Differential Protein Expression Analysis of Leishmania major Reveals Novel Roles for Methionine Adenosyltransferase and S-Adenosylmethionine in Methotrexate Resistance

Drummelsmith, Jolyne; Girard, Isabelle; Trudel, Nathalie; Ouellette, Marc

doi:10.1074/jbc.m405183200

Cited by 63 publications

(62 citation statements)

References 51 publications

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“…Under our experimental conditions, the RNA corresponding to MRPA could not be detected in wild-type cells. Increased expression in Leishmania, at least in the promastigote stage, is sometimes the result of gene amplification (2, 3), but clearly other mechanisms can also lead to an increase in RNA (9,17). The DNAs derived from L. infantum wild-type and Sb2000.1 cells were isolated, digested, blotted, and hybridized to MRPA, SAHH, and FPGS probes.…”

Section: Resultsmentioning

confidence: 99%

Role of the ABC Transporter MRPA (PGPA) in Antimony Resistance in Leishmania infantum Axenic and Intracellular Amastigotes

Fadili

Messier

Leprohon

et al. 2005

Antimicrob Agents Chemother

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133

127

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Antimonial compounds are the mainstay for the treatment of infections with the protozoan parasite Leishmania. We present our studies on Leishmania infantum amastigote parasites selected for resistance to potassium antimonyl tartrate [Sb(III)]. Inside macrophages, the Sb(III)-selected cells are cross-resistant to sodium stibogluconate (Pentostam), the main drug used against Leishmania. Putative alterations in the level of expression of more than 40 genes were compared between susceptible and resistant axenic amastigotes using customized DNA microarrays. The expression of three genes coding for the ABC transporter MRPA (PGPA), S-adenosylhomocysteine hydrolase, and folylpolyglutamate synthase was found to be consistently increased. The levels of cysteine were found to be increased in the mutant. Transfection of the MRPA gene was shown to confer sodium stibogluconate resistance in intracellular parasites. This MRPA-mediated resistance could be reverted by using the glutathione biosynthesis-specific inhibitor buthionine sulfoximine. These results highlight for the first time the role of MRPA in antimony resistance in the amastigote stage of the parasite and suggest a strategy for reversing resistance.Leishmania is a protozoan parasite affecting several million people throughout the world. The clinical manifestations of the infection depend on the species, the most life-threatening being visceral leishmaniasis caused by the Leishmania donovani complex. Treatment relies exclusively on chemotherapy, and pentavalent antimonials [Sb(V)] are still the mainstay against all forms of Leishmania infections (14, 18). While Sb(V) is used for treating patients, it is generally agreed that Sb(V) is reduced to trivalent antimony [Sb(III)], which constitutes the active form of the drug against the parasite. The exact site of drug reduction (inside the macrophages or inside the parasites) is not known, but activities were recently discovered in Leishmania that could be implicated in this reduction process (7,26,29,36). Resistance to Sb(V) is so widespread in part of India (33) that first-line treatment in this region is either based on miltefosine (31) or amphotericin B (32). Miltefosine is interesting because it can be taken orally, but single point mutations can lead to resistance (24), suggesting that resistance to this drug may occur rapidly.Leishmania has a relatively simple life cycle with two main stages, the flagellated promastigote in the insect stage and the intracellular amastigote living inside macrophages. Progress in culture techniques has allowed the growth of Leishmania amastigotes as part of axenic cultures. An increase in the temperature from 25°C to 37°C and a decrease in the pH of the culture medium to mimic the conditions encountered in the phagolysosome are the key parameters to transform promastigotes into amastigotes (reviewed in reference 37). It is nonetheless easier to grow promastigotes, and most of the work pertaining to resistance mechanisms to antimonials was performed in the insect stage of the parasit...

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Section: Resultsmentioning

confidence: 99%

Role of the ABC Transporter MRPA (PGPA) in Antimony Resistance in Leishmania infantum Axenic and Intracellular Amastigotes

Fadili

Messier

Leprohon

et al. 2005

Antimicrob Agents Chemother

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133

127

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“…The neomycin phosphotransferase (NEO) gene was amplified from the plasmid pSP␣NEO␣ using primers 9 and 10 and then fused to the upstream and downstream DNA fragments of AdoMetT1 using PCR. The same strategy was used to generate the ZEO cassette; primer pairs 5-11 and 12-8 were used to amplify the upstream and downstream fragments of AdoMetT1, whereas primers 13 and 14 were used to amplify the ZEO gene from the pSP␣ZEO␣ vector (29).…”

Section: Methodsmentioning

confidence: 99%

“…Cross-resistant to Sinefungin-Folic acid (FA) and AdoMet are the onecarbon metabolic donors in cells, and previous reports have demonstrated that AdoMet metabolism is potentially changed in Leishmania cells resistant to the FA analogue MTX (29,36). Indeed, the L. tarentolae MTX1000.6 mutant is resistant to MTX (Fig.…”

Section: Leishmania Methotrexate-resistant Mutant Ismentioning

confidence: 99%

High Affinity S-Adenosylmethionine Plasma Membrane Transporter of Leishmania Is a Member of the Folate Biopterin Transporter (FBT) Family

Dridi¹,

Ouameur²,

Ouellette

2010

Journal of Biological Chemistry

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S-Adenosylmethionine (AdoMet) is an important methyl group donor that plays a central role in many essential biochemical processes. The parasite Leishmania can both synthesize and transport AdoMet. Leishmania cells resistant to the antifolate methotrexate due to a rearrangement in folate biopterin transporter (FBT) genes were cross-resistant to sinefungin, an AdoMet analogue. FBT gene rearrangements were also observed in Leishmania major cells selected for sinefungin resistance. One of the rearranged FBT genes corresponded to the main AdoMet transporter (AdoMetT1) of Leishmania as determined by gene transfection and gene inactivation experiments. AdoMetT1 was determined to be a high affinity plasma membrane transporter expressed constitutively throughout the growth phases of the parasite. Leishmania cells selected for resistance or naturally insensitive to sinefungin had lower expression of AdoMetT1. A new function in one carbon metabolism, also a pathway of interest for chemotherapeutic interventions, is described for a novel class of membrane proteins found in diverse organisms.The parasite Leishmania is distributed globally and causes a variety of clinical symptoms ranging from self-healing cutaneous lesions to visceral infections that are usually fatal if left untreated (1, 2). Current first-line chemotherapy against leishmaniasis relies on a rather limited arsenal of drugs, including pentavalent antimonials, liposomal amphotericin B, or miltefosine (2). These drugs are associated with side effects, high costs, and drug resistance, and therefore, the search for new drugs and targets to control this parasite is warranted (3, 4).S-Adenosylmethionine (AdoMet or SAM) 5 is an important biological sulfonium compound recognized as the universal methyl donor for methylation of lipids, proteins, nucleic acids, and xenobiotics in living cells (5). AdoMet is also a donor of propylamine groups for the synthesis of polyamines and participates in the reverse trans-sulfuration pathway where AdoMet is converted into cysteine and glutathione and in Leishmania in the spermidine-glutathione conjugate trypanothione (6, 7). AdoMet is also used as a source of methylene groups, amino groups, and ribosyl groups in the synthesis of fatty acids, biotin, and the modified nucleoside epoxyqueusine of tRNAs (reviewed in Ref. 8).Most cells are capable of synthesizing AdoMet from L-methionine and ATP, in a process requiring the enzyme methionine adenosyltransferase (MAT). The gene coding for this enzyme is present in most sequenced organisms (reviewed in Ref. 9). However, in some Rickettsia strains, the MAT gene is inactivated by a mutation (10), and in the fungus Pneumocystis carinii, no MAT activity has been detected (11). These organisms have the rare distinction of meeting their AdoMet needs by importing it (12). The Rickettsia transporter is part of the drug/ metabolite transporter superfamily (10), and the identity of the Pneumocystis transporter is not known. Transport of AdoMet across the plasma membrane does not occur to a signifi...

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“…Fifteen protein species, encoded by 14 genes, could be identified; interestingly some protein species were only present in one resistant strain but not the other, e.g. pteridine reductase 1 and argininosuccinate synthase, in one, and acyl-coa-dehydrogenase in the other strain 46 . Over-expression of argininosuccinate synthase and acyl-coa-dehydrogenase was the result of gene locus amplification events of genes in close structural proximity linked to methotrexate resistance pteridine reductase 1 and DHFR-TS, respectively.…”

Section: Phosphoproteomics and Parasite Differentiationmentioning

confidence: 99%

“…Metabolomics on the other hand will support future proteomic studies and as an outcome novel drug targets are likely to be identified. Proteomic studies will also be useful to study further drug resistance in the field as it has already been shown to identify proteins 26,46,52,54 .…”

Section: Gel Free Versus Gel Based Proteomicsmentioning

confidence: 99%

Contribution of proteomics of Leishmania spp. to the understanding of differentiation, drug resistance mechanisms, vaccine and drug development

Paape

Aebischer

2011

Journal of Proteomics

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:Leishmania spp., protozoan parasites with a digenetic life cycle, cause a spectrum of diseases in humans. Recently several Leishmania spp. have been sequenced which significantly boosted the number and quality of proteomic studies conducted. Here a historic review will summarize work of the pre-genomic era and then focus on studies after genome information became available. Firstly works comparing the different life cycle stages, in order to identify stage specific proteins, will be discussed. Identifying of post-translational modifications by proteomics especially phosphorylation events will be discussed. Further the contribution of proteomics to the understanding of the molecular mechanism of drug resistance and the investigation of immunogenic proteins for the identification of vaccine candidates will be summarized. Approaches of how potentially secreted proteins were identified are discussed. So far 30 -35% of the total predicted proteome of Leishmania spp. has been identified. This comprises mainly the abundant proteins, therefore the last section will look into technological approaches how this coverage may be increased and what the gel-free and gel-based proteomics have to offer will be compared.

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Differential Protein Expression Analysis of Leishmania major Reveals Novel Roles for Methionine Adenosyltransferase and S-Adenosylmethionine in Methotrexate Resistance

Cited by 63 publications

References 51 publications

Role of the ABC Transporter MRPA (PGPA) in Antimony Resistance in Leishmania infantum Axenic and Intracellular Amastigotes

Role of the ABC Transporter MRPA (PGPA) in Antimony Resistance in Leishmania infantum Axenic and Intracellular Amastigotes

High Affinity S-Adenosylmethionine Plasma Membrane Transporter of Leishmania Is a Member of the Folate Biopterin Transporter (FBT) Family

Contribution of proteomics of Leishmania spp. to the understanding of differentiation, drug resistance mechanisms, vaccine and drug development

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