate dehydrogenase) which can be incorporated into the glycolysis pathway, as described in T. brucei [50,51]. We also observed increased uptake of glucose and glycerol by c14dm�mutants (Fig 6), a possible adaptive response to their high demand for carbon sources.…”

“…Recent studies have revealed that bloodstream forms of Trypanosoma brucei can use glycerol to support growth in the absence of glucose [50,51]. To test if Leishmania parasites can utilize glycerol effectively, we attempted to rescue c14dm�mutants with glycerol.…”

Cell-based and multi-omics profiling reveals dynamic metabolic repurposing of mitochondria to drive developmental progression of Trypanosoma brucei

“…For the LC-MS metabolomic analysis, the sample extraction was performed as described previously [81,82]. Briefly, 5 × 10 7 cells were used for each sample.…”

Glucose 6-Phosphate Dehydrogenase from Trypanosomes: Selectivity for Steroids and Chemical Validation in Bloodstream Trypanosoma brucei

“…Nonetheless, a significant amount of cytosolic NADPH has been shown to be produced by the PPP (ca. 25% in bloodstream T. brucei ) due to gluconeogenesis fueled by glycerol (in the mammalian stage) [ 12 ] or proline (in the insect stage) [ 13 ]. G6PDH [ 14 ] and 6PGDH [ 15 ] proved indispensable for the bloodstream form of T. brucei , though it remains unclear whether this is a consequence of a shortage of NAPDH and/or ribose 5-phosphate.…”