Adenosine Kinase Mediates High Affinity Adenosine Salvage in Trypanosoma brucei

Abstract

African sleeping sickness is caused by Trypanosoma brucei. This extracellular parasite lacks de novo purine biosynthesis, and it is therefore dependent on exogenous purines such as adenosine that is taken up from the blood and other body fluids by high affinity transporters. The general belief is that adenosine needs to be cleaved to adenine inside the parasites in order to be used for purine nucleotide synthesis. We have found that T. brucei also can salvage this nucleoside by adenosine kinase (AK), which has a higher affinity to adenosine than the cleavagedependent pathway. The recombinant T. brucei AK (TbAK) preferably used ATP or GTP to phosphorylate both natural and synthetic nucleosides in the following order of catalytic efficiencies: adenosine > cordycepin > deoxyadenosine > adenine arabinoside (Ara-A) > inosine > fludarabine (F-Ara-A). TbAK differed from the AK of the related intracellular parasite Leishmania donovani by having a high affinity to adenosine (K m ‫؍‬ 0.04 -0.08 M depending on [phosphate]) and by being negatively regulated by adenosine (K i ‫؍‬ 8 -14 M). These properties make the enzyme functionally related to the mammalian AKs, although a phylogenetic analysis grouped it together with the L. donovani enzyme. The combination of a high affinity AK and efficient adenosine transporters yields a strong salvage system in T. brucei, a potential Achilles' heel making the parasites more sensitive than mammalian cells to adenosine analogs such as Ara-A. Studies of wild-type and AK knockdown trypanosomes showed that Ara-A inhibited parasite proliferation and survival in an AK-dependent manner by affecting nucleotide levels and by inhibiting nucleic acid biosynthesis.Trypanosoma brucei is an extracellular parasite that is transmitted by tsetse flies and lives in the blood, lymph, and central nervous system of its mammalian hosts (1, 2). The parasite causes African sleeping sickness in humans and nagana in cattle. There are two variants of African sleeping sickness, a chronic form caused by the subspecies Trypanosoma brucei gambiense and an acute form caused by Trypanosoma brucei rhodesiense. Both variants are fatal, but the chronic form has a slower progress. Current treatment is unsatisfactory because of low efficacy and high toxicity. Therefore, there is a great need of new drugs to treat the disease, especially at later stages when the parasites infect the brain. Promising results with the adenosine analog cordycepin (3Ј-deoxyadenosine) on T. brucei-infected mice with brain infection suggest that adenosine analogs can be developed into new antitrypanosomal agents (3).Unlike mammalian cells, trypanosomes lack de novo purine biosynthesis, and they are therefore totally dependent on purine salvage (4). The major purine source in human blood is a matter of controversy; when the blood was directly mixed with an adenosine deaminase inhibitor to prevent purine degradation, adenosine was present at 2 M concentration, whereas hypoxanthine (0.7 M) and inosine (0.2 M) were minor sources (5). However, other r...

Trypanosoma brucei Methylthioadenosine Phosphorylase Protects the Parasite from the Antitrypanosomal Effect of Deoxyadenosine

“…The adenosine cleavage reaction is catalyzed by inosine-adenosineguanosine-nucleoside hydrolase (IAG-NH) 3 (11). Although the relative contribution of each pathway for adenosine salvage is not known, the much higher affinity of adenosine kinase than IAG-NH for this substrate suggests that it represents the major route (6). As a side activity, T. brucei adenosine kinase is also able to phosphorylate deoxyadenosine (6).…”

“…Although the relative contribution of each pathway for adenosine salvage is not known, the much higher affinity of adenosine kinase than IAG-NH for this substrate suggests that it represents the major route (6). As a side activity, T. brucei adenosine kinase is also able to phosphorylate deoxyadenosine (6). This reaction is det-rimental to the parasites because addition of 0.5 mM deoxyadenosine to the culture medium causes the parasites to die within 15 h with grossly elevated dATP levels and a concomitant decrease in ATP, which is consumed in the phosphorylation of the deoxynucleoside (12).…”

“…Nucleoside analogues need to be phosphorylated inside the cells into their nucleotide forms to inhibit transcription, replication, or other nucleotide-dependent processes. In contrast to mammalian cells, which have several kinases to phosphorylate nucleosides and deoxynucleosides, T. brucei has only two: thymidine kinase primarily phosphorylates thymidine and deoxyuridine (4,5), and adenosine kinase phosphorylates adenosine and to some extent deoxyadenosine (6).…”

“…brucei lacks de novo purine biosynthesis, and adenosine kinase is part of the efficient salvage pathways that enable the parasite to utilize nucleosides and bases from the host (6,7). Adenosine is one of the major purine sources in human blood where the concentration is 2 M (8).…”

“…Arabinoside analogues can be considered equivalent to deoxynucleoside analogues because they are generally phosphorylated by the same kinases as deoxynucleosides and primarily inhibit DNA synthesis and other deoxynucleotide-dependent reactions. Adenine arabinoside (Ara-A) is an example of one such analogue that in combination with deoxycoformycin inhibits T. brucei DNA synthesis and cures T. brucei-infected mice (6).…”