Abstract. Laboratório de Imunoparasitologia, Curso de Medicina, Universidade do Sul de Santa Catarina (UNISUL), Tubarão, SC, Brasil 2 - PDF

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Brazilian Journal of Medical and Biological Research (2006) 39: Protection of Leishmania by glutathione/trypanothione systems ISSN X 355 Glutathione and the redox control system trypanothione/trypanothione

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Brazilian Journal of Medical and Biological Research (2006) 39: Protection of Leishmania by glutathione/trypanothione systems ISSN X 355 Glutathione and the redox control system trypanothione/trypanothione reductase are involved in the protection of Leishmania spp. against nitrosothiol-induced cytotoxicity P.R.T. Romão 1, J. Tovar 2, S.G. Fonseca 3, R.H. Moraes 4, A.K. Cruz 5, J.S. Hothersall 6, A.A. Noronha-Dutra 6, S.H. Ferreira 4 and F.Q. Cunha 4 1 Laboratório de Imunoparasitologia, Curso de Medicina, Universidade do Sul de Santa Catarina (UNISUL), Tubarão, SC, Brasil 2 School of Biological Sciences, Royal Holloway University of London, Egham, UK 3 Laboratório de Imunologia, Instituto do Coração, Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP, Brasil 4 Departamento de Farmacologia, 5 Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, de São Paulo, Ribeirão Preto, SP, Brasil 6 The Institute of Urology and Nephrology, University College of London, London, UK Correspondence P.R.T. Romão Laboratório de Imunoparasitologia Curso de Medicina, UNISUL Rua José Acácio Moreira, 787, DEHON Tubarão, SC Brasil Fax: Research supported by FAPESP (No. 97/ ). P.R.T. Romão and S.G. Fonseca were recipients of FAPESP fellowships. F.Q. Cunha, S.H. Ferreira, and R.H. Moraes were recipients of CNPq fellowships. Received January 21, 2005 Accepted November 11, 2005 Abstract Glutathione is the major intracellular antioxidant thiol protecting mammalian cells against oxidative stress induced by oxygen- and nitrogen-derived reactive species. In trypanosomes and leishmanias, trypanothione plays a central role in parasite protection against mammalian host defence systems by recycling trypanothione disulphide by the enzyme trypanothione reductase. Although Kinetoplastida parasites lack glutathione reductase, they maintain significant levels of glutathione. The aim of this study was to use Leishmania donovani trypanothione reductase gene mutant clones and different Leishmania species to examine the role of these two individual thiol systems in the protection mechanism against S-nitroso-N-acetyl-D,L-penicillamine (SNAP), a nitrogen-derived reactive species donor. We found that the resistance to SNAP of different species of Leishmania was inversely correlated with their glutathione concentration but not with their total low-molecular weight thiol content (about 0.18 nmol/10 7 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/10 7 parasites, respectively. L. amazonensis, that have a higher level of glutathione, were less susceptible to SNAP (30 and 100 µm). The IC 50 values of SNAP determined to L. amazonensis, 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 decreased capacity to survive (~40%) in the presence of SNAP ( µm). In conclusion, the present data suggest that both antioxidant systems, glutathione and trypanothione/trypanothione reductase, participate in protection of Leishmania against the toxic effect of nitrogen-derived reactive species. Key words Leishmania Glutathione Trypanothione Trypanothione reductase Nitric oxide Free radicals 356 P.R.T. Romão et al. Introduction The trypanosomatids, members of the order Kinetoplastida, include parasitic protozoa of importance to public health such as Leishmania spp. Leishmania cause a spectrum of diseases ranging from self-healing ulcers to disseminated and often fatal infections, depending on the species involved and the host s immune response. Adequate vaccines against trypanosomatid infections have yet to be developed, and drugs currently available for chemotherapeutic intervention are mostly unsatisfactory mainly because of their lack of specificity, toxicity to humans, and, in many cases, developed parasite resistance (1). Thus, one of the priorities in tropical medicine research has been the identification and characterisation of parasite-specific biomolecules, which play relevant physiological roles and thus might be exploited as selective targets. Among many other metabolic distinctions, trypanosomatids maintain their intracellular redox balance by a mechanism that is different from that of their insect vectors and mammalian hosts. They lack glutathione reductase, which in nearly all other organisms is responsible for the maintenance of an intracellular thiol-reducing environment, and thus for the reduction of disulphides, detoxification of peroxides and synthesis of DNA precursors (2). Instead, they possess a unique system using trypanothione [T(SH) 2 ] that is the major reduced thiol of Kinetoplastida parasites (3) and comprises a spermidine moiety linked to two glutathione molecules (2). Together with three thiol-redox proteins, trypanothione reductase (TryR), tryparedoxin and tryparedoxin peroxidase (4,5), T(SH) 2 is thought to provide defence against oxidants, certain heavy metals (6) and xenobiotics (3). Thus, TryR has a vital physiological role in maintaining T(SH) 2 redox, particularly within the highly oxidative intracellular environment of the host cells which is generated during the antimicrobial defence response. In murine leishmaniasis, nitric oxide (NO) plays a crucial role in the killing of parasites both in vitro (7) and in vivo (8,9). In vitro macrophage microbicidal activity correlates with NO production, and both in vivo and in vitro microbicidal activities are completely inhibited by the NO synthase inhibitor L- arginine analogue N G -monomethyl-l-arginine (L-NMMA) but not by its enantiomer D-NMMA (8). In addition, the NO donors, S-nitroso-N-acetyl-D,L-penicillamine (SNAP) and 3-morpholino-sydnonimine hydrochloride are able to kill Leishmania parasites in a cell-free model system (10). We have reported that glutathione is involved in the protection of mammalian macrophages against the cytotoxic effects of NO. Furthermore, despite evidence that glutathione in Leishmania appears not to be the major antioxidant, we have demonstrated that it protects L. major from the toxic effects of NO (11). In the present study, we have extended these observations by comparing the glutathione levels and SNAP sensitivity of different Leishmania species: L. amazonensis, L. braziliensis, L. donovani, and L. major. Our results demonstrate that the sensitivity of distinct species of Leishmania to SNAP is inversely correlated with their glutathione concentration. When we extended our investigation to the role of TryR in the protection against SNAP using mutants of L. donovani for the TryR gene (tryr, formerly trya) (12), we found that, compared to control parasites (tryr genotype + / + / + ), a double mutant clone (tryr genotype - / - / + ) was significantly more sensitive to the cytotoxic effect of SNAP. Overall, these results demonstrate that glutathione as well as the T(SH) 2 /TryR redox system are essential protective components against NO cytotoxicity in Leishmania. Material and Methods Leishmania strains and culture conditions The Leishmania species used in this study Protection of Leishmania by glutathione/trypanothione systems 357 were L. braziliensis (MHOM/BR/75/M2904), L. amazonensis (MPRO/BR/72/M1841-LV- 79), L. major (LV-39, clone 5-Rho-SU/59/P), and L. donovani (clone LV9-3 from MHOM/ ET/67/HU3). The L. donovani clones used were: wild-type LV9-3, which possesses three copies of the tryr (formerly trya) gene (tryr +/+/+ ) and the mutants of TryR: clones H2-tryR -/+/+ (LV9-3 submitted to single replacement), clone HB3-tryR -/-/+ (LV9-3 submitted to double replacement (12) and clone HB3-pTTcTR (Tovar J and Fairlamb AH, unpublished results) that is identical to clone HB3 (tryr -/-/+ ) but harbours plasmid pttctr (13). Promastigote forms of all Leishmania species were grown in M199 medium supplemented with 40 mm HEPES, ph 7.4, 0.1 mm adenine, 7.7 mm hemin, 10% (v/v) heat-inactivated foetal calf serum, 50 U/mL penicillin, and 50 µg/ml streptomycin. Cultures were incubated at 26ºC, and cells were kept at densities ranging between 5 x 10 5 and 3 x 10 7 parasites/ml. Transfectants were cultured in the presence of selective drugs (12). The mutant H2-tryR -/+/+ was cultured in the presence of 16 µg/ml hygromycin B, the clone HB3- tryr -/-/+ in the presence of 16 µg/ml hygromycin B plus 2.5 µg /ml phleomycin, and the HB3-tryR -/-/+ clone plus episomal pttctr in the presence of 16 µg/ml hygromycin B, 25 µg/ml G418 and 2.5 µg/ ml phleomycin. Growth curves Promastigotes of wild-type or L. donovani mutants were cultured in M199 medium prepared as described previously (14). Cell density in the inoculum was 1 x 10 5 /ml. Viability was evaluated from motility and cell density was determined daily using a hemocytometer. Glutathione and non-protein low-molecular weight thiol measurement Non-protein low-molecular weight thiols and glutathione (reduced plus disulphide forms) were measured in lysates of promastigote forms (stationary phase) of different Leishmania species (L. amazonensis, L. braziliensis, L. donovani, and L. major) including the different clones of L. donovani. Lowmolecular weight thiols were measured using Ellman s reagent (15). To measure soluble thiols the samples were deproteinised with 1% sulfosalicylic acid in the presence of 5 mm EDTA. The concentration of SH groups was calculated from a standard curve of 0.01 to 2 nmol cysteine. Glutathione levels were measured by the glutathione reductase enzyme recycling method (16). These assays were adapted for use in a microtitre plate using a microplate spectrophotometer system spectra MAX 250 (Molecular Devices, Union City, CA, USA). Cells were lysed by the addition of 100 µl 1 mm EDTA to each well and freezing immediately. Following thawing, plates were shaken for 30 s and then sonicated for 60 s. Assays were carried out immediately (17). Although this assay provides a measure of both oxidised and reduced glutathione, in non-oxidative stress equilibrium, the cellular condition under which we have measured glutathione, the thiol content is 95-99% reduced glutathione. Cytotoxic effect of S-nitroso-N-acetyl-D,Lpenicillamine on Leishmania viability The direct cytotoxic effect of the nitrosothiol SNAP on Leishmania species was measured. Briefly, parasites (3 x 10 6 /well) were incubated in M199 medium supplemented with 10% heat-inactivated foetal calf serum in the presence or absence of SNAP ( µm) for 12 h. The incubation medium contains L-cystine which allows the membrane transport of SNAP into cells (18). Parasites were then pulsed with 1 µci/well [ 3 H]-thymidine, and the incorporation of radioactivity by viable parasites was determined after 24 h in a ß-counter (11). The 358 P.R.T. Romão et al. Figure 1. Intracellular glutathione (GSH) concentrations in different Leishmania species. Total glutathione (GSH + oxidized glutathione (GSSG)) was measured in lysates of 1 x 10 7 promastigotes of different Leishmania species (L. amazonensis, L. braziliensis, L. donovani, and L. major). Data are reported as means ± SEM of four replicate cultures and are representative of three experiments. P 0.05 compared with L. amazonensis; # P 0.05 compared with L. donovani; + P 0.05 compared with L. major (ANOVA followed by Bonferroni s t-test). Figure 2. Concentrations of nonprotein low-molecular weight thiols in different Leishmania species. Total non-protein thiols (glutathione, T(SH) 2, cysteine, and ovothiol) were measured in lysates of 1 x 10 7 promastigotes of Leishmania species (L. amazonensis, L. braziliensis, L. donovani, and L. major). Data are reported as means ± SEM of four replicate cultures and are representative of three experiments. 50% inhibitory concentration (IC 50 ) values for each Leishmania species were determined using Sigma-Plot software, Version 5.0. Statistical analysis Data are reported as means ± SEM and statistical significance (P 0.05) was assessed by ANOVA followed by Bonferroni s t-test. Results Non-protein low-molecular weight thiol and glutathione concentration in different Leishmania species Glutathione concentrations of different species of Leishmania (L. amazonensis, L. braziliensis, L. major, and L. donovani) are shown in Figure 1. Statistically significant differences in glutathione levels were observed between all Leishmania species, with promastigotes of L. braziliensis having the lowest level. Glutathione concentration was L. amazonensis L. donovani L. major L. braziliensis. No significant differences in the non-protein low-molecular weight thiol levels were detected between different Leishmania species (Figure 2). Cytotoxic effect of S-nitroso-N-acetyl-D, L- penicillamine on different Leishmania species To ascertain whether the glutathione levels in the different Leishmania species (Figure 1) correlate with their sensitivity to reactive nitrogen species, we investigated the cellular viability of L. amazonensis, L. braziliensis, L. donovani, and L. major after treatment with SNAP. The addition of a SNAP directly to promastigotes of different species of Leishmania resulted in dose-dependent parasite killing (Figure 3). The sensitivity of Leishmania species to SNAP correlated inversely with the glutathione levels. L. amazonensis, that has a higher glutathione concentration, was more resistant to the cytotoxic effect of SNAP at concentrations of 30 and 100 µm, with an IC 50 of ( µm). In contrast, L. braziliensis, that shows lower levels of glutathione, was more susceptible to the toxic effect of SNAP (IC 50 of 83 ( µm)). The IC 50 values determined for L. donovani and L. major were and µm, respectively. Effect of S-nitroso-N-acetyl-D,L-penicillamine on the viability of Leishmania donovani tryr gene mutants To ascertain whether the T(SH) 2 /TryR antioxidant system is involved in the protection of Leishmania against nitrogen-derived reactive species, we investigated the effect Protection of Leishmania by glutathione/trypanothione systems 359 of SNAP on the viability of targeted L. donovani TryR mutants that have been generated by gene disruption. Wild-type L. donovani (LV9-3, genotype of tryr +/+/+ ) and clones submitted to a single (clone H2 genotype of tryr -/+/+ ) or double (clone HB3 genotype of tryr -/-/+ ) experiment for the replacement of the tryr locus were utilised. It is important to mention that a null mutant has not yet been obtained for tryr (12,19). The addition of SNAP directly to promastigotes of different L. donovani clones resulted in partial parasite killing (Figure 4). Recombinant HB3 possessing only one tryr allele was more sensitive to the toxic effect of SNAP at concentrations of µm compared with recombinant H2 or L. donovani parental clone (LV9-3). The sensitivity to high concentration of SNAP (300 µm) was unchanged. To demonstrate that the decrease in the sensitivity was solely due to the disruption of the tryr gene, we tested the viability of clone HB3 (tryr -/-/+ ) supplemented with the plasmid pttctr that harbours a functional T. cruzi tryr gene (13). This recombinant strain, whose levels of TryR are higher than those of wild-type parasites, regained wild-type levels of resistance to SNAP-generated stress (Figure 4). Glutathione concentration in Leishmania donovani TryR mutants Our previous results (11) and the data presented here suggest that glutathione is involved in the protection of Leishmania against the toxic effects of SNAP. As TryR is the enzyme that maintains T(SH) 2 in its reduced form and plays a central role in oxidant detoxification through the enzymatic regeneration of the thiol pool, we examined whether the effect of the loss of the tryr copy in L. donovani mutant clones changed the glutathione levels. No significant differences in glutathione were detected between the wild-type and single- or double-targeted L. donovani tryr mutants or with the HB3- % of cellular viability relative to control L. amazonensis L. donovani L. major L. braziliensis Figure 3. Cytotoxic effect of S-nitroso-N-acetyl-D,L-penicillamine (SNAP) on different Leishmania species. Promastigote forms of L. amazonensis, L. braziliensis, L. donovani, and L. major (3 x 10 6 cells/well) were incubated in M199 medium (control) or M199 plus SNAP ( µm). The cultures were pulsed with 3 [H]-thymidine 12 h after SNAP treatment. Leishmania survival was determined after 24 h of further culture by the ability of residual live parasites to incorporate 3 [H]-thymidine. Data are reported are means ± SEM of four replicates and are representative of three experiments. Statistically significant different with P 0.05 compared with L. amazonensis, # P 0.05 compared with L. donovani, and + P 0.05 compared with L. major (ANOVA followed by Bonferroni s t-test). % of cellular viability relative to control # + # SNAP (µm) Figure 4. Cytotoxic effect of S-nitroso-N-acetyl-D,L-penicillamine (SNAP) on L. donovani viability. Effects of the number of tryr allele promastigotes (3 x 10 6 ) of wild-type L. donovani (LV9-3: tryr +/+/+ ) and clones H2 (tryr -/+/+ ), HB3 (tryr -/-/+ ) and HB3-pTTcTR (tryr -/-/+ plus ptextctr episomic) were incubated in M199 medium (control) or M199 plus SNAP ( µm) for 12 h. Leishmania survival was determined after 24 h of further culture by the ability of residual live parasites to incorporate 3 [H]-thymidine. Data are reported as means ± SEM of four replicates and are representative of three experiments. P 0.05 compared with the parental clone (LV9-3; ANOVA followed by Bonferroni s t-test) SNAP (µm) # + # LV9-3 ( tryr +/+/+ ) H2 (tryr -/+/+ ) HB3 (tryr -/-/+ ) HB3-pTTcTR 360 P.R.T. Romão et al. Figure 5. Intracellular glutathione (GSH) levels in L. donovani trypanothione reductase (tryr) mutants. Total glutathione (GSH + oxidized glutathione (GSSG)) was measured in lysates of 1 x 10 7 promastigotes of wild-type L. donovani (LV9-3: tryr +/+/+ ) and clones H2 (tryr -/+/+ ), HB3 (tryr -/-/+ ), and HB3-pTTcTR (tryr -/-/+ plus ptextctr episomic). Data are reported as means ± SEM of four replicate cultures and are representative of three experiments. pttctr transfected clone (Figure 5). Growth of Leishmania donovani clones Growth curves of both wild-type (LV9-3) and L. donovani mutants (clones H2-tryR -/+/+, Figure 6. Growth curve of wild-type L. donovani (LV9-3: tryr +/+/+ ) and clones H2 (tryr -/+/+ ), HB3 (tryr -/-/+ ), and HB3-pTTcTR (tryr -/-/+ plus ptextctr episomic). Each point represents the average of counts from two cultures, initially inoculated with 1 x 10 5 organisms/ml in M199 medium. HB3-tryR -/-/+ and HB3-pTTcTR) show the typical log and stationary growth phases. A similar pattern of continual growth was observed for all transfectants (Figure 6). Discussion The results presented in this study suggest that both glutathione and T(SH) 2 /TryR antioxidant systems are important components of the protective mechanisms of Leishmania against the cytotoxic effects of nitrogen-derived reactive species. This conclusion is supported by the following observations: a) the sensitivity of distinct species of Leishmania (L. amazonensis, L. braziliensis, L. donovani, and L. major) to SNAP, a nitrogen-derived reactive species donor, correlated inversely with their cellular glutathione levels but not with their total non-protein low-molecular weight thiol content; b) mutants of L. donovani possessing only one wild-type tryr allele, tryr -/-/+ (submitted to a double replacement of the tryr gene locus), had a decreased capacity to survive in the presence of SNAP. Nevertheless, under normal conditions of in vitro growth, the L. donovani wild-type and recombinant mutants have similar levels of glutathione. Our data are consistent with Lemesre et al. (20) who demonstrated that amastigote forms of L. amazonensis, which
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