Background: Deoxyhypusine synthase (DHS) catalyzes the spermidine-dependent modification of translation factor eIF5A.Results: Trypanosomatid DHS activity is increased 3000-fold by heterotetramer formation with a catalytically dead paralog, and both gene products are essential for parasite growth.Conclusion: Trypanosomatid DHS is a complex between catalytically impaired and inactive DHS subunits.Significance: This activation mechanism uniquely evolved for two independent enzymes within the trypanosomatid polyamine pathway.

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Summary Human African sleeping sickness (HAT) is caused by the parasitic protozoan Trypanosoma brucei. Polyamine biosynthesis is an important drug target in the treatment of HAT. Previously we showed that trypanosomatid S-adenosylmethionine decarboxylase (AdoMetDC), a key enzyme for biosynthesis of the polyamine spermidine, is activated by heterodimer formation with an inactive paralog termed prozyme. Furthermore, prozyme protein levels were regulated in response reduced AdoMetDC activity. Herein we show that T. brucei encodes three prozyme transcripts. The 3’UTRs of these transcripts were mapped and chloramphenicol acetyltransferase (CAT) reporter constructs were used to identify a 1.2 kb region that contained a 3’UTR prozyme regulatory element sufficient to up regulate CAT protein levels (but not RNA) upon AdoMetDC inhibition, supporting the hypothesis that prozyme expression is regulated translationally. To gain insight into trans-acting factors, genetic rescue of AdoMetDC RNAi knockdown lines with human AdoMetDC was performed leading to rescue of the cell growth block, and restoration of prozyme protein to wild-type levels. Polyamine and AdoMet metabolite analysis showed that prozyme protein levels were inversely proportional to intracellular levels of decarboxylated AdoMet (dcAdoMet). These data suggest that prozyme translation may be regulated by dcAdoMet, a metabolite not previously identified to play a regulatory role.

Background: eIF5A facilitates translation of mRNAs encoding proteins with polyprolyl repeats. Results: The protozoan parasite Trypanosoma brucei contains polyprolyl tracts in 15% of its proteome, and both eIF5A and its post-translational modification with deoxyhypusine are essential. Conclusion: Loss of eIF5A leads to abnormal cell morphology, abnormal cell division, and decreased flagellar attachment. Significance: Inhibitors of hypusine biosynthesis would disrupt multiple essential cellular processes.

Human African trypanosomiasis (HAT) is a debilitating and fatal vector-borne disease. Polyamine biosynthesis is the target of one of the key drugs (eflornithine) used for the treatment of late-stage disease, suggesting that the pathway might be exploited for the identification of additional drug targets. The polyamine spermidine is required in trypanosomatid parasites for formation of a unique redox cofactor termed trypanothione, which is formed from the conjugation of glutathione to spermidine. Here we characterize recombinant Trypanosoma brucei glutathione synthetase (TbGS) and show that depletion of TbGS in bloodform parasites using a regulated knockout strategy leads to loss of trypanothione and to cell death as quantified by fluorescenceactivated cell sorter (FACS) analysis. These data suggest that >97% depletion of TbGS is required before trypanothione is depleted and cell growth arrest is observed. Exogenous glutathione was able to partially compensate for the loss of TbGS, suggesting that parasites are able to transport intact glutathione. Finally, reduced expression of TbGS leads to increased levels of upstream glutathione biosynthetic enzymes and decreased expression of polyamine biosynthetic enzymes, providing evidence that the cells cross regulate the two branches of the trypanothione biosynthetic pathway to maintain spermidine and trypanothione homeostasis.