DOWN-REGULATION OF LEISHMANIA-DONOVANI TRYPANOTHIONE REDUCTASE BY HETEROLOGOUS EXPRESSION OF A TRANSDOMINANT MUTANT HOMOLOG - EFFECT ON PARASITE INTRACELLULAR SURVIVAL

A trans-dominant mutational strategy was used to down-regulate trypano thione reductase (TR) activity levels in Leishmania donovani, the caus ative agent of visceral leishmaniasis in humans. TR, regarded as an id eal drug target against trypanosomatid infections, is a homodimeric fl avoprotein oxidoreductase unique to these organisms that plays a centr al role in the enzymatic regeneration of the thiol pool, Extrachromoso mal, heterologous expression of a transdominant mutant version of the Trypanosoma cruzi enzyme in L, donovani resulted in the formation of i nactive cross-species heterodimers and in a dramatic decrease of endog enous TR activity levels. Recombinant cells depleted of up to 85% of T R activity were significantly impaired in their ability to regenerate dihydrotrypanothione from trypanothione disulfide following oxidation with diamide, Nonetheless trans-dominant mutant recombinants were stil l capable of maintaining a reduced intracellular environment during ce ll growth in culture and were able to metabolize hydrogen peroxide at wildtype rates in vitro, Importantly, however, cells expressing the tr ans-dominant mutant enzyme displayed a decreased ability to survive in side activated macrophages in a murine model of Leishmania infection, The apparent inability of Leishmania to modulate the expression of act ive TR homodimers in response to the expression of trans-dominant muta nt protein suggests that specific inhibitors of this enzyme should be useful anti-leishmanial agents.