Elevated levels of polyamines and trypanothione resulting from overexpression of the ornithine decarboxylase gene in arsenite‐resistant Leishmania

Abstract

SummaryThe levels of trypanothione, a glutathione±spermidine conjugate, are increased in the protozoan parasite Leishmania selected for resistance to the heavy metal arsenite. The levels of putrescine and spermidine were increased in resistant mutants. This increase is mediated by overexpression of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis. Gene overexpression is generally mediated by gene ampli®cation in Leishmania but, here, the mRNA and the enzymatic activity of ODC are increased without gene ampli®cation. This RNA overexpression is stable when cells are grown in the absence of the drug and does not result from gene rearrangements or from an increased rate of RNA synthesis. Transient transfections suggest that mutations in the revertant cells contribute to these elevated levels of RNA. Stable transfection of the ODC gene increases the level of trypanothione, which can contribute to arsenite resistance. In addition to ODC overexpression, the gene for the ABC transporter PGPA is ampli®ed in the mutants. The co-transfection of the ODC and PGPA genes confers resistance in a synergistic fashion in partial revertants, also suggesting that PGPA recognizes metals conjugated to trypanothione.

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

“…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 Table II.…”

Dual Action of Antimonial Drugs on Thiol Redox Metabolism in the Human Pathogen Leishmania donovani

“…A considerable body of evidence implicates trypanothione and glutathione (␥-L-glutamyl-L-cysteinylglycine) in the efflux-mediated detoxification of Sb III and other heavy metals in the laboratory-induced resistant lines of Leishmania (18,19). Overexpression of ␥-glutamylcysteine synthetase (20) and ornithine decarboxylase (21) (the rate-limiting steps in the biosynthesis of the respective GSH and spermidine moieties of T[SH] 2 ) and overexpression of PgpA (20,22) (an intracellular metal-thiol transporter) have all been demonstrated to play a role in resistance. However, there are no reports implicating these effects in the mode of action of Sb III .…”

Regulatory mechanisms of Leishmania Aquaglyceroporin AQP1

“…Ornithine decarboxylase (ODC) and γ-glutamylcysteine synthetase (γ-GCS) are the two rate-limiting enzymes present in the polyamine and glutathione biosynthetic pathways, respectively (Haimeur et al, 1999).…”

“…Reversion of metal resistance can be achieved by downregulation of TSH levels by specific γ-GCS or ODC inhibitors, such as L-buthionine-(SR)-sulphoxime (BSO) and DL-α-difluor-o-methylornithine (DFMO), respectively (Haimeur et al, 1999). Grondin et al (1993 reported an increase in thiol levels as a result of an overexpression of either ODC or γ-GCS in L. tarentolae wild-type cells.…”

“…In addition, the overexpression of these specific inhibitors was almost equivalent to resistant mutants with the difference that the cells did not show arsenite resistance. Whereas, when ODC or γ--GCS was cotransfected with MRPA in wild type cells, arsenite resistance was observed (Haimeur et al, 1999;. The acquired resistance was reversed when transfectants were treated with BSO-a thiol depletory (El Fadili et al, 2005).…”