The reason why Leishmania parasites are susceptible to organic antimonial drugs, the standard chemotherapeutic agents for over 50 years, apparently lies in the fact that the mammalian stage of the parasite reduces the pentavalent form of the administered drug to a trivalent form that causes parasite death. We have identified and characterized a parasite-specific enzyme that can catalyse the reduction of pentavalent antimonials and may therefore be central to the anti-parasite activity of the drug. The unusual protein, a trimer of two-domain monomers in which each domain has some similarity to the Omega class glutathione S-transferases, is a thiol-dependent reductase (designated TDR1) that converts pentavalent antimonials into trivalent antimonials using glutathione as the reductant. The higher abundance of the enzyme in the mammalian stage of the parasite could explain why this parasite form is more susceptible to the drug.
- glutathione S-transferase
- thiol-dependent reductase
Leishmaniases are a group of parasitic diseases that are prevalent in humans and widespread throughout tropical and subtropical regions (http://who.int/tdr/diseases/leish). Standard chemotherapeutic treatment for more than 50 years has been with pentavalent antimonial formulations (Pentostam, based on sodium stibogluconate, and Glucantime, based on N-methylglucamine antimonite). Although the mode of action of the drugs remains uncertain , the belief is that their toxicity is probably dependent on in vivo conversion of the relatively inert pentavalent form to a more toxic trivalent form [2–5]. Whereas trivalent antimonial compounds are toxic to both amastigotes (the mammalian stage) and promastigotes (the stage that occurs in the sandfly vector) of Leishmania, only amastigotes are susceptible to the pentavalent antimonial drugs [2–4,6–8]. This suggests that only the amastigotes encounter the drug in its toxic form.
There is continuing speculation as to whether activation of the antimonial drug occurs spontaneously, perhaps as a result of conditions existing either within the parasitophorous vacuole in which the amastigote form of the parasite resides or within the amastigote itself, or whether it is enzymically mediated by an enzyme produced by the amastigote. In support of the former suggestion, there have been reports that pentavalent antimonials can be spontaneously reduced in vitro to a trivalent form by low-molecular-mass thiols such as GSH and the parasite-specific trypanothione [9–11]. This reduction occurred most rapidly at low pH, similar to that of the parasitophorous vacuole and also the parasite's abundant lysosomes . Zilberstein and co-workers  reported that amastigotes of Leishmania donovani are themselves capable of mediating the reduction of pentavalent antimonials when incubated with the drugs in vitro. This activity was not detected with promastigotes and was also lacking in amastigotes of a line selected in vitro for resistance to sodium stibogluconate. Although these findings do not rule out non-enzymic activation, they are consistent with reduction and thereby with activation of the pentavalent drugs mediated by an amastigote-specific enzyme activity, the loss of which could constitute one mechanism of resistance to antimonial drugs.
Cellular metabolism of the semi-metal arsenic, which is closely related to antimony, has been studied quite extensively [13,14]. In many organisms, inorganic arsenic can be converted into arsenite [As(III)] by a specific arsenate reductase. Commonly, this conversion is a prelude to the excretion of arsenite from the cell, to avoid toxicity, via a transporter protein [15,16]. Such transporters have been identified in Leishmania and implicated in resistance to antimonials . However, in some organisms, arsenic reduction constitutes the first step in an arsenic biomethylation pathway by which inorganic arsenic is converted via sequential reduction and methylation reactions into di- and trimethylated species [13,14]. The function of this pathway remains uncertain; the original concept was that the pathway was a detoxification mechanism, but this idea has been questioned by the finding that many of the products are more toxic than the inorganic arsenic itself [18–20].
The human versions of the enzymes catalysing the arsenic biomethylation pathway have recently been isolated and identified [21–24]. Notably, the enzyme catalysing the rate-limiting step [monomethylarsenate (MMAV) reductase, which converts MMAV into monomethylarsenite (MMAIII) using glutathione as the reductant] has been shown to belong to the Omega class of GSTs (glutathione S-transferases) (GSTO, EC 22.214.171.124) and has been designated as hGSTO1 (human GSTO1) [21,25]. Unlike other classes of GSTs, which catalyse the conjugation of glutathione with electrophilic substrates as part of the phase II detoxification pathway, GSTOs use glutathione as a reducing agent and possess dehydroascorbate reductase and thiol transferase activities [25,26]. These activities are similar to those of glutaredoxins, enzymes that have also been noted to bear a distant relationship to some metal-binding proteins, including the arsenate reductase arsC .
We hypothesized that the activation of pentavalent antimonial drugs by amastigotes of Leishmania may involve an enzyme similar to MMAV reductase. In the present study, we report the identification and characterization of an enzyme (TDR1, thiol-dependent reductase) of L. major. It shows some similarity to hGSTO, but there are significant differences, and it has the ability to catalyse the reduction of anti-leishmanial pentavalent antimonials.
Sodium stibogluconate was obtained from GlaxoSmithKline, Glucantime was a gift from Professor S. L. Croft (London School of Hygiene and Tropical Medicine) and disodium methylarsenate (MMAV) was purchased from Greyhound Chromatography (Birkenhead, U.K.). Unless stated otherwise, all other reagents were obtained from Sigma.
Parasite cultivation and handling
Promastigotes of L. major (MHOM/JL/80/Friedlin) were grown as described previously . Amastigotes of L. major were grown in Balb/c mice and purified by the method described by Hart et al.  and modified by Mottram and Coombs .
Cloning of TDR1 of L. major
A partial sequence having similarity to GSTOs was identified by a BLAST search of the L. major genome database (www.genedb.org) with the sequence of hGSTO1 [EBI (European Bioinformatics Institute) accession no. AF212303]. This partial sequence (the C-terminal 225 amino acids of TDR1; see Figure 1) was cloned and expressed in Escherichia coli BL21 Codon Plus (DE3)RP cells (Stratagene) and the soluble protein purified by Ni+-affinity chromatography was used for antibody production (see below).
The initiation methionine of the full-length protein was determined by using 5′-RACE (rapid amplification of cDNA ends) on mRNA. Total RNA of L. major promastigotes was isolated using TRIzol® (Invitrogen, Paisley, U.K.). Gene-specific primers OGST1 (5′-AACAATCAGCTGCGACTGGTG-3′), OGST2 (5′-TACAGCCTCGCCCCTCG-3′) and OGST3 (5′-AATAGCGCAGGCACCGTATC-3′) were designed based on the gene sequence in the genome database. Using OGST1, gene-specific single-stranded cDNA was synthesized by reverse transcription with Superscript II reverse transcriptase (Invitrogen) according to the manufacturer's instructions. PCR amplification was performed on the cDNA, using the L. major splice leader primer SL1 (5′-TAACGCTATATAAGTATCAGTTTC-3′) and OGST2, using the Expand High Fidelity system (Roche). The product of this reaction was further amplified using OGST3 and the L. major nested splice leader primer SL2 (5′-AGTATCAGTTTCTGTACTTTATTG-3′) with Expand High Fidelity Pwo/Taq polymerase. The PCR-amplified 5′ region of the gene was cloned into the pGEM-T vector (Promega) and sequenced. This allowed elucidation of the complete coding sequence of TDR1 (gene encoding TDR1; EBI accession no. AJ582069).
Genomic DNA of L. major was isolated using TELT buffer [50 mM Tris/HCl, pH 8.0/62.5 mM EDTA/2.5 M LiCl/49% (v/v) Triton X-100] as described in . The open reading frame of L. major TDR1 was amplified from genomic DNA by PCR using the Expand High Fidelity system. For this, the 5′-perfect match primer FLOGST1 (5′-GACATATGGCCGCGCGCGCGCTAAAGCTGTACG-3′), together with the 3′-perfect match primer FLOGST2 (5′-TAGCGGCCGCTTACCCGCCCTGGGCCCTCCGTTG-3′), was used. An NdeI restriction site (underlined) was added to the 5′-end of FLOGST1 and a NotI restriction site (underlined) was added to FLOGST2 to facilitate cloning and purification. The PCR product was cloned into pGEM-T vector and sequenced. The pGEM-T vector containing TDR1 was digested with NdeI and NotI and ligated into the NdeI–NotI-digested pET28a+ vector (Novagen) to produce the pET construct pETLmajTDR1. The plasmid was used to transform E. coli BL21 Codon Plus (DE3)RP cells (Stratagene) for the production of recombinant TDR1.
Production of recombinant TDR1
Optimal expression of the soluble protein was obtained by induction with 2 mM isopropyl β-D-thiogalactoside for 16–18 h at 15 °C. The induced cells were pelleted and lysed by sonication (10×30 s at 22 μm) in 20 ml of buffer A (20 mM Tris/500 mM NaCl, pH 7.9) containing 5 mM imidazole. The lysate was centrifuged at 13000 g for 30 min at 4 °C and the soluble fraction was applied on to a 13 ml Ni2+-nitrilotriacetate column (bioCAD 700E workstation) preequilibrated in buffer A containing 5 mM imidazole. The column was washed first with 100 ml of buffer A containing 5 mM imidazole and then with 20 ml of buffer A containing 50 mM imidazole, and the His-tagged recombinant protein was eluted with 500 mM imidazole in buffer A. The peak binding fractions were pooled and buffer-exchanged into 25 mM Tris, pH 7.9 (buffer B) using an Amersham Biosciences PD10 column, and applied on to a POROS 20 HQ column (4.6 mm×100 mm) preequilibrated in buffer B. The column was washed with 5 column volumes of buffer B and then eluted with a 0–100% gradient of buffer B (10 column volumes) containing 2 M NaCl. TDR1 eluted as a single peak at approx. 0.25 M NaCl and fractions containing the enzyme were aliquoted and stored at −80 °C. Approx. 20 mg of purified recombinant protein was recovered per litre of E. coli culture.
The ability of TDR1 to conjugate GSH with the generic GST substrates CDNB (1-chloro-2,4-dinitrobenzene), ethacrynic acid (EA) and EPNP [1,2-epoxy-3(4-nitrophenoxy)propane] was assayed at 30 °C as described in . Thiol transferase activity was assayed using the synthetic disulphide HEDS (2-hydroxyethyldisulphide). The standard assay mixture contained, in a final volume of 1 ml, 50 mM Tris/HCl (pH 7.0), 5 mM EDTA, 300 μM NADPH, 1 mM GSH, 0.75 mM HEDS and 1 unit/ml GSH reductase. The assay components were preincubated for 10 min at 30 °C before the initiation of reaction by adding TDR1, and the activity was monitored according to the decrease in absorbance at 340 nm. Activities were calculated from the linear rate during the first 1–2 min of the reaction.
DHA (dehydroascorbate) reductase (DHAR) activity was measured according to the increase in absorbance at 265 nm as a result of formation of ascorbic acid. The standard assay mixture contained, in a final volume of 1 ml, 50 mM Tris/HCl (pH 7.0), 2 mM GSH and 1.5 mM DHA. The spontaneous reaction in the presence of DHA and GSH was followed for 1–2 min at 30 °C before the initiation of reaction by adding TDR1, and the activities were calculated from the initial rate during the first 30–60 s after subtraction of the spontaneous rate. Activity towards antimonials and sodium arsenate was measured via monitoring GSH oxidation by reducing the GSSG produced using GSH reductase and monitoring the concomitant NADPH oxidation. The standard assay mixture contained, in a final volume of 1 ml, 0.1 M Bis-Tris (pH 5.5), 5 mM EDTA, 300 μM NADPH, 1 unit/ml GSH reductase, 10 mM GSH and 10 mM antimony or arsenic species. The assay components were preincubated for 10 min at 30 °C before initiation of reaction by adding TDR1, and the activity was monitored according to the decrease in absorbance at 340 nm during the first 30–60 s. Assays were routinely performed at three different enzyme concentrations to ensure a linear relationship between TDR1 concentration and reaction rate.
Protein concentrations were determined using the Bio-Rad protein assay kit using BSA as the standard.
BPR (Bromopyrogallol Red) assay for trivalent antimony
SbIII determination was based on the method described by Ferreira et al. . Briefly, 200 μl of analyte solution containing 20 mM sodium phosphate (pH 6.8), 0.1% (w/v) tartaric acid and 70 μM BPR (Aldrich, Gillingham, Dorset, U.K.) was added to 20 μl of test solution in a microtitre plate. The absorbance was read at 540 nm in a plate reader and subtracted from that of a control sample containing no SbIII. Potassium antimonyl tartrate (0–0.5 mM in 20 μl) was used to construct a calibration curve for each experiment and tests were performed to ensure that GSH, GSSG and SbV did not interfere with the assay.
DTNB [5,5′-dithiobis-(2-nitrobenzoic acid)] assay for GSH
GSH concentrations were determined by reaction with DTNB as follows: 20 μl of sample containing 0–2 mM GSH was mixed with 200 μl of 50 mM Tris/HCl (pH 7.4) containing 5 mM EDTA and 0.6 mM DTNB in a microtitre plate. The absorbance was read at 414 nm and concentrations were estimated from a standard curve of 0–0.5 mM GSH (final concentration).
Isolation of TDR1 from parasite lysates using S-hexyl-GSH–agarose
S-hexyl-GSH-binding proteins were isolated from lysates of Leishmania cells by batchwise adsorption on to S-hexyl-GSH–agarose (Sigma). Amastigotes and promastigotes were lysed (1–5×109/ml) by sonication (4×10 s at 18 μm) in PBS (pH 7.3), containing proteinase inhibitors [10 μM E-64 (trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane), 1 μM pepstatin, 200 μM phenanthroline, 0.5 mM PMSF and 1 mM EDTA] and the lysate was clarified by centrifugation at 14000 g for 20 min at 4 °C. The resultant supernatant was mixed with approx. 100 μl/ml S-hexyl-GSH–agarose (pre-equilibrated in PBS, pH 7.3) and incubated for 2 h at room temperature with gentle mixing. The agarose was sedimented by a brief centrifugation (1 min at 2000 g) and the supernatant was removed. After washing three times with 1 ml of PBS, binding proteins were eluted by vortex-mixing with 50 μl of 10 mM S-hexyl-GSH (Sigma) in 50 mM Tris/HCl (pH 8.0).
Western-blot analysis and SDS/PAGE
Rabbit polyclonal antiserum was raised against the 225-amino-acid C-terminal fragment of TDR1 by the Scottish Antibody Production Unit (Carluke, U.K.) using standard methods. The final bleed antibodies were purified by affinity chromatography against purified recombinant TDR1 using Affigel 15 (Pierce) according to the manufacturer's instructions. Western-blot analysis was performed as described previously , with the purified antibodies diluted in Tris-buffered saline containing 1% (w/v) low-fat dried milk and 0.1% Tween 20. Bound antibody was detected using horseradish peroxidase-coupled secondary anti-rabbit antibodies (Santa Cruz Biotechnology) and ECL® Western blotting detection reagents (Amersham Biosciences).
Determination of native molecular mass by gel-filtration and cross-linkage analyses
Gel-filtration analysis was performed using a high-resolution Sephacryl S-300 column (1.6 cm diameter, 60 cm long, bed volume of 120 ml), which was equilibrated in 20 mM Tris and 100 mM NaCl (pH 7.4) and run at 0.5 ml/min. Cross-linkage analysis was performed as described in .
Identification and cloning of TDR1
A BLAST search of the L. major genome database revealed an apparent open reading frame coding for a 25 kDa polypeptide having 29% identity with hGSTO1. Subsequently, 5′-RACE revealed that the sequence was the C-terminal portion of a protein of approximately twice the size consisting of two similar halves (27.2% identity). TDR1 of L. major (EBI accession no. AJ582069) is predicted to encode a protein (TDR1) of 450 amino acids, having a subunit molecular mass of 49.9 kDa. Whereas both halves of the protein contain the CPF motif characteristic of GSTO  (see Figure 1), the motif in the N-terminal portion is followed by a second cysteine residue. This CPFC motif is characteristic of thioredoxin-like proteins, including glutaredoxin . TDR1 has an overall identity of 45.7% with a Trypanosoma cruzi protein designated Tc52 [36–39]; interestingly, the two putative active sites of the two proteins differ significantly (underlined in Figure 1). Partial sequences having high identity with the L. major TDR1 protein sequence were also identified in the L. infantum and L. brasiliensis databases (90 and 75% identity respectively; www.genedb.org). The L. infantum sequence has active-site sequences identical with that of TDR1; however, the partial L. brasiliensis sequence does not currently extend as far as the active-site motifs.
Production of recombinant TDR1
The full-length L. major TDR1 was cloned into pET28a+ and expressed in E. coli with an N-terminal His tag. Approx. 20 mg of soluble protein was expressed per litre of culture when induced overnight at 15 °C. To exclude the possibility of contamination with E. coli glutaredoxin 2 (which is known to have similar activities as GSTO ), the recombinant enzyme was purified first by passage through a nickel agarose column and then by binding to a HQ anion-exchange column at pH 7.9. It was found that E. coli glutaredoxin 2 (pI 9.17) does not bind to the HQ column at pH 7.9, whereas TDR1 (pI 6.3) bound and was subsequently eluted with NaCl (approx. 0.25 M). The resultant protein was >95% pure as determined by SDS/PAGE and could be stored in elution buffer for at least 6 months at −80 °C without loss of activity.
DHA reductase, thiol transferase and GSH-conjugating activities of TDR1
TDR1 was capable of using GSH as an electron donor to reduce DHA and HEDS. The specific activities for the two reactions were similar to each other and were significantly higher than those reported for the hGSTO assayed under similar conditions (Table 1). The enzyme catalysed the conjugation of GSH with the generic GST substrate CDNB at a low rate, but no activities were detected with EA or EPNP (Table 1).
The DHAR activity of TDR1 could be abolished effectively by preincubating the enzyme with iodoacetic acid for 30 min before the assay (IC50, 40 μM). This is consistent with there being one or more cysteine residue involved in the catalytic mechanism. In contrast, the activity was relatively insensitive to treatment with sodium arsenite and potassium antimonyl tartrate (IC50 value for each was greater than 10 mM). These species have an affinity for vicinal thiols and, therefore, may be expected to interact with the CPFC motif present in the N-terminal half of the enzyme.
Activity of TDR1 towards pentavalent antimony and arsenic species
The ability of TDR1 to use antimony and arsenic species as substrates was investigated by monitoring GSH oxidation in the presence of the species by linking the reaction to NADPH oxidation through the enzyme GSH reductase. Using this assay system, GSH oxidation was detected in the presence of the antimonial drugs sodium stibogluconate and glucantime and also sodium arsenate and MMAV (Table 2). The highest activities were with the arsenic species and, in all cases, the highest initial activities were obtained when the substrates were preincubated together for several minutes before the reaction was initiated by adding TDR1. This suggests that the enzyme may be working on a spontaneously formed intermediate between metal compounds and GSH rather than on the two compounds individually. A similar situation was seen with the HEDS-based thiol transferase assay and this result is consistent with previous reports  that the spontaneously formed 2-mercaptoethanol–glutathione disulphide, rather than HEDS itself, is the substrate for GSTs. In both reactions of TDR1, a good relationship between TDR1 concentration and reaction rate was obtained only when the substrates were preincubated before the reaction was initiated, implying that formation of the intermediate was otherwise limiting the reaction rate when TDR1 concentrations were high.
Maximal activity towards sodium stibogluconate was obtained at pH 5.5 (the activity at this pH being four times that at pH 8.0, which is the optimum pH reported for hGSTO1-1 ) in the presence of 5 mM EDTA. When different concentrations of sodium stibogluconate were preincubated with 10 mM GSH, the resultant activity curve was in accordance with Michalis–Menton kinetics and gave a Km (app) of 5.7 mM (n=2). This value is lower than that reported (23.5 mM) for hGSTO1-1 towards its substrate MMAV  and is similar to those reported for arsenate reductases towards sodium arsenate . However, this result should be treated with caution given the complexity of the reaction kinetics.
TDR1 activity towards stibogluconate yields a trivalent antimonial
The BPR assay  was used to show that trivalent antimony was a product of the reaction of TDR1 with the pentavalent antimonial sodium stibogluconate. Under the conditions used, this assay was shown to be specific for trivalent antimonial compounds and gave no reaction with sodium stibogluconate, GSH or GSSG. When used to quantify the amount of trivalent antimonial formed as a function of the GSH used, a stoichiometry of 2.15±0.39 (n=8) (GSH: SbIII) was obtained (Figure 2). Under the assay conditions used, a low level of spontaneous reduction of SbV to SbIII (approx. 2 μM/min) was routinely observed and subtracted from the rate measured in the presence of enzyme. The spontaneous reaction had the same stoichiometry as the reaction catalysed by TDR1.
Quantification of TDR1 in promastigotes and amastigotes of L. major
As has been reported for Tc52 and GSTOs [26,37,43], recombinant TDR1 did not interact with GSH–agarose but bound to S-hexyl-GSH–agarose and could be eluted with 10 mM S-hexyl-GSH (results not shown). This property was used to isolate TDR1 from lysates of L. major promastigotes and amastigotes using a batch-binding approach. A band corresponding to the molecular mass of TDR1 was the major binding protein in extracts of both the life-cycle stages and its identity was confirmed by Western-blot analysis using an antibody raised against the C-terminal half of the enzyme expressed in E. coli. Approx. ten times more TDR1 was recovered from an amastigote lysate than from an equivalent promastigote lysate (Figure 3). Attempts to isolate TDR1 from spent L. major promastigote culture medium using S-hexyl-GSH–agarose were unsuccessful, implying that TDR1 is probably not secreted by this stage of the parasite.
TDR1 exists as a trimer
The hGSTO is reported to form a homodimer in solution . Gel-filtration analysis indicated that TDR1 has a native molecular mass of approx. 155 kDa (Figure 4A), which is most consistent with the native protein being a trimer. Furthermore, when the protein was treated with glutaraldehyde for a limited time period to cross-link the associated subunits , the predominant species observed on SDS/PAGE corresponded to the molecular mass of a trimer (Figure 4B).
We have identified an enzyme of L. major that has the ability to reduce pentavalent antimonials to trivalent forms and is expressed in amastigotes at approx. 10-fold the level of expression in promastigotes. We suggest that this enzyme could be responsible for the activation of antimonials by Leishmania parasites and accounts, in part, for the specificity of the drug to the amastigote stage of the parasite.
It is generally accepted that anti-leishmanial antimonials act only against amastigotes, but it is less clear whether it is the amastigote itself that is susceptible or the existence within the acidic parasitophorous vacuole that is crucial. There are conflicting reports as to whether or not axenic amastigotes are susceptible to pentavalent antimonials [3,7,8,44]; hence, it remains uncertain whether activation occurs within the parasite or outside it. Independent of this, activation chemically involving thiols has been postulated and reported to occur in vitro. However, the reported rates of spontaneous conversion in vitro are relatively low and slow when compared with those obtained using TDR1. For example, Frezard et al.  reported that only approx. 30% of 2 mM SbV was converted into the trivalent form when incubated with 10 mM GSH at 37 °C for 3 days at pH 5.0, whereas in the presence of 1 μg/ml TDR1, the same reaction would occur within approx. 1 h under optimal assay conditions. Thus the potential role of TDR1 in drug reduction is undoubted.
It is possible that the higher expression of TDR1 in amastigotes does not by itself account for the increased susceptibility of this developmental stage of the parasite. Amastigotes of Leishmania species have been reported to be 50–600 times more sensitive to SbV-based antimonials when compared with promastigotes [2,4,7,8], whereas TDR1 levels are only approx. 10-fold higher in the latter. However, amastigotes have also been reported to possess a greater capacity than promastigotes for accumulating pentavalent antimonials  and also appear to be more sensitive than promastigotes to SbIII . Thus it is probable that a combination of factors contributes to the difference in sensitivity of the two parasite stages of antimonials, with the ability of TDR1 to reduce and therefore activate the pentavalent drugs being a key event.
Three classes of arsenate reductases have been reported previously  and although TDR1 has some similarities to these, such as a conserved active-site cysteine residue, it is the first arsenate reductase reported to use GSH directly as the reducing agent. Other classes use glutaredoxin and thioredoxin . In this context, the two-domain nature of TDR1 is intriguing, especially since the putative active-site residues differ between the two domains. It is notable that the N-terminal domain contains the CXXC motif characteristic of glutaredoxin and thioredoxin, whereas the C-terminal domain active site is more similar to that of GSTO. The possibility that the two domains function together as electron donor and reductase respectively deserves consideration.
The similarity between TDR1 and Tc52 of the related parasite T. cruzi is interesting, especially since the putative active-site residues of the two proteins are identical in the N-terminal domains (CPFC), but differ in the C-terminal domains (CPFV and SPFS). Both possess thiol transferase and DHA reductase enzymic activities (the present study and ), but Tc52 has not been assessed for metal reductase activity. The parasite itself, however, is not susceptible to pentavalent antimonials or arsenicals and it is tempting to speculate that this difference between the drug sensitivities of the two parasites reflects the difference between the active sites of TDR1 and Tc52.
Tc52 has been reported to be an excretory–secretory product having an important role in aiding the parasite to modulate the mammalian host's immune response to infection . Although we could not detect the release of TDR1 by promastigotes of L. major, the possibility remains that it is secreted by amastigotes of the parasite and, thus, contributes to activation of antimonials within the parasitophorous vacuole in which the amastigote form of the parasite resides. This localization is in agreement with the low optimum pH (pH 5.5) obtained for the reduction of sodium stibogluconate by the enzyme.
The physiological role of TDR1 remains unclear. Arsenate reductases have generally been suggested to play a role in the detoxification of arsenic , with the reduction being followed by excretion of the reduced species from the cell. Such a role for TDR1 cannot be excluded. Similarly, TDR1 could be part of a biomethylation pathway that has been reported to occur in mammalian cells [13,14]. The finding that there were several products of stibogluconate in amastigotes of L. donovani  is consistent with the presence of such a pathway. The role of the biomethylation pathway remains unclear. If it does play a part in detoxification, then up-regulation in Leishmania could also be a mechanism whereby the parasite could become resistant to the antimonial drugs used against it. Leishmania may contain other arsenate reductases in addition to TDR1; certainly, the genome database (www.genedb.org) contains sequences having apparent similarity to known arsenate reductases of other organisms.
The high DHAR activities of TDR1 and Tc52 may reflect the physiological roles of the proteins. It was recently reported that T. cruzi contains an ascorbate-dependent haemoperoxidase  and a sequence having similarity to this enzyme is also present in the L. major genome database. This enzyme requires ascorbate, which has been proposed to be produced from DHA non-enzymically . Tc52 and TDR1 could be involved in generating the ascorbate required by this enzyme. It is interesting to note that the DHAR activity of TDR1 is not sensitive to sodium arsenite or potassium antimonyl tartrate, which suggests that the N-terminal domain of TDR1 (which contains the CXXC motif generally bound by these compounds) may be unimportant for the DHAR activity of the enzyme. A pilot study of a recombinant form of the C-terminal domain alone has confirmed that it has DHAR enzymic activity (H. Denton and G. H. Coombs, unpublished work); unfortunately, the protein was too unstable for a fuller analysis. Many of these outstanding issues on the activities and role of TDR1 can be addressed by gene overexpression and deletion experiments and by site-directed mutagenesis studies, and these are in progress.
The very high identity between TDR1 of L. major and the homologues in L. infantum and L. brasiliensis suggests an important and conserved function. Notably, Tc52 has been reported to be essential . Similar two-domain molecules are not present in human genome or other mammalian genome databases. Interestingly, a second hGSTO (hGSTO2) has been found to be located 7.5 kb downstream of hGSTO1 , but sequence analyses do not particularly support the concept that gene fusion has occurred in Leishmania. Thus, irrespective of the potential importance of TDR1 in the anti-leishmanial activity of organic pentavalent antimonials, the parasite enzyme could itself represent a viable drug target for specific inhibitors.
This work was supported by the Wellcome Trust. We thank J.G. Lindsay for facilitating the biophysical studies and G.D. Westrop for expert technical advice.
Abbreviations: BPR, Bromopyrogallol Red; DHA, dehydroascorbate; DHAR, DHA reductase; DTNB, 5,5′-dithiobis-(2-nitrobenzoic acid); EA, ethacrynic acid; EPNP, 1,2-epoxy-3(4-nitrophenoxy)propane; GST, glutathione S-transferases; GSTO, Omega class GST; hGSTO, human GSTO; HEDS, 2-hydroxyethyldisulphide; MMAV, monomethylarsenate; RACE, rapid amplification of cDNA ends; TDR1, thiol-dependent reductase
- The Biochemical Society, London