RNA Interference-Mediated Silencing of Ornithine Decarboxylase and Spermidine Synthase Genes in Trypanosoma brucei Provides Insight into Regulation of Polyamine Biosynthesis

Abstract

Polyamine biosynthesis is a drug target for the treatment of African sleeping sickness; however, mechanisms regulating the pathway in Trypanosoma brucei are not well understood. Recently, we showed that RNA interference (RNAi)-mediated gene silencing or the inhibition of S-adenosylmethionine decarboxylase (AdoMetDC) led to the upregulation of the AdoMetDC activator, prozyme, and ornithine decarboxylase (ODC) proteins. To determine if this regulatory response is specific to AdoMetDC, we studied the effects of the RNAi-induced silencing of the spermidine synthase (SpdSyn) and ODC genes in bloodstream form T. brucei. The knockdown of either gene product led to the depletion of the polyamine and trypanothione pools and to cell death. Decarboxylated AdoMet levels were elevated, while AdoMet was not affected. There was no significant effect on the protein levels of other polyamine pathway enzymes. The treatment of parasites with the ODC inhibitor ␣-difluoromethylornithine gave similar results to those observed for ODC knockdown. Thus, the cellular response to the loss of AdoMetDC activity is distinctive, suggesting that AdoMetDC activity controls the expression levels of the other spermidine biosynthetic enzymes. RNAi-mediated cell death occurred more rapidly for ODC than for SpdSyn. Further, the ODC RNAi cells were rescued by putrescine, but not spermidine, suggesting that the depletion of both putrescine and spermidine is more detrimental than the depletion of spermidine alone. This finding may contribute to the effectiveness of ODC as a target for the treatment of African sleeping sickness, thus providing important insight into the mechanism of action of a key antitrypanosomal agent.

Spermidine Synthase Is Required for Virulence of Leishmania donovani

“…It should be noted that ⌬odc L. donovani promastigotes are (62). Although the cellular functions of polyamines are not completely understood, it appears to be evident that putrescine is more than just a precursor for the production of spermidine, which could account for the discrepancy in the magnitude of the virulence deficit caused by the ⌬odc and ⌬spdsyn lesions.…”

The silicon trypanosome

“…Preliminary attempts to model trypanothione metabolism have been made (Xu Gu, University of Glasgow PhD thesis, unpublished). Information available on the abundance of key metabolites measured in bloodstream form T. brucei grown in vitro (Fairlamb et al 1987) and in vivo (Xiao et al 2009), before and after exposure to eflornithine, was used to determine whether predicted behaviour under those perturbed conditions emulated the measured behaviour. The scarcity of kinetic data describing the whole pathway, however, has presented many challenges to constructing a model that captures observed behaviour.…”

Discovery of Potent and Selective Inhibitors of Trypanosoma brucei Ornithine Decarboxylase

“…1). There are no inter-conversion pathways, and transport of exogenous polyamines plays a lesser role (19). In addition, T. brucei ODC is much longer lived than its mammalian counterpart, and levels are not actively regulated (20,21).…”

“…Prozyme expression is in turn regulated in response to changes in polyamine levels (23). Depletion of polyamines either by inhibition of the biosynthetic enzymes or by gene knockdown leads to reduced trypanothione levels and to cell death (19,23,24). DFMO is clinically approved for the treatment of human African trypanosomiasis, and a new combination with nifurtimox is now the recommended frontline therapy for late stage Trypanosoma brucei gambiense (25).…”

“…Furthermore, DFMO is only effective against one of two subspecies of T. brucei causing human disease (27). As the parasite cannot increase ODC levels in response to polyamine depletion, the development of potent reversible inhibitors may allow inhibition of ODC to be fruitful in both subspecies (19).…”