Differential protein expression analysis of Leishmania major reveals novel roles for methionine adenosyltransferase and S-adenosylmethionine in methotrexate resistance.
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.
Drummelsmith
Jolyne
J
Infectious Diseases Research Centre, Laval University, Quebec City, Quebec G1V 4G2, Canada.
Girard
Isabelle
I
Trudel
Nathalie
N
Ouellette
Marc
M
D000818:Animals; D015152:Blotting, Northern; D015139:Blotting, Southern; D004351:Drug Resistance / Q000235:genetics*; D015180:Electrophoresis, Gel, Two-Dimensional; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D006911:Hydroxyprostaglandin Dehydrogenases / Q000235:genetics; D018320:Leishmania major / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D008716:Methionine Adenosyltransferase / Q000235:genetics / Q000502:physiology*; D008727:Methotrexate / Q000494:pharmacology; D009154:Mutation; D010751:Phosphopyruvate Hydratase / Q000235:genetics; D016133:Polymerase Chain Reaction; D015800:Protozoan Proteins / Q000235:genetics*; D012436:S-Adenosylmethionine / Q000502:physiology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D015800:Protozoan Proteins; D012436:S-Adenosylmethionine; D006911:Hydroxyprostaglandin Dehydrogenases; C031051:prostaglandin-F synthase; D008716:Methionine Adenosyltransferase; D010751:Phosphopyruvate Hydratase; D008727:Methotrexate