Proc. Natl. Acad. Sci. USA Vol. 95, pp. 5311–5316, April 1998 Microbiology Down-regulation of Leishmania donovani trypanothione reductase by heterologous expression of a trans-dominant mutant homologue: Effect on parasite intracellular survival JORGE TOVAR*, MARK L. CUNNINGHAM†,ADEN C. SMITH,SIMON L. CROFT, AND ALAN H. FAIRLAMB‡ Department of Tropical and Infectious Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, United Kingdom Edited by Anthony Cerami, The Kenneth S. Warren Laboratories, Tarrytown, NY, and approved February 12, 1998 (received for review December 4, 1997) ABSTRACT A trans-dominant mutational strategy was olites and enzymatic activities with potential as antiparasitic used to down-regulate trypanothione reductase (TR) activity drug targets. Although analogous to the glutathione system levels in Leishmania donovani, the causative agent of visceral that operates in humans and almost all other aerobic organisms leishmaniasis in humans. TR, regarded as an ideal drug target the trypanothione system offers a number of distinct features against trypanosomatid infections, is a homodimeric fla- that may be exploited for selective attack. One such example voprotein oxidoreductase unique to these organisms that is the pronounced difference in substrate specificity between plays a central role in the enzymatic regeneration of the thiol trypanothione reductase (TR) and its mammalian counter- pool. Extrachromosomal, heterologous expression of a trans- part, glutathione reductase (reviewed in ref. 3). The important dominant mutant version of the Trypanosoma cruzi enzyme in role of TR in thiol metabolism and its absence from human L. donovani resulted in the formation of inactive cross-species cells suggests that it may be ideally suited for development as heterodimers and in a dramatic decrease of endogenous TR a chemotherapeutic target. Attempts to validate this oxi- activity levels. Recombinant cells depleted of up to 85% of TR doreductase as a drug target by molecular genetics and bio- activity were significantly impaired in their ability to regen- chemical means have been reported recently (4–6). Biochem- erate dihydrotrypanothione from trypanothione disulfide fol- ical studies involving the overexpression of Leishmania TR in lowing oxidation with diamide. Nonetheless trans-dominant Leishmania donovani and Trypanosoma cruzi demonstrated mutant recombinants were still capable of maintaining a that regeneration of dihydrotrypanothione (T(SH)2) from reduced intracellular environment during cell growth in cul- trypanothione disulfide (T(S)2) is neither limiting in the ture and were able to metabolize hydrogen peroxide at wild- management of oxidative stress thought to be induced by type rates in vitro. Importantly, however, cells expressing the antiparasitic drugs such as nifurtimox, nitrofurazone, and trans-dominant mutant enzyme displayed a decreased ability gentian violet, nor in the metabolism of hydrogen peroxide to survive inside activated macrophages in a murine model of (H2O2), one of the reactive oxygen species toxic to Leishmania Leishmania infection. The apparent inability of Leishmania to (4, 7, 8). Attempts to down-regulate TR activity by antisense modulate the expression of active TR homodimers in response RNA expression in T. cruzi failed to decrease endogenous to the expression of trans-dominant mutant protein suggests activity levels apparently as a result of a specific sequence that specific inhibitors of this enzyme should be useful inversion in a proportion of antisense plasmid DNA molecules anti-leishmanial agents. (5). Gene targeting experiments have also failed to produce null mutants completely devoid of TR catalytic activity (ref. 6; Trypanosomatid protozoa are members of the order Kineto- J.T. and A.H.F., unpublished data). plastida that includes parasites of relevant medical and veter- Evaluation of the minimal levels of TR activity required for inary importance such as Leishmania spp. and Trypanosoma parasite survival is fundamental for the purpose of inhibitor spp. Their complex life cycles involve alternation between an design. Recently we created by site-directed mutagenesis a set insect vector and a vertebrate host and entail dramatic mor- of negative-complementing TR mutants that retain their nat- phological changes that are developmentally regulated (1). In ural folding and dimerization properties but which are devoid susceptible hosts trypanosomatids can cause severe illness and of catalytic activity (ref. 9; unpublished data). We have now in some cases death. Adequate protective vaccines against used these mutants to generate a trans-dominant mutant trypanosomatid infections have yet to be developed, and drugs version of the T. cruzi TR enzyme. Here we report on its currently available for chemotherapeutic intervention are heterologous expression in L. donovani and on some of the mostly unsatisfactory mainly because of their lack of specific- phenotypic consequences of the stepwise inactivation of en- ity, toxicity to humans, and, in many cases, to developed dogenous TR catalytic activity in Leishmania. parasite resistance (2). The search for parasite-specific, physiologically relevant MATERIALS AND METHODS cellular components that may be developed as drug targets and Parasites. A cloned promastigote line of L. donovani their validation by genetic means are the fundamentals of a (MHOMyETy67yHU3) was grown at 22–24°C in M199 me- rational approach to the design and discovery of drugs against trypanosomatid infections. The trypanothione system of trypanosomatid protozoa, a unique thiol-redox cycling system This paper was submitted directly (Track II) to the Proceedings office. that protects the parasite against damage by oxidants and toxic Abbreviations: TR, trypanothione reductase; tryA, trypanothione re- ductase-encoding gene; ALAT, alanine aminotransferase; T(S)2, heavy metals, represents a promising source of unique metab- trypanothione disulfide; T(SH)2, dihydrotrypanothione; H2O2, hydro- gen peroxide; IFN-g, interferon-g; LPS, lipopolysaccharide. The publication costs of this article were defrayed in part by page charge *To whom reprint requests should be addressed. e-mail: j.tovar@ lshtm.ac.uk. payment. This article must therefore be hereby marked ‘‘advertisement’’ in †Present address: Department of Molecular Microbiology, Washing- accordance with 18 U.S.C. §1734 solely to indicate this fact. ton University School of Medicine, St. Louis, MO 63110. © 1998 by The National Academy of Sciences 0027-8424y98y955311-6$2.00y0 ‡Present address: Department of Biochemistry, Wellcome Trust PNAS is available online at http:yywww.pnas.org. Building, Dundee University, Dundee DD1 4HN, Scotland, U.K. 5311 Downloaded by guest on September 24, 2021 5312 Microbiology: Tovar et al. Proc. Natl. Acad. Sci. USA 95 (1998) dium (GIBCOyBRL) supplemented with 40 mM Hepes (pH was monitored essentially as described (19) by using 1 3 107 21 m 7.4), 0.1 mM adenine, 7.7 mM haemin, 10% (volyvol) heat cells ml and an initial H2O2 concentration of 40 M; inactivated fetal calf serum, 50 unitsyml penicillin, and 50 incubations were carried out at 22°C. mgyml streptomycin. Cells were maintained by subculture and Cell Cultures and Infectivity Assays. Bone marrow-derived kept at densities ranging between 5 3 105 to 3 3 107 cells ml21. macrophages were obtained from 6- to 7-week-old female y Transfection of Parasites. Transfection of L. donovani was BALB c mice (Charles River) as described (20). Macrophages performed essentially as described (4). Late-log phase pro- were rested for 2 days in the absence of colony stimulating mastigotes were electroporated in the presence or absence of factor 1, harvested, and seeded into 16-chamber tissue culture 25 or 50 mg of supercoiled plasmid DNA. Cells were diluted slides (Nunc). Bone marrow-derived macrophages were in- 2-fold with culture medium and allowed to recover for 24 h at fected at 37°C with stationary-phase promastigotes of wild- type and recombinant L. donovani at a 1:20 cell-to-parasite 24°C. Recombinant clones were selected on M199 medium m y ratio. After 4 h, excess parasites were eliminated by vigorous solidified with 1% bactoagar and supplemented with 25 g ml washing with serum-free DMEM. Infected macrophages were G418. activated with 11 unitsyml murine recombinant interferon-g Plasmid Construction. Plasmid pC53A harbors a mutant (IFN-g; Genentech) in DMEM supplemented with 10% heat- version of the T. cruzi TR-encoding gene (tryA) in which the inactivated fetal calf serum and 10 ngyml lipopolysaccharide essential redox-active cysteine at position 53 has been replaced (LPS; Sigma). After incubation periods of 4, 24, 48, and 72 h, by an alanine residue (9). This plasmid was used as a template cultures were fixed in methanol, stained with Giemsa, and to introduce a second point mutation that changed the essen- examined microscopically. tial active site histidine at position 461 by a glutamine residue by using mutagenic oligonucleotide TcTRHQ (59-ATTGGT- GTGCAgCCCACAAGT-39). The presence of both mutations RESULTS in the resulting plasmid pC53AyH461Q was confirmed by Rational and Construction of Recombinant Clones. A sche- sequencing the entire tryA coding region. This gene was matic representation of the strategy used is shown in Fig. 1A. subsequently cloned into the EcoRV site of expression vector pTEX (10) by conventional methodology (11) to generate plasmid pTEXTcTRtdm. Enzymatic Assays. TR and alanine aminotransferase (ALAT)