Down-regulation of Leishmania donovani trypanothione reductase by heterologous expression of a trans-dominant mutant homologue: effect on parasite intracellular survival.
A trans-dominant mutational strategy was used to down-regulate trypanothione reductase (TR) activity levels in Leishmania donovani, the causative agent of visceral leishmaniasis in humans. TR, regarded as an ideal drug target against trypanosomatid infections, is a homodimeric flavoprotein oxidoreductase unique to these organisms that plays a central role in the enzymatic regeneration of the thiol pool. Extrachromosomal, heterologous expression of a trans-dominant mutant version of the Trypanosoma cruzi enzyme in L. donovani resulted in the formation of inactive cross-species heterodimers and in a dramatic decrease of endogenous TR activity levels. Recombinant cells depleted of up to 85% of TR activity were significantly impaired in their ability to regenerate dihydrotrypanothione from trypanothione disulfide following oxidation with diamide. Nonetheless trans-dominant mutant recombinants were still capable of maintaining a reduced intracellular environment during cell growth in culture and were able to metabolize hydrogen peroxide at wild-type rates in vitro. Importantly, however, cells expressing the trans-dominant mutant enzyme displayed a decreased ability to survive inside activated macrophages in a murine model of Leishmania infection. The apparent inability of Leishmania to modulate the expression of active TR homodimers in response to the expression of trans-dominant mutant protein suggests that specific inhibitors of this enzyme should be useful anti-leishmanial agents.
Tovar
J
J
Department of Tropical and Infectious Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, United Kingdom. j.tovar@1shtm.ac.uk
Cunningham
M L
ML
Smith
A C
AC
Croft
S L
SL
Fairlamb
A H
AH
D000818:Animals; D019281:Dimerization; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D005799:Genes, Dominant; D006579:Heterozygote; D006861:Hydrogen Peroxide / Q000378:metabolism; D007893:Leishmania donovani / Q000201:enzymology* / Q000254:growth & development; D008262:Macrophage Activation; D008264:Macrophages / Q000469:parasitology; D051379:Mice; D008807:Mice, Inbred BALB C; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics*; D010084:Oxidation-Reduction
D006861:Hydrogen Peroxide; D009247:NADH, NADPH Oxidoreductases; C049608:trypanothione reductase