Down-regulation of Leishmania donovani trypanothione reductase by heterologous expression of a trans-dominant mutant homologue: effect on parasite intracellular survival - PubMed (original) (raw)
Down-regulation of Leishmania donovani trypanothione reductase by heterologous expression of a trans-dominant mutant homologue: effect on parasite intracellular survival
J Tovar et al. Proc Natl Acad Sci U S A. 1998.
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.
Figures
Figure 1
(A) Representation of the trans-dominant mutational strategy used in this study. TR monomers from L. donovani and T. cruzi associated either as homodimers or heterodimers are illustrated showing the essential cysteine and histidine residues that comprise the trypanothione-binding region of the active site. (B) Effect of overexpression of trans-dominant mutants on levels of active enzyme in L. donovani. The extent of inactivation of endogenous TR (Ld/Ld) depends on the level of overexpression of mutant protein (Tc:Ld ratio, r) as described in the text. (Inset) Theoretical relative amounts of each dimer species as a function of heterologous protein overexpression.
Figure 2
Analysis of recombinant lines transfected with pTEX_TcTRtdm._ (A) Structure of expression plasmid. The tryA coding region, the bacterial neomycin phosphotransferase (neo) gene, and GAPDH untranslated regulatory sequences are represented by open, grey, and filled boxes, respectively. The location of polyadenylation (A+) and splice acceptor sites (SAS) within regulatory regions is indicated; also shown is the unique _Eco_RI restriction site. (B) DNA blot analysis. Total DNA isolated from indicated lines was digested with _Eco_RI, size-fractionated, and blotted onto nylon; the membrane was sequentially probed with radiolabeled neo and tryA sequences. Sizes of molecular markers are given in kilobases.
Figure 3
Heterodimer formation in L. donovani and T. cruzi. Cells that had been transfected with a pTEX plasmid containing either homologous or heterologous tryA were analyzed for TR content by anion exchange chromatography as described. (A) L. donovani promastigotes: wild-type control (•), transfected with pTEX_TcTR_ (○), and transfected with pTEX_LdTR_ (▵). (B) T. cruzi epimastigotes: wild-type control (•), transfected with pTEX_LdTR_ (○), ad transfected with pTEX_TcTR_ (▵). The identity of the homodimers (Tc/Tc or Ld/Ld) and heterodimer peaks (Tc/Ld) are indicated.
Figure 4
Trypanothione regeneration following diamide-induced oxidant stress in L. donovani promastigotes. Regeneration of T(SH)2 over time was monitored as described. Plotted values represent the percentage of T(SH)2 found in untreated control cells: wild-type (control line; ○), pTEX_TcTRtdm_ (TR underexpressing line; •), and pTEX_TcTR_ (TR overexpressing line; ▪).
Figure 5
Effect of TR down-regulation on L. donovani macrophage colonization. Untreated (A and C) or cytokine-activated macrophages (B and D) were infected as described with L. donovani: wild-type control (□), transfected with pTEX_TcTRtdm_ (⋄), and same recombinant line grown in the absence of plasmid-selection for a minimum of 60 generations (▪). Plotted values represent the percentage of infected macrophages (A and B) and the total number of parasites per 100 macrophages (C and D) as determined from three independent experiments.
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