RNA interference-mediated silencing of ornithine decarboxylase and spermidine synthase genes in Trypanosoma brucei provides insight into regulation of polyamine biosynthesis.
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 alpha-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.
Xiao
Yanjing
Y
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9041, USA.
McCloskey
Diane E
DE
Phillips
Margaret A
MA
D012437:Adenosylmethionine Decarboxylase / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D005978:Glutathione / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D009955:Ornithine Decarboxylase / Q000235:genetics* / Q000378:metabolism; D011073:Polyamines / Q000378:metabolism*; D015800:Protozoan Proteins / Q000235:genetics* / Q000378:metabolism; D034622:RNA Interference; D013095:Spermidine / Q000031:analogs & derivatives / Q000378:metabolism; D011436:Spermidine Synthase / Q000235:genetics* / Q000378:metabolism; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics; D014353:Trypanosomiasis, African / Q000469:parasitology
D011073:Polyamines; D015800:Protozoan Proteins; C044809:trypanothione; D011436:Spermidine Synthase; D009955:Ornithine Decarboxylase; D012437:Adenosylmethionine Decarboxylase; D005978:Glutathione; D013095:Spermidine