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

Antimicrob Agents Chemother. 2007 Nov;51(11):3895-901. doi: 10.1128/AAC.00458-07. Epub 2007 Aug 13.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine / analogs & derivatives
  • Adenosine / chemistry
  • Adenosine / pharmacology
  • Adenosine Kinase / genetics
  • Adenosine Kinase / metabolism*
  • Animals
  • Blotting, Northern
  • Blotting, Southern
  • Blotting, Western
  • Deoxyadenosines / pharmacology
  • Fluorescent Antibody Technique
  • Gene Silencing
  • Genetic Complementation Test
  • Mutation
  • Phylogeny
  • Protozoan Proteins / genetics
  • Protozoan Proteins / metabolism*
  • Protozoan Proteins / physiology
  • RNA Interference
  • Saccharomyces cerevisiae / enzymology
  • Saccharomyces cerevisiae / genetics
  • Trypanocidal Agents / chemistry
  • Trypanocidal Agents / pharmacology*
  • Trypanosoma brucei brucei / drug effects*
  • Trypanosoma brucei brucei / genetics
  • Trypanosoma brucei brucei / metabolism

Substances

  • Deoxyadenosines
  • Protozoan Proteins
  • Trypanocidal Agents
  • Adenosine Kinase
  • cordycepin
  • Adenosine