TY - JOUR
T1 - Down-regulation of Leishmania donovani trypanothione reductase by heterologous expression of a trans-dominant mutant homologue
T2 - Effect on parasite intracellular survival
AU - Tovar, Jorge
AU - Cunningham, M.L.
AU - Smith, Aden C.
AU - Croft, Simon L.
AU - Fairlamb, Alan H.
N1 - Medline is the source for the MeSH terms of this document.
PY - 1998/4/28
Y1 - 1998/4/28
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0032574808&partnerID=8YFLogxK
U2 - 10.1073/pnas.95.9.5311
DO - 10.1073/pnas.95.9.5311
M3 - Article
AN - SCOPUS:0032574808
SN - 0027-8424
VL - 95
SP - 5311
EP - 5316
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 9
ER -