Discussion

Ornithine is an essential precursor for the synthesis of polyamines in Trypanosoma brucei. Ornithine decarboxylase, an enzyme that converts ornithine into putrescine, is the target of eflornithine, an approved drug for therapy against sleeping sickness. To date, no ornithine carrier has been identified in T. brucei. Complementation of Saccharomyces cerevisiae amino acid transport mutants with putative amino acid transporters (AAT) from T. brucei identified TbAAT2 and TbAAT10 as potential histidine transporters. Interestingly, TbAAT2-mediated histidine uptake was inhibited by ornithine, suggesting that ornithine can also be transported. Systematic investigations using [³H]ornithine revealed that TbAAT10 and TbAAT2 mediate high-affinity ornithine transport (apparent K_m 4.3 and 4.0 µM), however, with an obvious difference in substrate specificity. While TbAAT10 was selective for ornithine, uptake of ornithine by TbAAT2 was significantly inhibited by histidine, suggesting that TbAAT2-mediated ornithine uptake is influenced by the extracellular histidine concentration. Moreover, RNAi knockdown of TbAAT10 caused a small but significant growth defect in T. brucei bloodstream forms, whereas TbAAT2 down-regulation did not affect cell proliferation in culture. Interestingly, drug sensitivity assays revealed opposing effects on suramin and eflornithine toxicity. RNAi against TbAAT10 caused a 2-fold increase in resistance to suramin and, in contrast, a 10-fold higher sensitivity to eflornithine. Together, our data provide insights on ornithine uptake and metabolism in T. brucei and its relevance for therapeutics against sleeping sickness.