Melaminophenyl arsenical drugs are a mainstay of chemotherapy against late-stage African sleeping sickness, but drug resistance is increasingly prevalent. We describe here the characterization of two genes encoding putative metal–thiol conjugate transporters from Trypanosoma brucei. The two proteins, TbMRPA and TbMRPE, were each overexpressed in trypanosomes, with or without co-expression of two key enzymes in trypanothione biosynthesis, ornithine decarboxylase and gamma-glutamyl-cysteine synthetase. Overexpression of gamma-glutamyl-cysteine synthetase resulted in a twofold increase in cellular trypanothione, whereas overexpression of ornithine decarboxylase had no effect on the trypanothione level. The overexpression of TbMRPA resulted in a 10-fold increase in the IC50 of melarsoprol. The overexpression of the trypanothione biosynthetic enzymes alone gave two- to fourfold melarsoprol resistance, but did not enhance resistance caused by MRPA. Overexpression of TbMRPE had little effect on susceptibility to melarsoprol but did give two- to threefold resistance to suramin.
The kinetoplastid protists, Trypanosoma brucei, Trypanosoma cruzi and Leishmania sp., cause severe diseases in Africa, South and Middle America and Asia, with some species extending up into the Mediterranean basin. Vaccines are not available, and the few existing chemotherapies have several disadvantages: unacceptable toxicity, mounting resistance incidence and poor delivery. For the late stages of T. brucei gambiense infection, two treatments are available. The arsenical drug melarsoprol causes reactive encephalitis in 5–10% of patients (Blum et al., 2001); about half these die. The alternative, difluoromethylornithine, suffers from limited availability, as the high manufacturing cost cannot be reflected in the purchase price in poor countries. For late-stage East African sleeping sickness (T. brucei rhodesiense), only arsenicals are effective. Unfortunately, melarsoprol is often ineffective against sleeping sickness in epidemic areas. In a recent study, eight out of 22 patients failed to respond to treatment, but the pharmacokinetics of melarsoprol did not differ significantly between these patients and those who were cured (Burri and Keiser, 2001). It is therefore suspected that the trypanosomes have become drug resistant.
Three possible mechanisms of resistance to arsenical and antimonial drugs have been found in laboratory studies of Trypanosoma brucei and Leishmania: loss of drug uptake, failure to activate the drug and active extrusion or sequestration of drug–thiol conjugates. The only mechanism pertaining specifically to anti-trypanosomal drugs concerns loss of uptake. A T. brucei line that was selected for arsenical resistance in the la-boratory was found to lack adenosine transporter activity and was also resistant to the diamidine drugs used to treat early-stage sleeping sickness. The transporter concerned, TbAT1, was required for uptake of arsenical drugs and diamidines into trypanosomes (reviewed by Barrett and Fairlamb, 1999). One drug-resistant field isolate from Sudan had an inactivating mutation in the TbAT1 gene (Mäser et al., 1999), but an alteration in TbAT1 was detected in only half of a panel of resistant Ugandan T. brucei gambiense isolates (Matovu et al., 2001a). Similar results were obtained using isolates from treated but relapsed patients from Uganda and Angola (Matovu et al., 2001b).
The two other mechanisms of resistance have been studied in Leishmania. One class of anti-Leishmanial drugs contains pentavalent antimony, but the effectiveness of these drugs against mammalian Leishmanias depends on their reduction to the trivalent form (Ephros et al., 1999). A Leishmania donovani line selected for resistance to the drug stibgluconate had lost this reducing activity (Shaked-Mishan et al., 2001).
The third mechanism, which is the one considered in this paper, has been studied mainly in the lizard parasite Leishmania tarentolae. L. tarentolae, Leishmania mexicana and Leishmania tropica that had been selected for resistance to trivalent antimony and/or arsenite had amplified a gene encoding an ABC transporter, PGPA (Detke et al., 1989; Légaréet al., 1997). In sequence compari-sons, LtPGPA groups with mammalian transporters of the multidrug resistance protein (MRP) family. These are ABC transporters that transport a variety of organic anions (Borst et al., 2000). Two of the human MRPs, MRPA and MRPE, can transport drugs conjugated with glutathione (Borst et al., 2000). Trypanothione, a conjugate of glutathione with spermidine, functionally replaces glutathione in kinetoplastids. In Leishmania, the degree of resistance that can be achieved by PGPA overexpression is limited by the intracellular trypanothione concentration (Mukhopadhyay et al., 1996; Legare et al., 1997). Gamma-glutamyl cysteine synthetase (GCS) and orni-thine decarboxylase (ODC) are the rate-limiting steps in the synthesis of glutathione and spermidine respectively. In L. tarentolae, maximal resistance to arsenite and antimony (and maximal trypanothione levels) were seen if GCS and ODC genes were co-amplified with LtPGPA (Grondin et al., 1997; Haimeur et al., 1999), but increased trypanothione alone could not mediate resistance (Grondin et al., 1997). The most recent results have demonstrated that LtPGPA indeed transports metal–trypanothione conjugates (Légaréet al., 2001).
The experiments in Leishmania involved metal salts rather than true chemotherapeutic agents, and mechanisms that may cause drug resistance in human African trypanosomiasis, apart from TbAT1 alteration, are not known. To find out whether MRP-like proteins are capable of mediating resistance to arsenical drugs in T. brucei, we have overexpressed two different MRPs, together with the cognate ODC and GCS genes, in trypanosomes.
Cloning and sequencing of TbMRPA and TbMRPE
The TbMRPA gene (accession number AJ318885) was obtained by screening a bacteriophage P1 genomic library (supplied by Dr S. Melville, Cambridge, UK) with a probe corresponding to the TbABC1 transporter gene identified by Mäser and Kaminsky (1998). The TbABC1 sequence is contained within TbMRPA. The TbMRPE gene (accession number AJ318886) was identified by a BLAST search of the unfinished genome sequence (TIGR, Rockville, MD, USA) and retrieved from the P1 library. The sequences were completed by a combination of database searches (TIGR and Sanger Centre, Hinxton, UK) and new sequencing. The 5′ ends were determined by reverse transcriptase–polymerase chain reaction (RT–PCR). Each gene was found to be present as a single copy by Southern blot analysis (not shown). Although the mRNAs from bloodstream trypanosomes were detected by RT–PCR, we were unable to visualize them on Northern blots.
The open reading frames (ORFs) of TbMRPA and TbMRPE predict proteins of 1581 amino acids (175 kDa) and 1759 amino acids (194 kDa) respectively. EMBL database searches, using the default parameters on TBLASTP, reveal that these proteins are members of the multidrug resistance protein (MRP) family of transporters (Fig. 1), so we have named them correspondingly. When compared with the L. tarentolae proteins, TbMRPA was most closely related to LtPGPA and TbMRPE to LtPGPE. Both TbMRPs have two nucleotide-binding domains like those of ABC transporters (Fig. 2), with the 13-amino-acid deletion in the first domain that is typical of MRP family proteins (Hipfner et al., 1999). In addition, each has an ≈ 200-amino-acid N-terminal extension relative to P-glycoproteins. Such extensions are characteristic of several mammalian MRP proteins. The extensions in some MRP family members are hydrophobic and include membrane-spanning domains (Borst et al., 2000), whereas the extensions in the trypanosomatid proteins are not hydrophobic (Fig. 2). The number and orientation of predicted membrane-spanning domains in the trypanosomatid proteins differs depending on the program used, but there are probably at least 10.
Overexpression of TbMRPs
To overexpress the MRPs in bloodstream T. brucei, we first cloned the complete ORFs into a vector designed for high expression of proteins in trypanosomes. This vector (pHD789) (Irmer and Clayton, 2001) contains a bacteriophage T7 promoter and appropriate trypanosome RNA processing signals and is designed to integrate into the silent RRNA spacer region. When this vector is transfected into trypanosomes expressing T7 polymerase, and integrated into the RRNA spacer, it yields 100 times more mRNA than a similar construct integrated into the tubulin locus and transcribed by RNA polymerase II (Irmer and Clayton, 2001). Plasmids containing either TbMRPA or versions bearing C-terminal epitope tags (TbMRPA-myc or TbMRPE-6His) were transfected into bloodstream-form T. brucei expressing bacteriophage T7 polymerase. Overexpression of the untagged gene was confirmed at the RNA level by Northern blotting (not shown), as we have so far been unable to generate specific antisera (see Experimental procedures). To confirm protein production, we examined cells expressing the tagged proteins (for examples, see Fig. 4). Expression of the MRPs had no effects on cell growth. The locations of the overexpressed tagged TbMRPA and TbMRPE were analysed by immunofluorescence (Fig. 3). TbMRPA-myc appeared to be associated with the plasma membrane, whereas TbMRPE-6His was in intracellular vesicles between the nucleus and the flagellar pocket. The latter is a region of the plasma membrane specialized for protein secretion and endocytosis (Overath et al., 1997). We have not examined this aspect in more detail, as the overexpression may be leading to mislocalization.
Overexpression of GCS and ODC
In L. tarentolae, the highest resistance to arsenite and antimony is seen when the MRP LtPGPA is co-expressed with GCS and ODC (see Introduction). We therefore over-expressed both enzymes, independently and together, in normal trypanosomes. We also transfected the expression plasmids into trypanosomes overexpressing MRPA-myc or MRPE-His. Overexpression of both enzymes was quali-tatively confirmed by Western blotting using antibodies to ODC and to the His tag on the cloned GCS (Fig. 4). In addition, the level of ODC was measured. The specific ODC activity of wild-type cells is ≈ 3–15 nmol of CO2 mg–1 protein h–1 (M. Phillips, personal communication), which is not detectable under our assay conditions. (The lower limit of detection was about 500 nmol of CO2 mg–1 protein h–1.) In our overproducing cells, activities ranged from 1.8 to 3.6 μmol of CO2 mg–1 protein h–1, indicating that the ODC activity was at least 100-fold the normal level. Correspondingly, the ODC in the wild-type cells was visible as only a very faint band in the Western blot, even on prolonged exposure, whereas the ODC in the overexpressing cells was easily detected (Fig. 4).
To determine the effects of ODC and GCS overproduction, we measured the levels of intracellular thiols. We observed very marked effects of cell density. When the parasites were in log phase (density about 5 × 105 cells ml–1), the concentration of glutathione was 700 μM (7 nmol per 108 cells) in wild-type cells and three times higher – 2.1 mM (21 nmol per 108 cells) – in cells transfected with GCS. Growth of the cells to near saturation (density about 3 × 106 cells ml–1) resulted in a seven- to eightfold reduction in glutathione, to 1 nmol per 108 cells in the wild-type and 2.7 nmol per 108 cells in cells overexpressing GCS. The levels of trypanothione in log-phase cells are shown in Fig. 5. Overexpression of GCS caused a doubling of steady-state trypanothione levels from 2 nmol per 108 cells to 4 nmol per 108 cells; additional overexpression of ODC gave only a marginal increase. Here, the density had less effect (two- to fourfold): at high density, trypanothione was about 0.6 nmol per 108 cells in wild-type and 2.6 nmol per 108 cells in cells overexpressing GCS. ODC overexpression had no effect on glutathione levels. These results indicate that, whereas in normal cells, GCS is limiting for trypanothione production, ODC is not really limiting even when GCS is overproduced. It is notable that the published levels of glutathione, which are for the 427 strain of T. brucei grown in rodents, are similar to those we measured in the near-saturated culture of normal 927 cells (Fairlamb and Cerami, 1992), whereas the published trypanothione levels are more similar to the levels we measured in log-phase cells. This discrepancy may reflect the tendency to grow parasites to very high densities in rodents in order to maximize the yield, or it may be a strain difference.
Interestingly, the cells overproducing ODC alone or in combination with MRPA grew slightly faster than the normal cells (not shown). Expression of GCS in addition to the other genes restored wild-type growth. These effects might be related to the requirement for polyamines in processes other than trypanothione synthesis.
Effect of MRP, GCS and ODC expression on sensitivity to anti-trypanosomal drugs
To find out whether overexpression of either of the MRP genes can cause drug resistance, we first tested the drug sensitivities of all cell lines in a standard 3 day microtitre plate assay. The results, as measured by the IC50, are shown in Table 1. There were no significant alterations in sensitivity to berenil. All the cloned transfected cell lines were two- to threefold more sensitive to pentamidine than the parent cell line, but as this was true irrespective of the inserted plasmid, we suspect it may be an artifact of prolonged culture or cloning. The most dramatic results were obtained in the cells treated with melarsoprol. Increased trypanothione, from either GCS or GCS+ODC, gave fourfold resistance. In the MRPA-myc transfectants, the IC50 was more than 10-fold the normal level. Overexpression of ODC and GCS in the MRPA-myc expressors did not significantly enhance resistance. To verify the results, we tested the cells expressing MRPA without the tag; the outcome was similar.
Table 1. Drug resistance profiles of overexpressing cell lines.
Melarsoprol IC50 (ng ml–1)
Berenil IC50 (ng ml–1)
Pentamidine IC50 (ng ml–1)
Suramin (IC50 ng ml–1)
IC50 values are shown as the average of two independent assays, measured in duplicate using a 590 nm filter and a twofold dilution series, except for the MRPA and MRPA+ODC+GCS lines, which were tested once under these conditions. The difference between the two values is shown in parentheses. Initial experiments were performed using a 570 nm filter (because the 590 nm filter was not available) using threefold dilution series. The absolute IC50 values using the 570 nm filter were about half those seen with the 590 nm filter, but the relative resistance factors (RRFs) were similar for both filters. The results shown for melarsoprol RRF are the mean and standard deviation for the two 590 nm assays and one 570 nm assay.
None (wild type)
ODC + GCS
4.06 ± 0.05
10.7 ± 1.5
MRPA + ODC + GCS
MRPA-myc + ODC + GCS
12.6 ± 4.2
MRPE-6His + ODC + GCS
6.8 ± 1.9
The most relevant measure of drug susceptibility from the point of view of treatment is the minimal inhibitory concentration (MIC). This is the dose at which no more normal cells are visible in the microscope, and provides some indication of the drug concentration required to kill all parasites in a patient. Again, the only major differences found were for melarsoprol. The MIC of melarsoprol was 2–4 ng ml–1 for the wild-type cells and about 60 ng ml–1 for the cells overproducing MRPA-myc (see Fig. 6). Overproduction of trypanothione alone caused a two- to threefold increase in the melarsoprol MIC, but did not enhance the resistance caused by MRPA overexpression. Notably, cells overproducing MRPA could survive the average level of drug normally attained in the cerebrospinal fluid (about 50 ng ml–1).
Overproduction of MRPE alone had no effect on sensitivity to any melarsoprol, berenil or pentamidine but did result in a threefold increase in the IC50 of suramin. There was also a two- to threefold increase in the suramin MIC (data not shown). We are not sure how significant this result is, but it may warrant more detailed investigation in future.
The results presented here show that overexpression of TbMRPA in bloodstream-form T. brucei results in 10-fold lowered sensitivity to melarsoprol. The MIC of melarsoprol determined for the wild-type trypanosomes was the same as that recorded for five Ugandan T. b. rhodesiense isolates (Matovu et al., 2001a). The MRPA overexpressors had MICs that were comparable with those of the most resistant Ugandan T. gambiense isolates, which had MICs in the range 36–72 ng ml–1 (Matovu et al., 2001a). The normal peak levels of melarsoprol in the cerebrospinal fluid of patients range between 40 and 60 ng ml–1 (Burri and Keiser, 2001). Even at this dose, side-effects are severe, so that a further dose increase cannot be contemplated. Thus, MRPA overexpression could clearly give clinical melarsoprol resistance.
The overexpression of GCS and ODC resulted in a doubling of trypanothione levels. In cells overexpressing MRPA, this caused only a marginal further increase in melarsoprol resistance. Nevertheless, trypanothione overproduction by itself or with MRPE did lower melarsoprol sensitivity slightly. There are two possible explanations. First, arsenical drugs are known to interact with thiols, both on proteins and on trypanothione and glutathione. An increase in trypanothione and glutathione might protect thiol proteins by competing for interaction with the drug (see Cunningham et al., 1994). Secondly, if melarsoprol or its active metabolites are exported by MRPA as thiol conjugates, overproduction of glutathione and trypanothione might enhance transport. Our results in MRPA-overproducing cells, however, show that GCS and ODC overexpression did not significantly increase resistance. Thus, in contrast to Leishmania, in which the rate-limiting step has been attributed to the formation of the metalloid–thiol pump substrates (Mukhopadhyay et al., 1996), in T. brucei, the synthesis of trypanothione seems not to be limiting. The reason for this difference may lie in the physiological cellular thiol concentrations. An arsenite-resistant clone of L. tarentolae had a 37-fold increase in the trypanothione level, which is much more dramatic than the twofold increase seen in trypanosomes. The normal trypanothione levels in the two parasite species are, however, very different. The basal trypanothione level in wild-type L. tarentolae is 0.1 nmol per 108 cells (Grondin et al., 1997), which is 20 times lower than the level in wild-type T. brucei. Thus, wild-type T. brucei already have almost as much trypanothione as over-producing L. tarentolae. It is therefore not surprising that trypanothione levels are not limiting for MRP function in trypanosomes.
Six different MRP-like genes have so far been found in Leishmania spp. (Légaréet al., 1994), and two from T. cruzi are also in the database. As the genome sequencing projects are incomplete, we do not know how many MRPs and other ABC transporters are present in the different trypanosomatids. Seven different MRPs have been characterised from human cells (Hopper et al., 2001). Some (though not all) of them can be distinguished from other P-glycoprotein-like ABC transporters by an N-terminal extension of 200 amino acid residues containing either three or five predicted transmembrane helices (for example, see Hipfer et al., 1997; Kast and Gros, 1997). The two trypanosome proteins described here (like the Leishmania homologues) have extensions, but these are hydrophilic. Mammalian MRPs are located on the plasma membrane and can be restricted to either the apical or the basolateral membrane of polarized cells (Keppler et al., 2000). Human MRPA and MRPE transport glutathione conjugates and other MRPs have a variety of other specificities (Borst et al., 2000; Keppler et al., 2000). Our results suggest that TbMRPA probably also transports arsenical drugs. Because little or no effect was seen on resistance when glutathione and trypanothione were overproduced in addition to TbMRPA, we cannot prove that the drug is transported as a conjugate with thiols. However, this is very likely to be the case, because the Leishmania homologue of TbMRPA, LtPGPA, has been convincingly demonstrated to transport thiol–metal conjugates (Légaréet al., 2001). TbMRPE overexpression was not able to confer resistance to arsenical drugs, although some resistance to suramin was seen. Amplification of the related LtPGPE gene in Leishmania did not confer resistance to hydrophobic drugs, methotrexate or arsenite (Légaréet al., 1994).
The physiological functions of TbMRPA and TbMRPE are not yet known. We do not even know at what stage of the life cycle they are normally expressed. It is most unlikely that these proteins arose in order to secrete heavy metals, which are presumably not normally encountered at toxic levels in either the mammalian or the tsetse fly host. Attempts to create homozygous deletion mutants have so far failed, but we suspect that this could be because of the very low level of gene expression, which could make it difficult to express selectable markers from the MRP loci. A homozygous deletion of LtPGPA rendered the parasites more sensitive to antimony and arsenite and less able to survive within mouse macrophages (Papadopoulou et al., 1996).
Epitope-tagged TbMRPA gave an immunofluorescence pattern indicating distribution all over the plasma membrane, which would be consistent with an ability to excrete trypanothione–drug conjugates. Tagged TbMRPE was in vesicles between the nucleus and flagellar pocket – most probably components of the secretory pathway. These results should, however, be viewed with caution, as it is not know whether the localization was influenced by overexpression or the presence of the tag. The localization of TbMRPE partially resembles that of tagged LtPGPA in Leishmania, in vesicles close to the flagellar pocket (Légaréet al., 2001). Sequestration of drug could occur into the vesicles, perhaps with occasional release into the flagellar pocket.
The precise nature and extent of melarsoprol resistance in the parasites causing the current trypanosomiasis epidemics in the Congo, Sudan and Angola remain unknown because the infectious parasites, of the T. brucei gambiense subspecies, are very difficult to isolate and transfer to the laboratory (Burri and Keiser, 2001), where it is again problematic to grow sufficient quantities for RNA and protein analysis (Matovu et al., 2001a,b). Our next efforts will have to concentrate on developing appropriate reagents to measure the level of expression of MRPs in very small samples of T. b. gambiense in order to see whether these proteins play any role in drug resistance in Africa.
Trypanosoma brucei strain 927 (van Duersen et al., 2001) bloodstream forms were grown in HMI9 containing 10% fetal calf serum (FCS) and with maximal cell densities of 3–5 × 106 cells ml–1. All cloning and transfection methods were as described previously (Biebinger et al., 1997). To express T7 polymerase in the trypanosomes, cells were transfected with pLEW13 (a kind gift from G. Cross and L. Wirtz, Rockefeller University). Drug selections were made with 0.5 μg ml–1 phleomycin (Cayla), 15 μg ml–1 hygromycin (Sigma), 1 μg ml–1 G418 (Gibco BRL) and 5 μg ml–1 blasticidin (Invitrogen).
Sequencing of the MRP genes was completed by TopLab or the ZMBH Sequencing Facility after subcloning using the genome priming system kit (New England Biolabs). The 5′-trans splicing sites were determined by reverse transcription and PCR using a 5′ spliced leader primer and bloodstream mRNA as template. The plasmids used in the experiments described here are listed in Table 2. To clone the complete MRP ORFs, two or three sections that contained convenient bordering restriction sites were amplified by PCR; then the complete ORFs were reconstructed by digestion and ligation. For tag addition, primers including tag sequences were used. The ORFs were verified by complete sequencing before subcloning into trypanosome expression vectors conferring hygromycin resistance. Plasmids containing the ORFs of TbODC and TbGCS (the latter with a His tag) were kindly provided by Dr M. Philips (University of Texas Southwestern Medical Center, TX, USA). TbGCS was cloned into pLEW81 (phleomycin resistance), and TbODC was inserted into a new T7 expression vector with blasticidin resistance (pHD 1177). More details, including reconstructed sequences, are available from the authors.
Table 2. Constructs used or made during the work described in this paper.
T7 RNA polymerase gene and neomycin resistance targeted to tubulin locus (Wirtz et al., 1998)
To measure ornithine decarboxylase (ODC) activity, 50 ml of parasite culture (106 cells ml–1) was harvested, washed and lysed by three freeze–thaw cycles in 100 μl of 50 mM Na2HPO4–NaH2PO4, pH 7.5, 2 mM dithiothreitol (DTT) containing 1 tablet/10 ml complete miniprotease inhibitor cocktail (Roche Molecular Biochemicals). Extracts were centrifuged (13 000 r.p.m., 30 min, 4°C), and the supernatants were stored at –80°C. The enzyme activity was followed at 35°C for 15 min using a CO2 detection kit (Sigma) in a total volume of 1 ml, in the presence of saturating (9–10 mM) ornithine. Recombinant T. brucei ODC (up to 1 μg; a gift from Dr M. Phillips) served as a positive control.
Concentrations of intracellular trypanothione and glutathione were measured in deproteinized cell extracts, using methods based on those of Fahey and Newton (1987), Fairlamb et al. (1987) and Newton and Fahey (1987). Cells were grown in HMI9 medium supplemented with 10% FCS to mid-exponential phase (≈ 5 × 105 cells ml–1). Trypanosomes (2–3 × 107 cells) were harvested by centrifugation (2000 r.p.m., 10 min, room temperature), washed twice with HMI9 medium (lacking FCS, 2-mercaptoethanol and L-cysteine) and finally once with PBS at 4°C. The pelleted cells were suspended in 100 μl of ice-cold 5% (w/v) trichloroacetic acid in 10 mM HCl. After 5 min on ice, denatured proteins and cell debris were removed by centrifugation at 4°C for 5 min at maximum speed in a bench-top centrifuge. The pellet was re-extracted with 50 μl of trichloroacetic acid, and the two supernatants were combined. Excess trichloroacetic acid was removed by four extractions with three volumes of ice-cold diethyl ether. Finally, 250 μl of 40 mM HEPES–KOH (pH 8.0) containing 4 mM diethylene triaminopentaacetic acid (DPTA) was added to the extracted supernatant.
Thiols in the extract were derivatized by adding 100 μl of 10 mM monobromobimane (Calbiochem; stock solution 200 mM in acetonitrile, prediluted to 10 mM in HEPES–KOH–DTPA buffer) to a final concentration of 2 mM. Tubes were incubated at room temperature in the dark for 15 min, followed by addition of 2.5 μl of 5 M methanesulphonic acid. Aliquots of the samples were stored at –80°C.
Samples were analysed by high-performance liquid chromatography (HPLC) using a reversed phase Vydac C18 column at 40°C with a constant flow rate (0.3 ml min–1) of solvent A [0.25% (w/v) D-camphor sulphonate Li-salt, pH 2.64] and/or solvent B [25% (v/v) 1-propanol in solvent A]. After injecting the sample, solvent A was applied for the first 5 min, followed by 90% of solvent A and 10% of solvent B for another 15 min. After that, a linear gradient of 10–20% solvent B was applied over 40 min. The column was washed and equilibrated by applying 100% solvent B for 10 min, followed by solvent A for 20 min. Micromolar solutions of derivatized glutathione (Roche Molecular Biochemicals), glutathionyl-spermidine (Bachem) and trypanothione (Bachem) were used as standards. Thiols were detected from bimane fluorescence with excitation at 390 nm and emission at 482 nm using an on-line fluorescence detector. Thiols were identified by their retention time relative to standards and quantified from the peak area.
Antisera, Western blots and immunofluorescence
In an attempt to generate antisera recognizing MRPA, rabbits were immunized with the peptides GNEKPPGRPGGNQKQPSPEC and CGNEPTQDGGNEGGEKKNTE from the MRPA sequence, coupled to keyhole limpet haemocyanin (Pierce). The resulting antisera did not recognize any specific bands either in wild-type trypanosomes or in cells over-expressing TbMRPA or TbMRPA-myc. As a control, we also attempted to overproduce full-length MRPA in Escherichia coli, without any success.
Antibodies to the His (Roche Molecular Biochemicals) and myc tags (Santa Cruz Biotechnology) were purchased, and the antibody to ODC was from Dr M. Phillips. The antibodies were used at 0.5 μg ml–1, 1:400 dilution and 1:1200 dilution, respectively, for Western blotting according to standard protocols (ECL kit, Amersham).
For immunolocalization of myc-tagged or His-tagged proteins, 0.5–1 × 106 cells per sample were washed in ice-cold TDB buffer (0.025 M KCl, 0.4 M NaCl, 0.005 M MgSO4.7H2O, 0.05 M Na2HPO4, 0.01 M NaH2PO4.2H2O, 0.1 M glucose), then fixed on poly L-lysine-coated glass slides with 3.7% formaldehyde in PBS for 15 min at room temperature. After washing with PBS, cells were permeabilized with 0.2% Triton X-100 in PBS for 10 min at room temperature. Samples were incubated for 1 h at room temperature with monoclonal anti-myc or anti-His6 antibodies diluted (1:100) in PBS–gelatin solution [0.5% (w/v) gelatin in PBS]. After three washes with PBS–gelatin solution, glass slides were reincubated with Cy3-conjugated goat anti-mouse IgG (Amersham-Buchler, 1:500 dilution in PBS–gelatin solution), washed again with PBS–gelatin and then rinsed in PBS. Nuclei and kinetoplasts were stained with DAPI (50 μg ml–1 in PBS). Digital images were captured using OPENLAB software.
Drug sensitivity assay
Drug sensitivity tests were set up in 96-well plates in a final volume of 100 μl per well, with serial twofold dilutions of drug and an initial trypanosome density of 3 × 103 cells ml–1. The plates were incubated for 70 h under standard culture conditions, then inspected under a microscope to ensure growth of the controls and sterility of the plate. A sample of 10 μl of Alamar blue (Trinova Biochem) was added to each well, and the plates were incubated for another 2 h. Reduction of Alamar blue (which is proportional to live cell counts) was read by a fluorescence scanner (excitation wavelength 530 nm, emission wavelength 590 nm), and the scanned images (signals) were quantified using IMAGEQUANT software. In earlier experiments, an emission wavelength of 570 nm was used, as the 590 nm filter was not available; some early experiments also involved higher starting cell densities. The IC50 was determined from the sigmoidal inhibition curve, and relative resistance factors (RRFs) were calculated by dividing the IC50 value for transgenic (transfected) cells by the IC50 value for wild-type (parental) cells.
The MICs (level at which no motile cells of normal morphology were present) were determined in six separate experiments over incubation periods of 10 days. Initially, two- to threefold drug dilutions were used, with selected cell lines, to obtain general estimates for the different drugs. The data illustrated were obtained using narrower dilution ranges as indicated by the preliminary results. Drugs used were melarsoprol (Arsobal; Specia), pentamidine (Pentacarinat; Rhone-Poulenc), suramin (Germanin; Bayer) and berenil and were kindly supplied by Dr R. Brun, Schweizer Tropeninstitut, Basel, Switzerland.
We thank Ronald Kaminsky (Swiss Tropical Institute, Basel, Switzerland) for providing the TbABC1 DNA probe, and Reto Brun (Swiss Tropical Institute) for supplying the anti-trypanosomal drugs and giving us the protocol for, and helping us to set up, the Alamar blue assay. We thank Margaret Phillips (University of Texas, Southwestern Medical Center, Dallas, TX, USA) for supplying the ODC and GCS clones, the anti-ODC antibody and the recombinant trypanosome ODC enzyme. We also thank George Cross and Elizabeth Wirtz (The Rockefeller University, New York, USA) for plasmid vectors. We are indebted to the Trypanosoma brucei sequencing consortium for both sequence and materials: the teams at the Sanger Centre and TIGR for sequence, and Sara Melville and coworkers for P1 filters and clones. We thank Professor Richard Herrmann and all members of the Clayton laboratory (ZMBH) for helpful discussions and suggestions, and Claudia Hartmann and Drifa Gundlsdotujr-Plank for technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (Graduiertenkolleg GK300 and SFB 544). DNA sequences described in this paper have accession numbers AJ318885 (TbMRPA) and AJ318886 (TbMRPE).