Phenotype of recombinant Leishmania donovani and Trypanosoma cruzi which over-express trypanothione reductase. Sensitivity towards agents that are thought to induce oxidative stress.
Trypanothione reductase is thought to be important in maintaining an intracellular reducing environment in trypanosomatids. To investigate the role of trypanothione reductase we transfected Leishmania donovani and Trypanosoma cruzi with an expression vector containing the L. donovani trypanothione reductase gene and achieved over-expression of enzyme activity (10-14-fold) in transformed cells. Following treatment of L. donovani cells with the thiol-oxidizing agent diamide, the ability to regenerate dihydrotrypanothione from trypanothione disulphide was considerably enhanced in cells which over-expressed trypanothione reductase. However, the growth of transformed and control cells was equally sensitive to inhibition by nifurtimox, nitrofurazone and gentian violet, drugs that are thought to act by inducing oxidative damage. Likewise, growth of transformed and control cells were equally susceptible to inhibition by hydrogen peroxide, and control and transformed L. donovani promastigotes metabolized hydrogen peroxide at comparable rates. Thus, these experiments suggest that the ability to regenerate dihydrotrypanothione from trypanothione disulphide is not a rate-limiting step in the metabolism of hydrogen peroxide.
Kelly
J M
JM
Department of Medical Parasitology, London School of Hygiene and Tropical Medicine, England.
Taylor
M C
MC
Smith
K
K
Hunter
K J
KJ
Fairlamb
A H
AH
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D017125:Genes, Protozoan; D005840:Gentian Violet / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000378:metabolism / Q000494:pharmacology; D007893:Leishmania donovani / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D009581:Nitrofurans / Q000494:pharmacology; D010084:Oxidation-Reduction; D010641:Phenotype; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014170:Transformation, Genetic; D014349:Trypanosoma cruzi / Q000187:drug effects / Q000235:genetics*
D017931:DNA Primers; D009581:Nitrofurans; D011994:Recombinant Proteins; D006861:Hydrogen Peroxide; D009247:NADH, NADPH Oxidoreductases; C049608:trypanothione reductase; D005840:Gentian Violet