Down-regulation of Leishmania donovani trypanothione reductase by heterologous expression of a trans-dominant mutant homologue: effect on parasite intracellular survival.

Abstract

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.

• Authors: J Tovar, M L Cunningham, A C Smith, S L Croft, A H Fairlamb
• Journal: Proceedings of the National Academy of Sciences of the United States of America (Proc Natl Acad Sci U S A) Vol. 95 Issue 9 Pg. 5311-6 (Apr 28 1998) ISSN: 0027-8424 [Print] United States
• PMID: 9560272 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Hydrogen Peroxide
• NADH, NADPH Oxidoreductases
• trypanothione reductase

Topics

• Animals
• Dimerization
• Female
• Gene Expression Regulation, Enzymologic
• Genes, Dominant
• Heterozygote
• Hydrogen Peroxide (metabolism)
• Leishmania donovani (enzymology, growth & development)
• Macrophage Activation
• Macrophages (parasitology)
• Mice
• Mice, Inbred BALB C
• NADH, NADPH Oxidoreductases (genetics)
• Oxidation-Reduction