Adenosine kinase of Trypanosoma brucei and its role in susceptibility to adenosine antimetabolites.

Trypanosoma brucei cannot synthesize purines de novo and relies on purine salvage from its hosts to build nucleic acids. With adenosine being a preferred purine source of bloodstream-form trypanosomes, adenosine kinase (AK; EC 2.7.1.20) is likely to be a key player in purine salvage. Adenosine kinase is also of high pharmacological interest, since for many adenosine antimetabolites, phosphorylation is a prerequisite for activity. Here, we cloned and functionally characterized adenosine kinase from T. brucei (TbAK). TbAK is a tandem gene, expressed in both procyclic- and bloodstream-form trypanosomes, whose product localized to the cytosol of the parasites. The RNA interference-mediated silencing of TbAK suggested that the gene is nonessential under standard growth conditions. Inhibition or downregulation of TbAK rendered the trypanosomes resistant to cordycepin (3'-deoxyadenosine), demonstrating a role for TbAK in the activation of adenosine antimetabolites. The expression of TbAK in Saccharomyces cerevisiae complemented a null mutation in the adenosine kinase gene ado1. The concomitant expression of TbAK with the T. brucei adenosine transporter gene TbAT1 allowed S. cerevisiae ado1 ade2 double mutants to grow on adenosine as the sole purine source and, at the same time, sensitized them to adenosine antimetabolites. The coexpression of TbAK and TbAT1 in S. cerevisiae ado1 ade2 double mutants proved to be a convenient tool for testing nucleoside analogues for uptake and activation by T. brucei adenosine salvage enzymes.

Molecular interactions underlying the unusually high adenosine affinity of a novel Trypanosoma brucei nucleoside transporter.

Trypanosoma brucei encodes a relatively high number of genes of the equilibrative nucleoside transporter (ENT) family. We report here the cloning and in-depth characterization of one T. brucei brucei ENT member, TbNT9/AT-D. This transporter was expressed in Saccharomyces cerevisiae and displayed a uniquely high affinity for adenosine (Km = 0.068 +/- 0.013 microM), as well as broader selectivity for other purine nucleosides in the low micromolar range, but was not inhibited by nucleobases or pyrimidines. This selectivity profile is consistent with the P1 transport activity observed previously in procyclic and long-slender bloodstream T. brucei, apart from the 40-fold higher affinity for adenosine than for inosine. We found that, like the previously investigated P1 activity of long/slender bloodstream trypanosomes, the 3'-hydroxy, 5'-hydroxy, N3, and N7 functional groups contribute to transporter binding. In addition, we show that the 6-position amine group of adenosine, but not the inosine 6-keto group, makes a major contribution to binding (DeltaG0 = 12 kJ/mol), explaining the different Km values of the purine nucleosides. We further found that P1 activity in procyclic and long-slender trypanosomes is pharmacologically distinct, and we identified the main gene encoding this activity in procyclic cells as NT10/AT-B. The presence of multiple P1-type nucleoside transport activities in T. brucei brucei facilitates the development of nucleoside-based treatments for African trypanosomiasis and would delay the onset of uptake-related drug resistance to such therapy. We show that both TbNT9/AT-D and NT10/AT-B transport a range of potentially therapeutic nucleoside analogs.

Molecular pharmacology of adenosine transport in Trypanosoma brucei: P1/P2 revisited.

Trypanosoma brucei are unicellular parasites that cause sleeping sickness in humans and nagana in livestock. Trypanosomes salvage purines from their hosts through a variety of transporters, of which adenosine permeases deserve particular attention because of their role in drug sensitivity. T. brucei possess two distinct adenosine transport systems, P1 and P2, the latter of which also mediates cellular uptake of the drugs melarsoprol and pentamidine. Loss or mutation of P2 has been associated with drug resistance and sleeping sickness treatment failures. However, genetic disruption in Trypanosoma brucei brucei of the gene encoding P2, TbAT1, reduced the susceptibility to melarsoprol and pentamidine by only a factor of approximately 2. In this study, we show stronger phenotypes of the tbat1 null mutant with respect to its sensitivity toward toxic adenosine analogs. Compared with parental TbAT1+/+ trypanosomes, the tbat1-/- mutant is 77-fold less sensitive to tubercidin and 14-fold less sensitive to cordycepin. Resistance is further increased by the addition of inosine but is reverted by adenine. It is surprising that the tbat1-/- mutant grows faster than TbAT1+/+ trypanosomes and that it overexpresses genes of the TbNT cluster encoding P1-type transporters. These unexpected phenotypes show that there are conditions other than drug pressure under which loss of P2 may confer a selective advantage to bloodstream-form trypanosomes. Overexpression of P1 by trypanosomes after loss of P2 indicates that combinatorial chemotherapy with trypanocidal P1 and P2 substrates may be a promising strategy to prevent drug resistance in sleeping sickness.

Drug transport and drug resistance in African trypanosomes.

Drug resistance in African trypanosomes has been studied for almost a hundred years. Beginning with Paul Ehrlich's work that led to the chemoreceptor hypothesis, reduction of net drug uptake has emerged as the most frequent cause of resistance. This review, therefore, focuses on trypanosomal drug transporter genes. TbAT1 encodes purine permease P2, which mediates influx of melarsoprol and diamidines. Disruption of TbAT1 in Trypanosoma brucei reduced sensitivity to these trypanocides. TbMRPA encodes a putative trypanothione-conjugate efflux pump, and overexpression of TbMRPA in T. brucei causes melarsoprol resistance. It will be important to determine the role of TbAT1 and TbMRPA in sleeping sickness treatment failures.