Brazilian Journal of Medical and Biological Research (2006) 39: 355-363 Protection of Leishmania by glutathione/trypanothione systems 355 ISSN 0100-879X Glutathione and the redox control system trypanothione/trypanothione reductase are involved in the protection of Leishmania spp. against nitrosothiol-induced cytotoxicity P.R.T. Romão1, 1Laboratório de Imunoparasitologia, Curso de Medicina, J. Tovar2, S.G. Fonseca3, Universidade do Sul de Santa Catarina (UNISUL), Tubarão, SC, Brasil R.H. Moraes4, 2School of Biological Sciences, Royal Holloway University of London, Egham, UK A.K. Cruz5, J.S. Hothersall6, 3Laboratório de Imunologia, Instituto do Coração, Faculdade de Medicina, A.A. Noronha-Dutra6, Universidade de São Paulo, São Paulo, SP, Brasil S.H. Ferreira4 4Departamento de Farmacologia, 5Departamento de Biologia Celular e Molecular e and F.Q. Cunha4 Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, de São Paulo, Ribeirão Preto, SP, Brasil 6The Institute of Urology and Nephrology, University College of London, London, UK Abstract Correspondence Glutathione is the major intracellular antioxidant thiol protecting Key words P.R.T. Romão mammalian cells against oxidative stress induced by oxygen- and • Leishmania Laboratório de Imunoparasitologia nitrogen-derived reactive species. In trypanosomes and leishmanias, • Glutathione Curso de Medicina, UNISUL trypanothione plays a central role in parasite protection against mam- • Trypanothione Rua José Acácio Moreira, 787, malian host defence systems by recycling trypanothione disulphide by • Trypanothione reductase DEHON • the enzyme trypanothione reductase. Although Kinetoplastida para- Nitric oxide 88704-900 Tubarão, SC • Free radicals Brasil sites lack glutathione reductase, they maintain significant levels of Fax: +55-48-621-3067 glutathione. The aim of this study was to use Leishmania donovani E-mail: [email protected] trypanothione reductase gene mutant clones and different Leishmania species to examine the role of these two individual thiol systems in the Research supported by FAPESP protection mechanism against S-nitroso-N-acetyl-D,L-penicillamine (No. 97/01938-8). P.R.T. Romão and (SNAP), a nitrogen-derived reactive species donor. We found that the S.G. Fonseca were recipients of FAPESP fellowships. F.Q. Cunha, resistance to SNAP of different species of Leishmania was inversely S.H. Ferreira, and R.H. Moraes were correlated with their glutathione concentration but not with their total 7 recipients of CNPq fellowships. low-molecular weight thiol content (about 0.18 nmol/10 parasites, regardless Leishmania species). The glutathione concentration in L. amazonensis, L. donovani, L. major, and L. braziliensis were 0.12, 0.10, 0.08, and 0.04 nmol/107 parasites, respectively. L. amazonensis, Received January 21, 2005 that have a higher level of glutathione, were less susceptible to SNAP Accepted November 11, 2005 (30 and 100 µM). The IC50 values of SNAP determined to L. amazo- nensis, L. donovani, L. major, and L. braziliensis were 207.8, 188.5, 160.9, and 83 µM, respectively. We also observed that L. donovani mutants carrying only one trypanothione reductase allele had a de- creased capacity to survive (~40%) in the presence of SNAP (30-150 µM). In conclusion, the present data suggest that both antioxidant systems, glutathione and trypanothione/trypanothione reductase, par- ticipate in protection of Leishmania against the toxic effect of nitro- gen-derived reactive species. Braz J Med Biol Res 39(3) 2006 356 P.R.T. Romão et al. Introduction plays a crucial role in the killing of parasites both in vitro (7) and in vivo (8,9). In vitro The trypanosomatids, members of the or- macrophage microbicidal activity correlates der Kinetoplastida, include parasitic protozoa with NO production, and both in vivo and in of importance to public health such as Leish- vitro microbicidal activities are completely mania spp. Leishmania cause a spectrum of inhibited by the NO synthase inhibitor L- diseases ranging from self-healing ulcers to arginine analogue NG-monomethyl-L-argi- disseminated and often fatal infections, de- nine (L-NMMA) but not by its enantiomer pending on the species involved and the host’s D-NMMA (8). In addition, the NO donors, immune response. Adequate vaccines against S-nitroso-N-acetyl-D,L-penicillamine (SNAP) trypanosomatid infections have yet to be de- and 3-morpholino-sydnonimine hydrochlo- veloped, and drugs currently available for che- ride are able to kill Leishmania parasites in a motherapeutic intervention are mostly unsat- cell-free model system (10). isfactory mainly because of their lack of speci- We have reported that glutathione is in- ficity, toxicity to humans, and, in many cases, volved in the protection of mammalian mac- developed parasite resistance (1). Thus, one of rophages against the cytotoxic effects of the priorities in tropical medicine research has NO. Furthermore, despite evidence that glu- been the identification and characterisation of tathione in Leishmania appears not to be the parasite-specific biomolecules, which play major antioxidant, we have demonstrated relevant physiological roles and thus might be that it protects L. major from the toxic ef- exploited as selective targets. fects of NO (11). Among many other metabolic distinctions, In the present study, we have extended trypanosomatids maintain their intracellular these observations by comparing the glutathi- redox balance by a mechanism that is different one levels and SNAP sensitivity of different from that of their insect vectors and mamma- Leishmania species: L. amazonensis, L. brazi- lian hosts. They lack glutathione reductase, liensis, L. donovani, and L. major. Our results which in nearly all other organisms is respon- demonstrate that the sensitivity of distinct spe- sible for the maintenance of an intracellular cies of Leishmania to SNAP is inversely cor- thiol-reducing environment, and thus for the related with their glutathione concentration. reduction of disulphides, detoxification of per- When we extended our investigation to the oxides and synthesis of DNA precursors (2). role of TryR in the protection against SNAP Instead, they possess a unique system using using mutants of L. donovani for the TryR trypanothione [T(SH)2] that is the major re- gene (tryR, formerly tryA) (12), we found duced thiol of Kinetoplastida parasites (3) and that, compared to control parasites (tryR geno- comprises a spermidine moiety linked to two type+/+/+), a double mutant clone (tryR geno- glutathione molecules (2). Together with three type-/-/+) was significantly more sensitive to thiol-redox proteins, trypanothione reductase the cytotoxic effect of SNAP. Overall, these (TryR), tryparedoxin and tryparedoxin per- results demonstrate that glutathione as well as oxidase (4,5), T(SH)2 is thought to provide the T(SH)2/TryR redox system are essential defence against oxidants, certain heavy metals protective components against NO cytotoxic- (6) and xenobiotics (3). Thus, TryR has a vital ity in Leishmania. physiological role in maintaining T(SH)2 re- dox, particularly within the highly oxidative Material and Methods intracellular environment of the host cells which is generated during the antimicrobial Leishmania strains and culture conditions defence response. In murine leishmaniasis, nitric oxide (NO) The Leishmania species used in this study Braz J Med Biol Res 39(3) 2006 Protection of Leishmania by glutathione/trypanothione systems 357 were L. braziliensis (MHOM/BR/75/M2904), ols and glutathione (reduced plus disulphide L. amazonensis (MPRO/BR/72/M1841-LV- forms) were measured in lysates of promas- 79), L. major (LV-39, clone 5-Rho-SU/59/P), tigote forms (stationary phase) of different and L. donovani (clone LV9-3 from MHOM/ Leishmania species (L. amazonensis, L. bra- ET/67/HU3). The L. donovani clones used ziliensis, L. donovani, and L. major) includ- were: wild-type LV9-3, which possesses ing the different clones of L. donovani. Low- three copies of the tryR (formerly tryA) gene molecular weight thiols were measured us- (tryR+/+/+) and the mutants of TryR: clones ing Ellman’s reagent (15). To measure H2-tryR-/+/+ (LV9-3 submitted to single re- soluble thiols the samples were deproteinised placement), clone HB3-tryR-/-/+ (LV9-3 sub- with 1% sulfosalicylic acid in the presence mitted to double replacement (12) and clone of 5 mM EDTA. The concentration of SH HB3-pTTcTR (Tovar J and Fairlamb AH, groups was calculated from a standard curve unpublished results) that is identical to clone of 0.01 to 2 nmol cysteine. Glutathione lev- HB3 (tryR-/-/+) but harbours plasmid pTTcTR els were measured by the glutathione reduc- (13). Promastigote forms of all Leishmania tase enzyme recycling method (16). These species were grown in M199 medium supple- assays were adapted for use in a microtitre mented with 40 mM HEPES, pH 7.4, 0.1 mM plate using a microplate spectrophotometer adenine, 7.7 mM hemin, 10% (v/v) heat-inac- system spectra MAX 250 (Molecular De- tivated foetal calf serum, 50 U/mL penicillin, vices, Union City, CA, USA). Cells were and 50 µg/mL streptomycin. Cultures were lysed by the addition of 100 µL 1 mM EDTA incubated at 26ºC, and cells were kept at to each well and freezing immediately. Fol- densities ranging between 5 x 105 and 3 x 107 lowing thawing, plates were shaken for 30 s parasites/mL. Transfectants were cultured in and then sonicated for 60 s. Assays were the presence of selective drugs (12). The mu- carried out immediately (17). Although this tant H2-tryR-/+/+ was cultured in the presence assay provides a measure of both oxidised of 16 µg/mL hygromycin B, the clone HB3- and reduced glutathione, in non-oxidative tryR-/-/+ in the presence of 16 µg/mL stress equilibrium, the cellular condition hygromycin B plus 2.5 µg /mL phleomycin, under which we have measured glutathione, and the HB3-tryR-/-/+ clone plus episomal the thiol content is 95-99% reduced glutathi- pTTcTR in the presence of 16 µg/mL one. hygromycin B, 25 µg/mL G418 and 2.5 µg/ mL phleomycin. Cytotoxic effect of S-nitroso-N-acetyl-D,L- penicillamine on Leishmania viability Growth curves The direct cytotoxic effect of the nitroso- Promastigotes of wild-type or L.