Abstract
Leishmania is a protozoan parasite responsible for significant morbidity and mortality worldwide. Few parasites have been subjected to proteomic analysis to date, but a genome sequencing project for Leishmania major is currently underway, making these studies possible. Here we present a high resolution proteome for L. major comprising almost 3700 spots, making it the most complete two-dimensional gel representation of a parasite proteome generated to date. We have identified a number of landmark proteins by mass spectrometry and show that several of these are valid for the related species Leishmania donovani infantum. We have also observed several forms and fragments of alpha- and beta-tubulins and show that the number and amount of these fragments increase with the age of the parasite culture. Trypanothione reductase (TRYR), which replaces glutathione reductase in trypanosomatid parasites, is an essential protein specific to these parasites and as such is under considerable scrutiny as a drug target. Two-dimensional gel analysis of a L. major strain overexpressing TRYR revealed increased amounts of five spots, all at the predicted molecular weight for TRYR and differing by 0.08 pH units in pI. Mass spectrometry identified four of these as TRYR, leading to the novel suggestion that it could be post-translationally modified. Finally quantitative comparative analysis of a methotrexate-resistant mutant of L. major generated in vitro found that a known primary resistance mediator, the pteridine reductase PTR1, was overexpressed. This constitutes the first proteomic analysis of drug resistance in a parasite and also the clearest identification of a primary drug resistance mechanism using this approach. Together these results provide a framework for further proteomic studies of Leishmania species and demonstrate that these tools are valuable for the essential study of potential drug targets and drug resistance mechanisms.
Highlights
Leishmania is a protozoan parasite responsible for significant morbidity and mortality worldwide
It is estimated that the Leishmania genome contains 8000 genes, and while numerous posttranslational modifications are known to exist in this organism, 146 Molecular & Cellular Proteomics 2.3
Our step was to identify a number of these spots, both to serve as landmark proteins and to determine the likelihood of obtaining identifications by MALDI-TOF MS, given the incomplete state of the genome sequencing project and the fact that few curated translations from the Leishmania genome project have been submitted to public databases. 62 spots of varying intensity were excised and digested with trypsin, and the resulting peptides were subjected to MALDI-TOF analysis
Summary
Cell Culture—L. major Friedlin, LV39, L. donovani infantum, and Leishmania donovani donovani were grown in M199 medium (Invitrogen) supplemented with 10% fetal calf serum (Wisent) and 5 g/ml hemin (ICN Biochemicals). Leishmania tarentolae TARII and L. major MHOM/IL/67/JERICHO II (50122) transfected with the trypanothione reductase (TRYR) gene [7] were grown in SDM-79 medium [8] supplemented with 10% fetal calf serum and hemin. Methotrexate (MTX, Sigma) or G418 (Invitrogen) was added. Samples were run on 18-cm Immobiline Dry Strips (pH 4.0 –5.0, 4.5–5.5, 5.0 – 6.0, 5.5– 6.7, and 6.0 –9.0; Amersham Biosciences) on an IPGphor isoelectric focusing system as recommended by the manufacturer with modifications as described in Ref. 9 for samples run on pH 6 –9 strips. 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 in equilibration buffer containing 25 mg/ml iodoac-
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