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

Jorge Tovar, M.L. Cunningham, Aden C. Smith, Simon L. Croft, Alan H. Fairlamb

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    149 Citations (Scopus)

    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 transdominant 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 wildtype 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.
    Original languageEnglish
    Pages (from-to)5311-5316
    Number of pages6
    JournalProceedings of the National Academy of Sciences of the United States of America
    Volume95
    Issue number9
    DOIs
    Publication statusPublished - 28 Apr 1998

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