Abstract
Trypanosoma brucei is the causative agent of African sleeping sickness. The polyamine biosynthetic pathway has the distinction of being the target of the only clinically proven anti-trypanosomal drug with a known mechanism of action. Polyamines are essential for cell growth, and their metabolism is extensively regulated. However, trypanosomatids appear to lack the regulatory control mechanisms described in other eukaryotic cells. In T. brucei, S-adenosylmethionine decarboxylase (AdoMetDC) and ornithine decarboxylase (ODC) are required for the synthesis of polyamines and also for the unique redox-cofactor trypanothione. Further, trypanosomatid AdoMetDC is activated by heterodimer formation with a catalytically dead homolog termed prozyme, found only in these species. To study polyamine regulation in T. brucei, we generated inducible AdoMetDC RNAi and prozyme conditional knockouts in the mammalian blood form stage. Depletion of either protein led to a reduction in spermidine and trypanothione and to parasite death, demonstrating that prozyme activation of AdoMetDC is essential. Under typical growth conditions, prozyme concentration is limiting in comparison to AdoMetDC. However, both prozyme and ODC protein levels were significantly increased relative to stable transcript levels by knockdown of AdoMetDC or its chemical inhibition. Changes in protein stability do not appear to account for the increased steady-state protein levels, as both enzymes are stable in the presence of cycloheximide. These observations suggest that prozyme and ODC are translationally regulated in response to perturbations in the pathway. In conclusion, we describe the first evidence for regulation of polyamine biosynthesis in T. brucei and we demonstrate that the unique regulatory subunit of AdoMetDC is a key component of this regulation. The data support ODC and AdoMetDC as the key control points in the pathway and the likely rate-limiting steps in polyamine biosynthesis.
Highlights
Human African trypanosomiasis is a neglected disease of subSaharan Africa caused by the protozoan parasite Trypanosoma brucei
The only safe treatment for latestage disease is an inhibitor of an essential metabolic pathway that is involved in the synthesis of small organic cations termed polyamines
By modulating the protein levels of a trypanosome-specific activator of polyamine biosynthesis, the parasite has developed a mechanism to regulate pathway output. We demonstrate that this pathway activator is essential to parasite growth
Summary
Human African trypanosomiasis is a neglected disease of subSaharan Africa caused by the protozoan parasite Trypanosoma brucei. Without treatment the disease is always fatal and available drug therapy is limited by toxicity, difficult dosing regimes, and emerging resistance [2]. Eflornithine (D,L-adifluoromethylornithine), a suicide inhibitor of ODC, is one of only two drugs available for the treatment of the late-stage disease, and its effectiveness has focused attention on the importance of the polyamine biosynthetic pathway for parasite growth. Polyamines are essential organic cations found in all species, and the metabolic pathway has been extensively studied as a potential target for the development of drugs to treat infectious and proliferative diseases [3,4]. In most eukaryotes the diamine putrescine is synthesized from L-ornithine by ODC, and it serves as the precursor for the formation of the longer chain amine spermidine (Figure 1). AdoMetDC catalyzes the formation of decarboxylated S-adenosylmethionine required as the aminopropyl group donor in the formation of the longer chain polyamines. Spermidine is conjugated to glutathione (GSH) to produce trypanothione, which is required in cellular redox reactions and necessary for nucleotide synthesis [5]
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