Cytosolic NADPH Homeostasis in Glucose-starved Procyclic Trypanosoma brucei Relies on Malic Enzyme and the Pentose Phosphate Pathway Fed by Gluconeogenic Flux

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 H 2 O 2 stress management and parasite growth. Blocking both pathways resulted in ∼10-fold increase of susceptibility to H 2 O 2 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 Δ mec /Δ mec null background, ( ii ) an ∼10-fold increase of susceptibility to H 2 O 2 stress observed for the Δ mec /Δ mec / RNAi PGI double mutant when compared with the single mutants, and ( iii ) the 13 C enrichment of glycolytic and PPP intermediates from cells incubated with [U- 13 C]proline, in the absence of glucose. Gluconeogenesis-supported NADPH supply may also be important for nucleotide and glycoconjugate syntheses in the insect host. 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.