Disruption of the trypanothione reductase gene of Leishmania decreases its ability to survive oxidative stress in macrophages
1997; Springer Nature; Volume: 16; Issue: 10 Linguagem: Inglês
10.1093/emboj/16.10.2590
ISSN1460-2075
Autores Tópico(s)Parasites and Host Interactions
ResumoArticle15 May 1997free access Disruption of the trypanothione reductase gene of Leishmania decreases its ability to survive oxidative stress in macrophages Carole Dumas Carole Dumas Centre de Recherche en Infectiologie du CHUL and Département de Microbiologie, Faculté de Médecine, Université Laval, Québec, Canada Search for more papers by this author Marc Ouellette Marc Ouellette Centre de Recherche en Infectiologie du CHUL and Département de Microbiologie, Faculté de Médecine, Université Laval, Québec, Canada Search for more papers by this author Jorge Tovar Jorge Tovar Department of Medical Parasitology, London School of Hygiene and Tropical Medicine, University of London, UK Search for more papers by this author Mark L. Cunningham Mark L. Cunningham Department of Medical Parasitology, London School of Hygiene and Tropical Medicine, University of London, UK Search for more papers by this author Alan H. Fairlamb Alan H. Fairlamb Department of Medical Parasitology, London School of Hygiene and Tropical Medicine, University of London, UK Search for more papers by this author Samira Tamar Samira Tamar Centre de Recherche en Infectiologie du CHUL and Département de Microbiologie, Faculté de Médecine, Université Laval, Québec, Canada Search for more papers by this author Martin Olivier Martin Olivier Centre de Recherche en Infectiologie du CHUL and Département de Microbiologie, Faculté de Médecine, Université Laval, Québec, Canada Search for more papers by this author Barbara Papadopoulou Corresponding Author Barbara Papadopoulou Centre de Recherche en Infectiologie du CHUL and Département de Microbiologie, Faculté de Médecine, Université Laval, Québec, Canada Search for more papers by this author Carole Dumas Carole Dumas Centre de Recherche en Infectiologie du CHUL and Département de Microbiologie, Faculté de Médecine, Université Laval, Québec, Canada Search for more papers by this author Marc Ouellette Marc Ouellette Centre de Recherche en Infectiologie du CHUL and Département de Microbiologie, Faculté de Médecine, Université Laval, Québec, Canada Search for more papers by this author Jorge Tovar Jorge Tovar Department of Medical Parasitology, London School of Hygiene and Tropical Medicine, University of London, UK Search for more papers by this author Mark L. Cunningham Mark L. Cunningham Department of Medical Parasitology, London School of Hygiene and Tropical Medicine, University of London, UK Search for more papers by this author Alan H. Fairlamb Alan H. Fairlamb Department of Medical Parasitology, London School of Hygiene and Tropical Medicine, University of London, UK Search for more papers by this author Samira Tamar Samira Tamar Centre de Recherche en Infectiologie du CHUL and Département de Microbiologie, Faculté de Médecine, Université Laval, Québec, Canada Search for more papers by this author Martin Olivier Martin Olivier Centre de Recherche en Infectiologie du CHUL and Département de Microbiologie, Faculté de Médecine, Université Laval, Québec, Canada Search for more papers by this author Barbara Papadopoulou Corresponding Author Barbara Papadopoulou Centre de Recherche en Infectiologie du CHUL and Département de Microbiologie, Faculté de Médecine, Université Laval, Québec, Canada Search for more papers by this author Author Information Carole Dumas1, Marc Ouellette1, Jorge Tovar2, Mark L. Cunningham2, Alan H. Fairlamb2, Samira Tamar1, Martin Olivier1 and Barbara Papadopoulou 1 1Centre de Recherche en Infectiologie du CHUL and Département de Microbiologie, Faculté de Médecine, Université Laval, Québec, Canada 2Department of Medical Parasitology, London School of Hygiene and Tropical Medicine, University of London, UK *Corresponding author. E-mail: [email protected] The EMBO Journal (1997)16:2590-2598https://doi.org/10.1093/emboj/16.10.2590 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Parasitic protozoa belonging to the order Kinetoplastida contain trypanothione as their major thiol. Trypanothione reductase (TR), the enzyme responsible for maintaining trypanothione in its reduced form, is thought to be central to the redox defence systems of trypanosomatids. To investigate further the physiological role of TR in Leishmania, we attempted to create TR-knockout mutants by gene disruption in L.donovani and L.major strains using the selectable markers neomycin and hygromycin phosphotransferases. TR is likely to be an important gene for parasite survival since all our attempts to obtain a TR null mutant in L.donovani failed. Instead, we obtained mutants with a partial trisomy for the TR locus where, despite the successful disruption of two TR alleles by gene targeting, a third TR copy was generated as a result of genomic rearrangements involving the translocation of a TR-containing region to a larger chromosome. Mutants of L.donovani and L.major possessing only one wild-type TR allele express less TR mRNA and have lower TR activity compared with wild-type cells carrying two copies of the TR gene. Significantly, these mutants show attenuated infectivity with a markedly decreased capacity to survive intracellularly within macrophages, provided that the latter are producing reactive oxygen intermediates. Introduction Parasitic protozoa of the order Kinetoplastida are the causative agents of several medically important tropical diseases including sleeping sickness (Trypanosoma brucei rhodesiense and T.b.gambiense), Chagas disease (T.cruzi) and visceral (kala-azar) and cutaneous (oriental sore) leishmaniasis (e.g. Leishmania donovani and L.major, respectively). Among many other metabolic peculiarities, trypanosomatids maintain their intracellular redox balance by a mechanism that is completely different from that of their insect vectors and mammalian hosts. They lack glutathione reductase (GR) which in nearly all other organisms is responsible for the maintenance of an intracellular reducing environment important for the reduction of disulfides, the detoxification of peroxides and the synthesis of DNA precursors (Fairlamb et al., 1985; Schirmer and Schulz, 1987). Instead, they possess a unique system using N1, N8-bis(glutathionyl)spermidine (trypanothione) and its metabolic precursor N1-glutathionylspermidine as their main thiols. These glutathione conjugates are kept in the reduced state by trypanothione reductase (TR) (Fairlamb et al., 1985; Shames et al., 1986; Fairlamb and Cerami, 1992). TRs are members of the NADPH-dependent flavoprotein oxidoreductase family and are structurally and mechanistically related to GRs (Shames et al., 1986; Krauth-Siegel et al., 1987). Human GR and all parasite TRs have mutually exclusive substrate specificities (Shames et al., 1986; Henderson et al., 1987; Krauth-Siegel et al., 1987; Cunningham and Fairlamb, 1995; Krauth-Siegel and Schöneck, 1995), providing a route for the design of selective inhibitors against the parasite enzymes. During its infective cycle in the vertebrate host, Leishmania must survive the rigorous oxidizing environment of the macrophage. TR and its subordinate thiols are proposed to play a vital role in maintaining an intracellular reducing environment and in protecting these parasites against oxidative damage, arising both internally as a result of their aerobic metabolism and externally by the immune response of the mammalian host (Fairlamb et al., 1985; Shames et al., 1986). Selective inhibition of TR is therefore potentially an attractive strategy to incapacitate these parasites. In view of the pivotal role and uniqueness of TR in the management of oxidative stress, we have attempted to generate attenuated Leishmania strains with decreased or null TR activity that could eventually be useful for the development of live vaccines. In order to generate attenuated strains and to investigate further the physiological role of TR, we have genetically engineered TR mutants by gene disruption in pathogenic Leishmania species and analysed the phenotype of the resulting mutants with respect to intracellular survival of the parasites within macrophages. Results Inactivation of the L.donovani TR gene by gene disruption The TR gene is single copy in the Leishmania genome (Taylor et al., 1994) and, since Leishmania is generally considered to be diploid and no sexual crosses have been achieved, two successive rounds of gene targeting should be sufficient to create null mutants for TR. In an attempt to inactivate both TR alleles in L.donovani, we have created two disruption constructs. These consist of the insertion of the neomycin phosphotransferase (neo) or the hygromycin phosphotransferase (hyg) expression cassettes (Papadopoulou et al., 1994a) into the unique BalI site of the 1.25 kb PCR fragment of the TR coding region subcloned into the pSP72 vector (Promega) in order to form TR::neo and TR::hyg, respectively (see Figure 1A). Figure 1.Inactivation of L.donovani TR alleles. (A) Schematic drawing of the TR locus in L.donovani and its relevant restriction sites (S, SacII; Bl, BalI). Integration of the hyg cassette into the TR gene must generate two additional SacII fragments of 1.3 and 2.83 kb. Upon integration of the neo disruption construct at the homologous locus, the genomic SacII fragment would increase from 2.9 to 3.8 kb. The 3.1 kb S–S′ fragment corresponds to a new restriction fragment generated following genomic rearrangement leading to a translocation of part of the TR locus on a 1200 kb chromosome (see C and text). (B) Southern blot analysis of L.donovani wild-type and TR single and double targeted mutants. DNAs were digested with SacII, electrophoresed through an agarose gel and hybridized independently with TR-, hyg- and neo-specific probes. Lanes 1, L.donovani wild-type; 2, L.donovani TR/TR::hyg mutant; 3, L.donovani TR/TR::neo; and 4, L.donovani TR/TR::hyg/TR::neo double targeted transfectant. (C) Molecular karyotype of L.donovani TR mutants. Chromosomes were separated by pulsed-field gel electrophoresis using a Bio-Rad CHEF-DR III apparatus at 5 V/cm, 120° separation angle and switch times varying from 35 to 120 for 30 h. Blots were hybridized to the same probes as above. Lanes 1 and 2 are as in (B) and lane 3 corresponds to the L.donovani TR/TR::hyg/TR::neo mutant. Download figure Download PowerPoint In a first round of targeting, the TR::hyg disruption construct was transfected into L.donovani. Cells growing in the presence of hygromycin B were cloned on semi-solid agar and the DNA of clones was digested with SacII and analysed by Southern blots hybridized to specific TR and hyg probes. Two SacII fragments of 2.6 and 2.9 kb were recognized by the TR probe in wild-type L.donovani (Figure 1B, lane 1). Integration of the hyg cassette into the TR gene should replace one of the 2.9 kb fragments with two new restriction fragments of 1.3 and 2.83 kb (see Figure 1A) while leaving the 2.6 kb fragment intact. If only one allele is disrupted, then the 2.6 and 2.9 kb bands corresponding to the second non-targeted TR allele should be visible as well. Hybridization to TR and hyg probes (Figure 1B, lanes 2) confirmed that the expected single disruption with the hyg gene had occurred. Likewise, transfection of wild-type cells with the TR::neo construct resulted in successful disruption of one TR allele, generating the predicted 3.8 kb SacII fragment (Figure 1B, lanes 3). A clone of the TR/TR::hyg transfectant was selected for the second round of targeting using the TR::neo disruption construct. Transfectants resistant to G418 and hygromycin B were obtained and clones were analysed by hybridization with probes specific for TR, neo and hyg. The neo gene was indeed integrated into the TR locus because the predicted 3.8 kb SacII fragment was obtained following hybridization to neo and TR probes (Figure 1B, lanes 4). Although TR gene disruption by neo and hyg took place, as clearly shown from the hybridization studies, one TR allele remained intact since the 2.9 kb genomic SacII fragment was still present in the double targeted mutant (Figure 1B, lane 4). Moreover, a novel 3.1 kb SacII band hybridizing to the TR probe was detected in this transfectant (Figure 1B, lane 4), most likely resulting from a genomic rearrangement. Digestions with other restriction enzymes also revealed new fragments hybridizing to the TR probe that did not co-migrate with bands present in the wild-type strain (not shown), indicating that a genomic rearrangement must have occurred upstream of the TR gene. To determine how these rearranged fragments might have arisen, chromosomes of the TR/TR::hyg/TR::neo transfectant were resolved by CHEF electrophoresis and the blot was hybridized to the appropriate probes. In wild-type L.donovani, TR is located on a 520 kb chromosome (Figure 1C), which is in contrast to the 1.1 Mb chromosome reported for another isolate of L.donovani (Taylor et al., 1994). In addition to a 520 kb chromosome on which the TR gene is normally located, a new chromosome of ∼1200 kb hybridized to a TR-specific probe (Figure 1C, lane 3). The newly generated TR allele was the one targeted by the neo gene (see Figure 1C, lane 3), thus leaving an intact TR allele at the original chromosomal location (520 kb). In additional independent transfection experiments, we were again unable to generate a null mutant, as the TR locus became trisomic (not shown). Thus, our attempts to generate a TR null mutant in L.donovani have, therefore, failed. These results suggest that the TR gene is possibly essential for survival of L.donovani promastigotes. In order to explain the events leading to genomic translocation and aneuploidy for TR and to determine the size of the translocated region, we have hybridized a CHEF blot of wild-type L.donovani and TR disruption mutants to several probes covering a region of ∼35 kb containing the TR locus. As a first probe, we used a 4.1 kb ClaI–EcoRI fragment (probe 1, Figure 2A) containing a large part of the TR gene and the putative sequences used for the rearrangement at the 5′ end as indicated from the SacII digestion (see Figures 1B and 2A). Even at relatively high stringency, a positive hybridization signal was obtained at the level of a 1200 kb chromosome in the L.donovani wild-type and TR/TR::hyg strains (Figure 2C, lanes 1 and 2), indicating that sequences homologous to those present at the TR locus on the 520 kb chromosome were also found on a 1200 kb chromosome. Figure 2.Translocation of part of the TR locus in L.donovani TR/TR::hyg/TR::neo mutant leading to partial trisomy for TR. (A) A restriction map of a genomic region of a 45 kb part of a 520 kb chromosome containing the TR locus. Restriction sites are indicated as C for ClaI, RI for EcoRI, S for SacII and X for XhoI. The two boxes located upstream and downstream of the TR gene represent the putative sequences used during translocation leading to the generation of a third TR allele. The upstream box is part of the 2.6 kb SacII restriction fragment (see Figure 1B) although its exact position is unknown. (B) Schematic drawing of a region of at least 35 kb within the TR locus translocated from the 520 kb (thin line) into a 1200 kb chromosome (thick line). S′, X′ and RI′ represent novel restriction sites generated following the upstream genomic rearrangement. (C) Presence of homologous sequences in the 520 kb TR locus-containing chromosome and in the 1200 kb chromosome where translocation occurred. Chromosomes were separated by CHEF electrophoresis as described in Materials and methods and hybridized to probe 1 (see A). Lanes 1, L.donovani wild-type; 2, L.donovani TR/TR::hyg; and 3, L.donovani TR/TR::hyg/TR::neo. (D) Southern blot of total genomic DNA digested with XhoI and hybridized to probe 1. Lanes are as in (C). (E) A CHEF blot hybridizing to probe 2 consisting of a 23 kb EcoRI–EcoRI fragment located downstream of the TR gene to look for the second rearrangement site. Lanes are as in (C). Download figure Download PowerPoint We tested the possibility of whether homologous genomic repeated sequences could also be used for the downstream rearrangement site. No homology between the upstream and the putative downstream point of rearrangement has been detected. Indeed, the only rearranged fragment hybridizing to probe 1 (even at longer exposure) was the 3.3 kb XhoI–XhoI′ representing the upstream rearrangement in the double TR-targeted mutant (Figure 2B and D, lane 3). To try to pinpoint the putative downstream rearrangement site, we used a large probe of 23 kb located 9 kb downstream of the TR gene (probe 2, Figure 2A). No rearranged fragments were observed using probe 2 in the double targeted TR mutant compared with wild-type (data not shown), suggesting that the second rearrangement occurred further downstream. The intense hybridization signal (Figure 2E, lane 3) observed at the level of the 1200 kb chromosome for the TR/TR::hyg/TR::neo mutant indicated that the region covered by probe 2 was translocated along with TR. Decreased intracellular survival of the L.donovani TR/TR::hyg and TR/TR::hyg/TR::neo mutants Not only the single but also the double targeted L.donovani TR mutants that we have generated by gene disruption contain one wild-type TR allele. To look at the effect of the loss of one TR copy in our mutants, we first examined TR mRNA levels in both promastigote and amastigote stages of the parasite. L.donovani stationary phase promastigotes were differentiated in vitro into amastigotes as described in Materials and methods. Differentiation into amastigotes was monitored by hybridizing RNAs (not shown) to an amastigote A2-specific probe (Charest and Matlashewski, 1994). Northern blot analysis of total RNAs isolated from L.donovani wild-type and TR mutants hybridized to a TR-specific probe showed that parasites either with the TR/TR::hyg or the TR/TR::hyg/TR::neo background produce lower amounts of TR mRNA than wild-type cells (Figure 3A). Densitometric scanning of the RNA bands and normalization with the tubulin RNA levels used as control indicated that there is 3- to 5-fold less RNA in the TR mutants than in wild-type Leishmania. Furthermore, no stage-specific expression of the TR gene was observed in wild-type Leishmania (see Figure 3A). Figure 3.TR mRNA expression in the wild-type and single and double TR targeted mutants of L.donovani. (A) Northern blot of total RNAs isolated from the promastigote and amastigote stages of L.donovani wild-type and transfectants and hybridized to a TR-specific probe. Each track contains ∼10 μg of total RNA. Lanes 1, L.donovani wild-type; 2, TR/TR::hyg single knockout mutant; and 3, TR/TR::hyg/TR::neo double targeted transfectant. (B) The same blot was stripped off and re-hybridized with the T.brucei α-tubulin probe to monitor the amount of RNA layered in each lane. Download figure Download PowerPoint To ascertain whether decreased levels of TR mRNA in the disruption mutants correlated with decreased TR activity, we have measured enzymatic activities in control and TR mutant strains. A 56% decrease of TR enzymatic activity was detected in the single and the double targeted L.donovani TR mutants compared with the control cells (see Table I). As TR is the enzyme that maintains trypanothione in its reduced form (Fairlamb et al., 1985), we examined whether a decrease in TR activity correlates with an alteration in thiol levels. No statistically significant differences in levels of trypanothione, glutathionylspermidine or glutathione were observed between controls and TR-targeted mutants (Table I). Table 1. Trypanothione reductase (TR) activity and thiol content in Leishmania control and TR mutants Cells Genotype Enzyme activity (U/mg) Ratio TR/ALAT Thiol content (nmol/108 cells) TR ALATa GSHb GspdSHc T[SH]2d L.donovani TR/TR 0.212 ± 0.023 0.117 ± 0.007 1.81 3.10 ± 0.33 1.67 ± 0.12 5.78 ± 1.33 L.donovani TR/TR::hyg 0.090 ± 0.005 0.112 ± 0.023 0.80 3.55 ± 1.65 1.91 ± 0.26 4.91 ± 1.40 L.donovani TR/TR::hyg/TR::neo 0.077 ± 0.016 0.097 ± 0.003 0.79 2.90 ± 0.24 1.30 ± 0.17 5.30 ± 0.17 L.major TR/TR 0.437 ± 0.041 0.337 ± 0.022 1.30 NDe ND ND L.major TR/TR::hyg 0.096 ± 0.005 0.114 ± 0.002 0.84 ND ND ND a ALAT, alanine aminotransferase used as an internal control; b GSH, glutathione; c GspdSH, glutathionylspermidine; d T[SH]2, trypanothione; e ND, not determined. Given the putative importance of TR in the detoxification of lethal oxygen metabolites that are produced by the macrophage following infection, we investigated whether our TR disruption mutants with less TR activity than wild-type cells have the same ability to survive intracellularly. To test this, we used an in vitro system to infect either murine or human macrophages with Leishmania promastigotes. Both the L.donovani TR/TR::hyg and TR/TR::hyg/TR::neo mutants were tested. In the case of human monocytes differentiated into macrophages, a significant decrease in the percentage of parasitized cells was observed with the single and double targeted TR mutants as early as 24 h following infection (Figure 4A). Moreover, although the infection rate was maintained throughout the experiment for the control L.donovani-neo transfectant, this was not the case for the TR/TR::hyg and TR/TR::hyg/TR::neo mutants where infectivity levels decreased steadily to reach only 12% after 72 h (Figure 4A). Similarly, the number of amastigotes per macrophage decreased drastically from nine at 6 h infection to ∼0.5 amastigote/cell after 72 h for the TR/TR::hyg and TR/TR::hyg/TR::neo mutants (see Figure 4B). Indeed, the loss of one TR allele has dramatic consequences on the intracellular viability of the parasite. To prove that the observed phenotype was due solely to the disruption of TR, we have transfected into the L.donovani TR/TR::hyg a neo expression vector carrying a 2.2 kb insert containing the TR gene of L.donovani infantum and 650 bp of upstream sequence (see Materials and methods). This recombinant strain regained its ability to survive inside macrophages to approximately wild-type levels (Figure 4), strongly suggesting that the loss of one TR copy was responsible for the decreased intracellular survival observed in the mutants. Figure 4.Infectivity of the L.donovani TR disruption mutants toward human monocytes differentiated into macrophages. Human macrophages were incubated with stationary phase Leishmania parasites (20:1, parasite to cell ratio) for 6 h as described in Materials and methods. After this initial incubation, free parasites were washed and incubation was followed for 24, 48 and 72 h. At these fixed time points, cell cultures were dried and stained with Diff Quick in order to determine the level of infection. ▪ Infection with L.donovani-neo, a transfectant containing a neo vector used as a control, ● L.donovani TR/TR::hyg single disruption mutant, ⋄ L.donovani TR/TR::hyg/TR::neo double targeted mutant, ▵ L.donovani TR/TR::hyg transfected with a neo expression vector carrying a 2.2 kb SphI–SphI genomic fragment containing only the TR gene (see Materials and methods) to revert the mutant phenotype. (A) The percentage of infected macrophages and (B) the total number of amastigotes within 100 macrophage cells with time. The average and standard deviation of four independent experiments are shown. Download figure Download PowerPoint The intracellular survival of L.donovani TR/TR::hyg and TR/TR::hyg/TR::neo mutants was also studied in the murine macrophage-like J774 cell line. However, in this cell line, the mutant's intracellular survival was not affected and parasites grew similarly to the control (not shown). To try to explain this intriguing difference, we looked at macrophage function related to intracellular parasite killing that could influence the phenotype of the TR mutants, such as the production of superoxide anion. The oxidation of cytochrome c was measured in the supernatants of cells stimulated with Leishmania, or phorbol myristate acetate as a positive control, as described in Materials and methods. Our results indicate that, following stimulation with Leishmania cells, the murine macrophage-like J744 cell line shows no detectable levels of superoxide anion, in contrast to the human macrophages that produce high amounts of this reactive oxygen metabolite (see Table II). Thus, the intracellular survival of Leishmania with diminished TR activity seems to decrease only in macrophages capable of producing superoxide anions. Table 2. Production of superoxide anion (nmol O2−/h/106 cells) by murine (J774) and human macrophages following infection with L.donovani 1 h 2 h 4 h 6 h Human J774 Human J774 Human J774 Human J774 PMA 11.2a <0.001 4.09 <0.001 1.28 <0.001 0.32 <0.001 L.donovani 13.1 <0.001 5.03 <0.001 0.82 <0.001 0.17 <0.001 L.donovani TR/TR::hyg/TR::neo 7.4 <0.001 4.89 <0.001 1.37 <0.001 0.11 <0.001 a Average of three independent experiments. Phenotype of L.major TR/TR::hyg mutant To test whether the results observed in L.donovani would be similar in other pathogenic species, we have inactivated one allele of the L.major TR gene by targeted gene disruption. Since isogenic DNA appears to be necessary for efficient gene targeting in Leishmania (Papadopoulou and Dumas, in preparation), we amplified the TR gene of L.major by PCR and introduced the hyg expression cassette into the BalI unique site of the gene. The resulting construct was then transfected into L.major. As shown by Southern blot hybridization of total XhoI DNA digests of L.major wild-type and TR/TR::hyg transfectant, the integration of the hyg gene into one TR allele leads to an increase in size of one genomic fragment from 8 to 9.2 kb (Figure 5B, lane 2). Less TR activity was found in this mutant as observed with L.donovani disruption mutants (Table I). The L.major TR/TR::hyg disruption mutant was tested further for its capacity to infect human macrophages. As observed for L.donovani, the L.major transfectant missing one TR allele showed an important decrease in parasite infectivity. Indeed, 1.1 amastigotes/cell were detected at 72 h post-infection in L.major TR/TR::hyg mutant compared with five amastigotes/cell at the beginning of infection (Figure 5C). Figure 5.Phenotype of L.major TR/TR::hyg mutant. (A) Schematic drawing of the L.major TR locus with its XhoI restriction sites. (B) Generation of a L.major TR disruption mutant by gene targeting. Southern blot analysis of total DNAs from L.major wild-type (lane 1) and L.major TR/TR::hyg (lane 2) digested with XhoI and hybridized to TR- and hyg-specific probes. (C) Infection of human macrophages with L.major in vitro. This was done as described in Figure 4 and in Materials and methods. The left panel corresponds to the total number of amastigotes in 100 macrophage cells and the right panel to the percentage of infected macrophages with time. The average and standard deviation of four independent experiments are shown. Download figure Download PowerPoint Discussion Trypanothione and its related enzymes are thought to be involved in many cellular functions similar to those of glutathione in mammalian cells, such as maintenance of an intracellular reducing environment and defence against damage by oxidants, certain heavy metals (Mukhopadhyay et al., 1996) and possibly xenobiotics (for a review, see Fairlamb and Cerami, 1992). The role of TR in L.donovani and T.cruzi has been investigated previously by overexpressing TR using transfections (Kelly et al., 1993). Such transfectants were equally susceptible to inhibition by hydrogen peroxide (H2O2) as controls, and metabolized H2O2 at comparable levels, suggesting that the ability to regenerate trypanothione from trypanothione disulfide is not the rate-limiting step in the metabolism of H2O2 (Kelly et al., 1993). To investigate further the physiological role of TR, we attempted to generate TR-knockout mutants by gene disruption. Recently, the function of other Leishmania genes has been established using gene targeting procedures (Bello et al., 1994; Papadopoulou et al., 1994b, 1996; Mottram et al., 1996). In this work, we have generated double and single TR disruption mutants for L.donovani and L.major, but were unable to obtain TR null mutants. Instead, the TR locus became trisomic (Figure 1). Recombinant parasites possessing only one TR allele produce less mRNA (Figure 3A) which correlates with lower TR activity when compared with wild-type levels (Table I). Despite a marked decrease in TR activity in the mutants, we found no differences in trypanothione levels between wild-type and mutants (Table I), and this is in accordance with previous results reported by Kelly et al. (1993). Reduced TR activity does not affect the growth of Leishmania in culture media, nor does it influence the transformation to amastigotes in vitro (not shown). The loss of one TR allele by gene disruption is not compensated by either an increase in TR copy number or RNA production (Figure 3A). These observations are in agreement with two alternative approaches to modulating TR activity that involve either the extrachromosomal expression of dominant-negative TR mutants of T.cruzi (Borges et al., 1995) in Leishmania cells (J.Tovar, M.L.Cunningham and A.H.Fairlamb, unpublished) or the homologous overexpression of antisense RNA for TR in T.c
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