Validation of spermidine synthase as a drug target in African trypanosomes.
The trypanocidal activity of the ODC (ornithine decarboxylase) inhibitor DFMO (difluoromethylornithine) has validated polyamine biosynthesis as a target for chemotherapy. As DFMO is one of only two drugs used to treat patients with late-stage African trypanosomiasis, the requirement for additional drug targets is paramount. Here, we report the biochemical properties of TbSpSyn (Trypanosoma brucei spermidine synthase), the enzyme immediately downstream of ODC in this pathway. Recombinant TbSpSyn was purified and shown to catalyse the formation of spermidine from putrescine and dcSAM (decarboxylated S-adenosylmethionine). To determine the functional importance of TbSpSyn in BSF (bloodstream form) parasites, we used a tetracycline-inducible RNAi (RNA interference) system. Down-regulation of the corresponding mRNA correlated with a decrease in intracellular spermidine and cessation of growth. This phenotype could be complemented by expressing the SpSyn (spermidine synthase) gene from Leishmania major in cells undergoing RNAi, but could not be rescued by addition of spermidine to the medium due to the lack of a spermidine uptake capacity. These results therefore genetically validate TbSpSyn as a target for drug development and indicate that in the absence of a functional biosynthetic pathway, BSF T. brucei cannot scavenge sufficient spermidine from their environment to meet growth requirements.
Taylor
Martin C
MC
Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WC1E 7HT, UK.
Kaur
Harparkash
H
Blessington
Bernard
B
Kelly
John M
JM
Wilkinson
Shane R
SR
D000595:Amino Acid Sequence; D000818:Animals; D000518:Eflornithine / Q000494:pharmacology; D008954:Models, Biological; D008969:Molecular Sequence Data; D034622:RNA Interference; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013095:Spermidine / Q000378:metabolism; D011436:Spermidine Synthase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D014344:Trypanocidal Agents / Q000627:therapeutic use; D014346:Trypanosoma brucei brucei / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development; D014353:Trypanosomiasis, African / Q000188:drug therapy*
D011994:Recombinant Proteins; D014344:Trypanocidal Agents; D011436:Spermidine Synthase; D013095:Spermidine; D000518:Eflornithine