The Adenosine Analog Tubercidin Inhibits Glycolysis in Trypanosoma brucei as Revealed by an RNA Interference Library

Abstract

We used an RNA interference (RNAi) library in a forward genetic selection to study the mechanism of toxicity of tubercidin (7-deazaadenosine) to procyclic Trypanosoma brucei. Following transfection of cells with an RNAibased genomic library, we used 5 M tubercidin to select a drug-resistant cell line. Surprisingly, we found in these resistant cells that the hexose transporters had been silenced. We subsequently found that silencing of hexokinase, a glycolytic enzyme, also yielded tubercidinresistant parasites. These observations suggested that glycolysis could be a target of tubercidin action and that RNAi silencing of glycolytic enzymes was gradual enough to allow the parasites to adapt to alternative sources of energy. Indeed, adaptation of procyclic trypanosomes to a glucose-independent metabolism by reduction of glucose in the culture medium caused tubercidin resistance. High pressure liquid chromatography analysis of glycolytic intermediates from parasites treated with tubercidin showed a dose-dependent increase in concentration of 1,3-bisphosphoglycerate, a substrate of phosphoglycerate kinase. Furthermore, tubercidin triphosphate inhibited recombinant T. brucei phosphoglycerate kinase activity in vitro with an IC 50 of 7.5 M. We conclude that 5 M tubercidin kills trypanosomes by targeting glycolysis, especially by inhibition of phosphoglycerate kinase.

Transposon Mutagenesis of Trypanosoma brucei Identifies Glycosylation Mutants Resistant to Concanavalin A

“…The change was shown to be due to the RNAiinduced silencing of the hexokinase gene, a totally unanticipated result (45). A screen for tubercidin resistance also produced an unexpected result that led directly to the demonstration of phosphoglycerate kinase as a target for tubercidin in T. brucei (46). Both of these reports emphasize the importance of applying forward genetics to T. brucei.…”

Hydrolysis products of cAMP analogs cause transformation ofTrypanosoma bruceifrom slender to stumpy-like forms

“…Although it currently is not possible to completely rule out any role for cAMP in this differentiation process, the present data throw into question whether the slender-tostumpy conversion is initiated by cAMP at all. The molecular mechanisms by which different adenosine analogs alter differentiation and proliferation are more difficult to interpret because adenosine analogs are known to cause such effects as triggering of cell-cycle arrest (39), inhibition of glycolysis (40,41), and alteration of purine transport or salvage (42). Similarly, AMP kinases are known targets in mammals but are as-yet-unknown in trypanosomes (43).…”

Glycerol 3-Phosphate Alters Trypanosoma brucei Hexokinase Activity in Response to Environmental Change

“…Abundance-PF trypanosomes can adapt their metabolism to conditions of low glucose, presumably by up-regulation of amino acid metabolism (18,19). Because of this dynamic metabolism, PF parasites can grow normally in the near absence of glucose.…”