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Originally published In Press as doi:10.1074/jbc.M405183200 on June 9, 2004

J. Biol. Chem., Vol. 279, Issue 32, 33273-33280, August 6, 2004
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Differential Protein Expression Analysis of Leishmania major Reveals Novel Roles for Methionine Adenosyltransferase and S-Adenosylmethionine in Methotrexate Resistance*

Jolyne Drummelsmith, Isabelle Girard, Nathalie Trudel, and Marc Ouellette{ddagger}

From the Infectious Diseases Research Centre, Laval University, Quebec City, Quebec G1V 4G2, Canada

Received for publication, May 10, 2004 , and in revised form, June 4, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Leishmania is a trypanosomatid parasite causing serious disease and displaying resistance to various drugs. Here, we present comparative proteomic analyses of Leishmania major parasites that have been either shocked with or selected in vitro for high level resistance to the model antifolate drug methotrexate. Numerous differentially expressed proteins were identified by these experiments. Some were associated with the stress response, whereas others were found to be overexpressed due to genetic linkage to primary resistance mediators present on DNA amplicons. Several proteins not previously associated with resistance were also identified. The role of one of these, methionine adenosyltransferase, was confirmed by gene transfection and metabolite analysis. After a single exposure to low levels of methotrexate, L. major methionine adenosyltransferase transfectants could grow at high concentrations of the drug. Methotrexate resistance was also correlated to increased cellular S-adenosylmethionine levels. The folate and S-adenosylmethionine regeneration pathways are intimately connected, which may provide a basis for this novel resistance phenotype. This thorough comparative proteomic analysis highlights the variety of responses required for drug resistance to be achieved.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Leishmania, a protozoan parasite transmitted by the bite of a sandfly, can cause a species-dependent spectrum of disease ranging from cutaneous lesions (Leishmania major) to potentially fatal visceral infections (Leishmania donovani). With an estimated 12 million cases of leishmaniasis worldwide and 1.5-2 million new cases reported each year (1), this parasite has a significant impact on human populations. Leishmania is transmitted to the host as a motile, elongated promastigote, whereupon it is engulfed by a macrophage and transforms into a round, non-motile amastigote able to spread within the host. The parasite is primarily found in South America, Asia, southern Europe, and Africa, but recent reports have confirmed the visceral form of the disease in dogs in 21 U. S. states and in Canada (2).

Antimony-containing compounds are by far the most commonly prescribed antileishmanial therapies, but in some regions up to 80% of cases exhibit clinical resistance to these drugs (3). New targets for the design of novel therapeutics are vital. The folate biosynthetic and reduction pathways in Leishmania contain several elements not found in mammalian cells and are under intense scrutiny for drug development. Antifolates, such as the folate analogue methotrexate (MTX),1 have been successful in the treatment of several diseases as well as some cancers (for review, see Ref. 4) but have not yet been successful against Leishmania. One of the reasons for this is the ease with which the parasite is able to manipulate expression of its genes. Leishmania strains insensitive to MTX display a variety of primary resistance mechanisms. These include amplification of the dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene (5) encoding the primary target of the drug, amplification of the gene encoding PTR1 (6, 7), a pteridine reductase able to partially replace the function of DHFR-TS (8, 9), deletion of the folate transporters that allow MTX to enter the cell (10, 11), or overexpression of the gene coding for the biopterin transporter BT1 (12).

The 34-megabase genome of the human pathogen L. major Friedlin has been sequenced by a consortium of laboratories (www.genedb.org). Many of its genes have no homologues in other organisms, and these may yield Leishmania-specific drug targets. Because of the number of unique open reading frames found in this organism as well as the complexity of the mechanisms of resistance exhibited by the parasite, a global analysis of the phenomenon of drug resistance is warranted. In a previous preliminary study we used a comparative proteomic approach to show increased levels of PTR1 in a L. major strain selected for MTX resistance in vitro (13). This was the first identification of a primary resistance mechanism using a proteomic approach and as such validated the use of two-dimensional gel electrophoresis in further studies of resistance.

Here we describe the results of detailed proteomic analyses of MTX exposure and resistance in L. major. Through these studies we have found altered expression of proteins involved in the stress response and in primary resistance mechanisms but also of proteins with less easily predicted roles in resistance. One of these corresponds to the methionine adenosyltransferase (MAT) protein, which is overexpressed both in sensitive cells shocked with MTX as well as in mutants resistant to the drug. We present evidence that the MAT protein and its product, S-adenosylmethionine (SAM), play previously unappreciated roles in MTX resistance.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—L. major LV39, mutants MTX60.2 (13), and MTX60.4 (14), and transfectants were grown in M199 medium (Invitrogen) supplemented with 10% fetal calf serum (Wisent) and 5 µg/ml hemin (ICN). Where appropriate, MTX, paromomycin (Sigma), SbIII, and/or zeocin (Invitrogen) were added. Cultures were incubated at 25 °C.

Two Dimensional Sample Preparation and Electrophoresis—Cultures were grown to late log phase as determined by optical density at 600 nm. Cells were harvested by centrifugation at 2500 x g, washed twice in HEPES-NaCl buffer, and resuspended in two-dimensional lysis buffer (7 M urea, 2 M thiourea, 40 mM Tris, 4% CHAPS, 0.1 mg/ml phenylmethylsulfonyl fluoride). Lysis was allowed to proceed for 2 h at room temperature, and samples were centrifuged to remove insoluble material. Protein concentration was assayed using the 2D Quant Kit (Amersham Biosciences). Proteins were separated into aliquots and stored at -80 °C.

In the first dimension samples were run on 18-cm Immobiline Dry-Strips (Amersham Biosciences) on an IPGPhor isoelectric focusing system as recommended by the manufacturer. Strips were equilibrated in equilibration buffer (50 mM Tris-Cl, pH 8.8, 6 M urea, 30% glycerol, 2% SDS, trace of bromphenol blue) containing 10 mg/ml dithiothreitol for 15 min and then in equilibration buffer containing 25 mg/ml iodoacetamide for 15 min and sealed to 12% acrylamide gels using 0.5% agarose in standard Tris-glycine electrophoresis buffer. Second dimension SDS-PAGE was run in a Hoefer DALT apparatus (Amersham Biosciences) at 40 mA/gel and 15 °C until the tracking dye was run off the gel.

Gel Staining, Imaging, and Image Analysis—Proteins were visualized by Sypro Ruby fluorescence (Molecular Dynamics). Gels were fixed overnight in 40% methanol, 7% acetic acid. Gels were stained overnight and then destained in 10% methanol, 7% acetic acid for 3 x 1 h. Gels were imaged with the ProXpress scanner (PerkinElmer Life Sciences) at 100-µm resolution using 480-nm excitation and 620-nm emission filters. Two-dimensional gels of four independent samples per strain or condition were averaged and compared using Progenesis software (Non-linear Dynamics). Significance of differences observed were determined by the Student's two-tailed t test in Microsoft Excel.

Mass Spectrometry—Gel plugs containing the proteins of interest were excised manually and sent for peptide mass fingerprinting (Eastern Quebec Proteomics Centre, Centre Hospitalier de l'Université Laval, Québec). Samples were prepared as described previously (13). Mass spectra were acquired on a Voyager-DE PRO MALDI-TOF mass spectrometer (Applied Biosystems) operating in the positive-ion reflector delayed-extraction mode. Protein identifications were obtained using MASCOT (MatrixScience) and searching for matching peptide mass fingerprints in a protein data base (Leishpep 300104) generated by annotation of L. major Friedlin sequence information, available through the GeneDB website (www.genedb.org). The search criteria used were complete carboxamidomethylation of cysteine, partial methionine oxidation, and mass deviation smaller than 60 ppm. A score of greater than 51 was considered significant (p < 0.05).

Peptide tandem mass spectra were obtained by capillary liquid chromatography coupled to an LCQ DecaXP (ThermoFinnigan, San Jose, CA) quadrupole ion trap mass spectrometer with a nanospray interface as described previously (13). The resulting peptide MS/MS spectra were interpreted using the SEQUEST algorithm (15) and searched against proteins in the Leishpep data base. Partial carboxamidomethylation of cysteine and oxidation of methionine were considered in the search. A protein was considered a good match if at least two peptides were confidently identified. Confident identification of a peptide required a cross-correlation score of 1.9, 2.5, and 3.7 for singly, doubly, and triply charged peptides, respectively. Each peptide identification was confirmed by manual inspection of the spectrum.

DNA Methods—Genes of interest were amplified by PCR from L. major LV39 genomic DNA using the proofreading polymerase Pwo (Roche Applied Science). Primers used were: for MAT, JD6, 5'-CCT TCC GCA CTA GTC ACA GTC C-3', and JD7, 5'-CGG CAC ACC TCG AATTCG AT-3'; for ARGG, JD 17, 5'-ACA CTT CCC CAT TTC TAG ACT CC-3', and JD 18, 5'-GGG CGC AAG CTT TGT AAG TAG T-3'; for PGFS, JD15, 5'-CGC GAG CTT GTT GAA AGC TTA A-3', and JD16, 5'-TCC CTT TTC TAG AAT CGT TGA CTG-3'; for MORN, JD19, 5'-CTG TCC CTC CTC TAG ATC CCT ATT T-3', and JD20, 5'-GCG ACC GAT GAT AAA GCT TGA G-3'; for acyl-CoA dehydrogenase, LmjF06.0880F, 5'-CGC CAA GGA GAA GAT GAT CC-3', and LmjF06.0880R, 5'-CAT CTT GGA GCC GTT GAT GA-3'. Nucleotides that have been altered to incorporate restriction sites are underlined. The products MAT, PGFS, and MORN were cloned into the Leishmania expression vector pSP{alpha}NEO{alpha}, encoding paromomycin/G418 resistance (6), or pSP{alpha}ZEO{alpha}, encoding zeomycin resistance. The resulting plasmids were transfected into L. major LV39 by electroporation as described previously (6) and maintained with 1 mg/ml paromomycin or 5 µg/ml zeocin. Transfections were confirmed by Southern hybridization.

Southern and Northern Hybridization—Genomic DNA was prepared from Leishmania cells using DNAzol (Invitrogen). DNA was digested with appropriate restriction enzymes (New England Biolabs) according to the manufacturer's recommendations, and the resulting fragments were separated on a 0.7% agarose gel using digoxigenin (DIG)-labeled DNA Molecular Weight Marker II (Roche Applied Science) as a size standards. RNA for northern blotting was run on denaturing agarose gels containing formaldehyde and formamide. RNA samples were prepared from Leishmania cells using RNAzol (Invitrogen) and were denatured at 65 °C for 15 min and chilled on ice before loading. Probes were generated by PCR and random prime-labeled with DIG-High Prime (Roche Applied Science) according to the manufacturer's directions. The gels were blotted to positively charged nylon membrane (Roche Applied Science), UV-cross-linked using a Spectrolinker XL-1000 cross-linker (Spectronics), hybridized with the appropriate probe, detected with anti-DIG-AP Fab fragments and CDP-Star (Roche Applied Science), and exposed to x-ray film, all according to the manufacturer's directions. Blots to be reprobed were stripped by pouring boiling 0.1% SDS on the membranes and shaking at room temperature for 10 min. Membranes were rinsed and stored wet.

SAM Extraction and HPLC Analysis—Leishmania cultures were harvested in late log phase and washed in HEPES-NaCl. Three independent cultures were grown for each condition. Cell pellets were weighed, resuspended in 2 volumes of 0.4 M HClO4, and incubated on ice for 20 min. Supernatants were passed through a 0.45-µm filter, separated into aliquots, and stored at -80 °C.

Metabolites were separated by HPLC by a modification of the method of Wang et al. (16) using a Schimadzu HPLC system and Prevail C18 column (250 x 4.6-mm inner diameter, 5-µm particle size; Alltech) and detected at 254 nm. The mobile phase consisted of two solvents: solvent A, 8 mM octanesulfonic acid and 25 mM NaH2PO4, pH 3.0; solvent B, 100% HPLC-grade methanol. Solvents were passed through a 0.45-µm filter before use. The column was equilibrated at 72% solvent A, 28% solvent B before use. Samples (20 µl) were injected, and conditions were held at 28% solvent B for 3 min. Then, solvent B was increased to 35% over 3 min and held there for 19 min before initial conditions were re-established. SAM was identified by co-chromatography with a SAM standard. A standard curve for quantitation was achieved by integration of peak areas of known quantities of SAM, and experimental peak areas were compared with this curve to determine pmol of SAM/mg of wet cell weight. Differences were analyzed using either parametric or non-parametric Student's 2-tailed t tests, the choice of which was determined by the F-test for equality of variances.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Comparative Two-dimensional Gel Electrophoresis Analysis of L. major LV39 Wild Type and in Vitro-derived MTX-resistant Mutants—Two independent mutants of L. major, MTX60.2 and MTX60.4, were selected for resistance to MTX. These strains were able to grow in 60 µM MTX, whereas the EC50 for the wild type strain is 0.05-0.1 µM. Initial two-dimensional gel electrophoresis analysis identified one highly overexpressed protein in mutant MTX60.2 that corresponded to PTR1, a known primary MTX resistance mechanism (6, 7).

The success of these initial analyses prompted us to perform a more detailed comparison of these mutants using narrow-range IPG strips. Gels of four independent samples were run and averaged and compared using the two-dimensional gel analysis software package Progenesis. Representative gels of two different pH ranges analyzed, pH 4.5-5.5 and 5.0-6.0, are shown in Fig. 1A. Fig. 1B shows a difference found using gels of pH range 3-10. Differences of greater than 2-fold identified by the software were individually validated and are marked. Spots of interest were excised from the gels and identified by mass spectrometry, and the results are listed in Table I. Quantitative differences in mutant strains are mostly due to protein overexpression as opposed to down-regulation. Only spot 1173 decreased in the mutants (Table I, Fig. 1A). Also, several of the proteins identified, such as chaperonins, antioxidants, and heat-shock proteins may have been involved in response to stress induced by drug selection. Finally, some proteins, such as PTR1 or the argininosuccinate synthase ARGG, are exclusively found in the mutant MTX60.2, whereas acyl-CoA dehydrogenase is expressed only in MTX60.4. However, most of the differences noted such as MAT and enolase are found in both strains, indicating that even in cases where distinct primary resistance mechanisms have evolved, other responses to MTX are conserved.



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  		FIG. 1.
Representative two-dimensional gels comparing sensitive and resistant strains of L. major LV39. A, gels covering pH 4.5-5.5 and 5.0-6.0. B, section of a pH 3-10 gel. Spot labels are as in Table I. Spots with significant differences in expression (Table I) were excised, and MALDI-TOF or MS/MS identification was attempted. wt, wild type.

 


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  		TABLE I
Proteins differentially expressed in LV39 mutants resistant to MTX versus the sensitive parent as identified by MALDI-TOF or MS/MS

MS ID refers to the labels in Fig. 1. Accession numbers are from the L. major database, GeneDB, version 300104. MASCOT scores above 51 were considered significant (p < 0.05). p values for -fold differences were calculated in Excel using the normalized volumes for individual spots calculated by Progenesis. exp/pred, experimental/predicted. AdoMetS, S-adenosylmethionine.

 
Analysis of the Initial Responses of L. major to MTX by Two-dimensional Gel Electrophoresis—The above analysis of MTX-resistant mutants raised the possibility that several of the differences noted could in fact be due to exposure to MTX as opposed to resistance per se. Such changes in protein expression could also represent initial steps in the acquisition of resistance or perhaps an early protective response that allows time for the parasite to express an effective resistance mechanism. To visualize these changes two analyses were carried out. In the first, wild type cultures were grown to mid-log phase and then exposed to 1000x the EC50 of MTX (100 µM) for 24 h. In the second proteins were extracted from wild type cells grown in the presence of 1x the EC50 of MTX (100 nM MTX). In both cases samples were compared with proteins extracted from wild type cells grown in the absence of drug exposure. Because this exposure to drug causes a decrease in growth rate, care was taken to attempt to harvest cells from the same phase of growth. Representative gels (spanning pH 4-6.7) from this analysis are shown in Fig. 2. Several differences between spots that had been identified in previous analyses (Hsp70, Cpn60) were noted (Fig. 2). Spots whose identity was unknown or whose positions made matches uncertain were excised from the gels and subjected to mass spectrometric identification and are shown in Table II. No trace of PTR1 was seen in these gels, suggesting that although this is a primary resistance mechanism, it is not involved in the initial response to MTX. Expression levels for most of the spots altered in these analyses showed the same trends in both treatments, although this was not always the case. Interestingly, several of the protein changes noted in this analysis were the same as those seen in the analysis of resistant mutants (Table I). This was expected in the case of stress response proteins such as heat-shock proteins, antioxidants, and chaperonins but was somewhat unexpected in the case of MAT.



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  		FIG. 2.
Representative two-dimensional gels comparing MTX-sensitive L. major LV39 in the absence and presence of MTX. Spot labels are as in Table II. Spots with significant differences in expression (Table II) were excised, and MALDI-TOF identification was attempted. wt, wild type.

 


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  		TABLE II
Proteins differentially expressed in LV39 wild type cells either shocked with high levels of MTX (shock) or grown in the EC50 of the drug (growth) as identified by MALFI-TOF MS

MS ID refers to labels in Fig. 2. Accession numbers are from the L. major GeneDB, version 300104. MASCOT scores above 51 were considered significant (p < 0.05). p values for -fold differences were calculated in Excel using the normalized volumes for individual spots calculated by Progenesis. exp/pred, experimental/predicted;. AdoMetS, S-adenosylmethionine; u, unique spot.

 
Overexpression by Gene Amplification and Linkage to Resistance Mediators—ARGG (spots 1168 and 1174) catalyzes the penultimate step in arginine biosynthesis. This protein is highly overexpressed in mutant MTX60.2 but not in mutant MTX60.4 (Table I). Leishmania often uses gene amplification to respond to drug pressure (17, 18). Southern hybridization revealed that the ARGG gene was greatly amplified in strain MTX60.2 compared with the wild type (Fig. 3A). The PTR1 protein overexpression observed in MTX60.2 is also correlated to amplification of the gene (Fig. 3B). This raised the possibility that there are multiple amplicons in strain 60.2. However, it has been known for a long time that ARGG and PTR1 are physically linked in Leishmania tarentolae (19). Sequencing of the L. major genome indicated that ARGG and PTR1 genes are also closely linked on chromosome 23 in this species and are in fact separated by a segment of only 4.4 kilobases. Therefore, ARGG is most likely overexpressed due to the proximity of its structural gene to that of PTR1 and, thus, its inclusion on the same DNA amplicon. Acyl-CoA dehydrogenase overexpression is seen solely in mutant MTX60.4 (Table I). This overexpression is also correlated to gene amplification (Fig. 3C). Interestingly, in L. major this gene is 4.6 kilobases downstream of DHFR-TS on chromosome 6 (www.genedb.org). The DHFR-TS gene, coding for the primary target of MTX, was also found to be amplified in this mutant (Fig. 3D).



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  		FIG. 3.
Southern hybridization of genomic DNA of L. major LV39 wild type (wt), MTX60.2 and MTX60.4. Probes were specific for ARGG (A), PTR1 (B), acyl-CoA dehydrogenase (C), and DHFR-TS (D). Equivalent loading was determined by ethidium bromide staining (not shown).

 
Prostaglandin F Synthase, MAT, MORN, and Enolase Are Overexpressed in Both Resistant Mutants—Several proteins were found to be overexpressed in both independent MTX mutants studied. One of these, enolase, is part of the glycolytic pathway. Overexpression of several glycolytic enzymes, including enolase, has been observed to result from various stresses such as heat shock or osmotic stress (20-22), and thus, its presence is probably not related specifically to MTX resistance. There were also several proteins whose presence could not be accounted for by stress responses. Prostaglandin F synthase (PGFS) activity has only recently been described in Leishmania (23), and its biological relevance is unclear. The PGFS protein was found to be overexpressed 4.0- and 3.7-fold in mutants MTX60.2 and MTX60.4, respectively, whereas a protein of unknown function but with several MORN repeat motifs encoded by the open reading frame LmjF30.3310 but which we will herein call MORN was overexpressed by 10.4- and 6.6-fold in the two mutants. This is a protein of unknown function but which has homologues in several organisms including other parasites such as Cryptosporidium parvum (www.genedb.org) and Plasmodium falciparum (24) as well as bacteria, plants, and mammals. The L. major MORN protein contains 15 putative MORN repeat motifs. These motifs are found in junctophilins, which are present in junctional complexes between the plasma membrane and the endoplasmic reticulum (25). They are also found in phosphatidylinositol-4-phosphate 5-kinases, which are involved in many cellular processes including cytoskeleton assembly, vesicular trafficking, signal transduction, and DNA synthesis (reviewed in Loijens et al. (26)). Finally, MAT was overexpressed in both resistant mutants (Table I) and, interestingly, was also increased in sensitive cells exposed to MTX (Table II). This suggested to us that it may have an important role in initial cellular responses to MTX in Leishmania. MAT catalyzes the synthesis of SAM (for review, see Refs. 27 and 28). This metabolite is involved in many essential cellular pathways, such as one-carbon metabolism, sulfuration, and the biosynthesis of polyamines. In L. major, two copies of this gene are present within a 7-kilobase region of chromosome 30 and are separated by a hypothetical open reading frame (www.genedb.org). A similar organization is seen in Leishmania infantum (29).

Southern hybridization indicated that unlike in the cases of PTR1, DHFR, ARGG, and acyl-CoA dehydrogenase, overexpression is not mediated by DNA amplification of their respective genes (Fig. 4A). Therefore, we explored other mechanisms that could have led to increased levels of these proteins. Although occurring less frequently than gene amplification, RNA overexpression without gene amplification has been observed in drug-resistant Leishmania (30). Previously, DNA microarray data and northern hybridization experiments had indicated that the MAT transcript was overexpressed in the strain MTX60.2 (14). Fig. 4B shows that this is also the case in mutant MTX60.4. Using a probe specific for the MORN gene, a similar trend was seen (Fig. 4B). Unexpectedly, however, northern blotting with a PGFS probe revealed that mRNA levels of this gene were unchanged in both mutants and the parent strain (Fig. 4B).



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  		FIG. 4.
Southern (A) and Northern (B) hybridization of DNA or RNA prepared from L. major LV39 wild type (wt), MTX60.2, and MTX60.4 to probes specific for MAT, MORN, and PGFS. Equivalent loading for Northern hybridization was confirmed by hybridization to an {alpha}-tubulin ({alpha}-tub) probe.

 
To evaluate the potential role, if any, of the PGFS and MORN proteins in MTX resistance, their respective genes were amplified by PCR and independently cloned into Leishmania expression vectors, producing plasmids pSP{alpha}ZEO{alpha}PGFS and pSP{alpha}ZEO{alpha}MORN, respectively. These plasmids as well as the vector pSP{alpha}ZEO{alpha} were independently transfected into L. major LV39. The abilities of the vector, PGFS, and MORN transfectants to grow in increasing levels of MTX were assessed. Growth curves showed no differences in MTX resistance levels between these strains (results not shown).

The MAT Protein and MTX Resistance—The MAT gene was similarly amplified by PCR and used to generate pSP{alpha}NEO{alpha}MAT. Both this plasmid and the vector pSP{alpha}NEO{alpha} were independently transfected into L. major LV39. Initially, the ability of vector and MAT transfectants to grow in the presence of increasing levels of MTX was assessed. No difference in MTX EC50 was seen between these strains (Fig. 5A), indicating that MAT is not directly responsible for resistance. However, we postulated that MAT overexpression might aid in the acquisition of resistance, since it was found to be overexpressed during initial growth and shock with MTX (Table II) as well as in mutants (Table I). To investigate this possibility we attempted to judge the ease with which the MAT transfectant was able to become resistant to MTX. Because resistance is due to independent genetic events whose effects cannot be averaged, this type of analysis was not done in a quantitative manner. However, common trends might be expected to emerge. After an initial period of growth in relatively low levels of MTX (100 nM), no differences in growth were noted, which was consistent with our initial growth curve measurement. However, a second passage into varying concentrations of MTX made it clear that cells overexpressing MAT were able to grow at much higher levels of MTX that those carrying the vector alone (Fig. 5B). This test was replicated a total of six times. Cells carrying vector alone showed modest increases in EC50 of 5-10-fold. However, 4 of 6 strains overexpressing MAT showed highly elevated EC50 for the drug (80-200x), one did not show elevated resistance, and one was even able to grow well beyond 10 µM MTX (200x the EC50). The differences in growth characteristics observed among these strains suggest that distinct changes may be at play, and as such, they have been presented independently. We also tested whether MORN or PGFS could be implicated in a similar phenotype, but this did not appear to be the case (results not shown).



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  		FIG. 5.
MTX sensitivity of L. major LV39 transfected with either pSP{alpha}NEO{alpha} (vector) or pSP{alpha}NEO{alpha}MAT. A, results of the first passage in MTX. After the cultures grown at 0.1 µM MTX had grown sufficiently, equal numbers of cells were used for a second growth curve experiment, shown in B. Squares represent the vector transfectant, whereas triangles represent the MAT transfectant.

 
These results raised the question as to whether the protective effect conferred on sensitive Leishmania cells by MAT was specific for MTX treatment or more general in nature. To investigate this possibility, growth curves for the pSP{alpha}NEO{alpha} and pSP{alpha}NEO{alpha}MAT transfectants were carried out in the presence of increasing concentrations of trivalent antimony. Although in this case, cells were passaged four times, no difference in growth between the two strains was apparent (results not shown).

The Effects of MTX Treatment and Resistance on SAM Levels—The implication of MAT in the MTX resistance phenotype prompted us to test whether the observed differences in levels of the MAT protein were reflected in terms of its product, SAM. HPLC analysis of cellular extracts of L. major LV39 wild type and MTX60.4 revealed that levels of SAM were approximately doubled in the mutant strain (Fig. 6A, p = 0.034). Overexpression of the MAT protein alone lead to a modest but statistically insignificant increase in SAM levels even though the gene was present in multicopy (Fig. 6B, vector versus MAT). However, analysis of a MAT transfectant that had acquired MTX resistance after growth in 0.1 µM MTX and subsequently 10 µM MTX (from Fig. 5B) showed a statistically significant increase in SAM levels (Fig. 6B, vector versus MAT plus MTX; p = 0.025). Thus, an increase in SAM is correlated to the selection and emergence of MTX resistance in Leishmania.



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  		FIG. 6.
SAM levels in various cell lines as determined by HPLC analysis. SAM was quantitated by comparison of integrated peak area to a standard curve and is expressed as pmol of SAM/mg wet weight. A, LV39 wt, L. major LV39; MTX60.4, L. major MTX60.4; B, vector control, L. major LV39 (pSP{alpha}NEO{alpha}); MAT, L. major LV39 (pSP{alpha}NEO{alpha}MAT); MAT+MTX, L. major LV39 (pSP{alpha}NEO{alpha}MAT) Data shown are from growth in 0.1 µM MTX and subsequently in 10 µM MTX (from Fig. 5B). Bars with different letters are statistically different (p < 0.05).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The ability of our proteomic analyses to identify primary resistance mechanisms has already been proven. Indeed, we had identified a primary MTX resistance mechanism, namely overexpression of the PTR1 protein, in an L. major LV39 MTX-resistant mutant using a proteomic approach (13). Previously, DNA microarray analyses of mutant MTX60.4 revealed the overexpression of DHFR-TS (14), which is a known primary resistance mechanism to MTX in Leishmania (5, 31). Our proteomic analysis of mutant MTX60.4 failed to detect this primary resistance mechanism. However, we identified acyl-CoA dehydrogenase as a unique spot in this mutant. The structural gene for this protein is closely linked (4.6 kilobases) to that of DHFR-TS. Thus, our proteomic approach can lead to the localization of a primary resistance protein even when it is not detected directly. A similar situation was seen in mutant MTX60.2 for ARGG (Fig. 3), the gene for which is adjacent to the PTR1 gene. Leishmania are well known for their ability to overexpress a particular protein by amplifying relatively large sections of chromosomal DNA, and this has been found to be involved in resistance of this organism to several classes of drug (19). This phenomenon could prove to be very useful in the search for resistance mechanisms. The discovery of an overexpressed spot could suggest the presence of an amplicon that could, in turn, point to the presence of a primary resistance mediator in that region. Gene amplification is a frequent event implicated in drug resistance and tumor progression in cancer cells (32, 33), and a proteomic approach as described here could point to proteins that, by virtue of gene proximity, might be implicated in the phenotype being studied.

During the course of the analysis presented here, many differences in protein expression were found, and among those identified were proteins involved in the stress response (chaperonins, heat-shock proteins, possibly enolase; Tables I and II). These resistant mutants display slightly longer doubling times that the wild type, suggesting that although they have been passaged for more than a year in high levels of MTX they have not yet fully adapted to these conditions. This observation could reflect either an inability of the resistance mechanisms being expressed to completely abrogate the effects of the drug or a significant energy cost associated with the expression of resistance for which the cells have not yet compensated. Gels of proteins extracted from these mutants grown in the absence of MTX for several rounds show that some of these spots are decreased although not down to wild type levels.2 In one study, some heat shock proteins remain overexpressed in cells completely adapted to trivalent antimony and grown for numerous passages in the absence of any obvious stress (34). It is, thus, possible that remnants of the stress response expressed during drug selection remain in adapted, resistant mutants or that resistant cells are under continuous stress.

Although our proteomic efforts to this point had led to primary resistance mechanisms and an appreciation for the role of the stress response in resistance, our goal for these studies is to identify novel elements involved in resistance, its acquisition, or its maintenance. Thus, we turned our attention to overexpressed proteins for which we could not easily provide a rationale. Among these, we chose to concentrate on the PGFS and MORN proteins, which are both overexpressed to a relatively high degree in the two independent MTX mutant strains (Table I) as well as MAT, which is overexpressed in both mutants and was also identified in our analysis of the initial responses to MTX (Tables I and II).

Although gene amplification is a frequent mode of gene and indirectly of protein overexpression, we found that the structural genes for these proteins were not amplified at the DNA level but that MAT and MORN mRNA levels increased in the mutants (Fig. 4). The levels of PGFS transcript appeared to be unchanged in MTX-resistant mutants (Fig. 4). It would therefore appear that a translational mechanism of regulation is at play in the case of PGFS overexpression. Very little is known concerning transcriptional and translational control in Leishmania and related parasites. In several cases, mRNA amounts are often affected by differential mRNA stability conferred by untranslated regions (35-37). In other cases, mRNA levels are unable to account for protein amount (38, 39), but as yet no mechanism for translational regulation has been described. Further study of PGFS expression and also the mechanisms by which MAT and MORN mRNAs are increased might provide new insight into poorly understood post-transcriptional regulatory mechanisms in this important class of pathogen.

No role in MTX resistance for MORN or PGFS was found by overexpressing their cognate genes in sensitive L. major cells. Neither of these proteins was induced upon exposure to MTX, but both were overexpressed in resistant mutants. It is possible that these proteins either contribute to a small degree to MTX resistance or are required for partial compensation of growth defects caused by such resistance. The growth curves used to assess the impact of overexpression of these genes on MTX resistance levels might not be sensitive enough to detect small changes. Equally possible is that alone, these proteins do not cause a measurable resistance phenotype. Further experiments, for example by co-transfecting these genes into strains carrying multicopy PTR1 or DHFR-TS and assessing any synergistic effects, might help to elucidate any role they may have in resistance.

Unlike MORN and PGFS, the MAT protein was found overexpressed not only in resistant mutants but upon exposure to MTX. This raised the possibility that MAT overexpression might be required for acquisition of MTX resistance or might have a protective effect that would give the cells time to express more effective resistance mechanisms. The growth curve experiments presented here support this hypothesis. In general, MTX resistance selection takes numerous passages in slowly increasing concentrations of drug. Here, however, cells overexpressing MAT were able to withstand initial drug treatment and quickly became capable of growing in fairly high levels of MTX (Fig. 5).

Although this was a novel and unexpected finding, there is evidence that MTX can affect SAM levels. The folate and SAM pathways intersect through the enzyme methionine synthase, which regenerates methionine from homocysteine, a metabolite produced via S-adenosylhomocysteine through the use of SAM in various transmethylation reactions (for review, see Refs. 40 and 41; Fig. 7). Methionine synthase requires 5-methyltetrahydrofolate (5-MeTHF) as a methyl donor. In mammalian tissues, the presence of MTX inhibits DHFR-TS, which leads to increases in dihydrofolate (DHF) pools (42). This phenomenon is also true in L. major.3 MTX reduces levels of 5,10-methylenetetrahydrofolate (5,10-MeTHF) (43), and high levels of DHF reduce the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate (44). Thus, the availability of this cofactor to methionine synthase is diminished, which should result in an imbalance of SAM/S-adenosylhomocysteine in the cell. This balance is critical to several metabolic functions in the cell. Depletion of the SAM pool has been shown to be a cause of MTX-associated hepatotoxicity during cancer treatment (42). MTX treatment in cancer and psoriasis patients has in fact been shown to increase levels of homocysteine (45) and decrease cellular methylation (46).



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  		FIG. 7.
Intersection of folate and S-adenosylmethionine regeneration pathways. MTX blocks action of DHFR-TS, and high levels of DHF inhibit the production of 5-methyltetrahydrofolate (5-MeTHF). Enzymes labeled are DHFR-TS, methionine synthase (MS), and MAT. Metabolites are THF, DHF, 5,10-methylenetetrahydrofolate (5,10-MeTHF), 5-methyltetrahydrofolate (5-MeTHF), methionine (Met), SAM, S-adenosylhomocysteine (SAH), and homocysteine (hCys).

 

Our proteomic analyses suggest that MAT levels are increased early during exposure to MTX (Fig. 2, Table II) and together with metabolite analyses (Fig. 6) suggest that increased MAT and SAM facilitate the emergence of MTX resistance. SAM levels are significantly increased in a mutant strain highly resistant to MTX (Fig. 6A) even when compared with MAT transfectants (Fig. 6B), implying that further changes occur during resistance acquisition and adaptation that act with MAT overexpression to increase SAM levels. There are numerous candidates for these changes. For example, mammalian MAT activity is regulated by phosphorylation (47), which could lead to quick changes in MAT activity. Post-translational modification of MAT may also exist in Leishmania. Furthermore, increased levels of methionine synthase activity might be required to provide substrate for the MAT enzyme (Fig. 7). The predicted methionine synthase enzyme in L. major is quite large (138.5 kDa); thus, it is unlikely that it would have been visualized by the two-dimensional gel method employed here. However, transcriptomic analysis of strain MTX60.4 did not see an increase in methionine synthase mRNA (14). Another potential mechanism to explain increased intracellular SAM levels is increased transport. In the related parasite Trypanosoma brucei, SAM uptake was found to account for 90% of the intracellular pool (48), suggesting that exogenous SAM is the primary source of this metabolite. Leishmania has SAM transport activity (49), but the identity of the transporter is unknown.

An aid to dissecting the role of SAM in MTX resistance would be to greatly perturb SAM levels. Unfortunately, given that MAT overexpression had minimal effects on SAM levels (Fig. 6), it is not likely that disrupting some or all of the MAT genes would provide the desired background. The complex nutritional requirements for in vitro culture of Leishmania preclude growth in a SAM-depleted medium, and since the transporter is unknown we cannot perturb this activity. We are, however, in the process of attempting to identify this transporter, which may allow us to further characterize the effect of SAM levels on MTX resistance.

MAT overexpression may contribute to MTX resistance by compensating for some of the deleterious effects of MTX itself or might have a more direct role in resistance. Either way, the implication of MAT and SAM in MTX resistance could prove to have a profound effect on drug design. MTX, for instance, has thus far proven ineffective in the treatment of leishmaniasis. The high level of drug required to affect the parasite in vivo is toxic for the host, and on top of this the pathogen quite easily acquires resistance. A combination of inhibitors of key enzymes of the folate and SAM pathways could prove to have a synergistic effect, thereby decreasing the levels of MTX or other antifolates required for treatment. In addition, inhibiting a process required for the acquisition of resistance, such as increased MAT activity or SAM uptake, might significantly reduce or even eliminate its development. These results could also be applicable to mechanisms of MTX resistance in cancer cells.

There have been comparatively few global analyses of the effects of antimicrobial treatment and resistance. In this analysis we have shown that a proteomic approach can directly or indirectly reveal primary resistance mechanisms. It also shows that stress proteins can be induced not only during the first response to drug treatment but for much longer periods. Finally, our proteomic approach has identified a previously unknown role for the MAT protein in antifolate resistance in Leishmania. Although other studies have succeeded in finding quantitative differences between mRNA or protein levels associated with drug exposure or resistance (50-54), here we have identified and characterized a novel resistance-associated mechanism. This highlights the ability of global proteomic approaches to discover subtle, yet potentially important protein and metabolic changes involved in drug resistance.


   FOOTNOTES
 
* This work was supported by a Canadian Institutes of Health Research (CIHR) fellowship (to J. D.) and CIHR group and operating grants (to M. O.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} A Burroughs Wellcome Fund Scholar in Molecular Parasitology. This author holds a Canada Research Chair in antimicrobial resistance. To whom correspondence should be addressed: Infectious Diseases Research Centre, CHUQ, pavillon CHUL, 2705 Boul. Laurier, Ste.-Foy, QC, G1V 4G2. Tel.: 418-654-2705; Fax: 418-654-2715; E-mail: marc.ouellette{at}crchul.ulaval.ca.

1 The abbreviations used are: MTX, methotrexate; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MS/MS, tandem mass spectrometry; DHFR-TS, dihydrofolate (DHF) reductase-thymidylate synthase; MAT, methionine adenosyltransferase; SAM, S-adenosylmethionine; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; DIG, digoxigenin; HPLC, high performance liquid chromatography; PGFS, Prostaglandin F synthase. Back

2 J. Drummelsmith and M. Ouellette, unpublished information. Back

3 J. Drummelsmith, I. Girard, and M. Ouellette, unpublished information. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Christian Brochu, Gaetan Roy, Vicky Brochu, René Paradis, and Dr. Joanna Hunter for the assistance for experimental aspects of this study and Dr. Chris Peacock and Dr. Al Ivens from the Sanger Institute for the L. major annotated protein data base.



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 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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