title: Comparative proteomics profiling of a gentamicin-attenuated Leishmania infantum cell line identifies key changes in parasite thiol-redox metabolism.
abstract: We have previously described an attenuated line of Leishmania infantum (H-line), selected by culturing promastigotes in vitro in the presence of gentamicin. To elucidate the molecular basis for this attenuation, we undertook a comparative proteomic analysis using multiplex 2-dimensional (2D) difference gel electrophoresis. Eighteen proteins that showed significant and reproducible changes in expression were identified. Many of these were components of the thiol-redox control system in Leishmania and this observation, validated by Western blot, prompted us to investigate the sensitivity of the attenuated line to oxidative stress. The attenuated line was found to be significantly more susceptible to hydrogen peroxide, a change which may explain the loss of virulence. In a direct assay of trypanothione-dependent peroxidase activity, hydrogen peroxide metabolism in the H-line was significantly lower than in wild type. Furthermore, trypanothione reductase activity was significantly lower in the H-line, suggesting that gentamicin selection may result in pleiotropic affects on thiol metabolism in Leishmania. A putative RNA-binding protein was very strongly up-regulated in the attenuated line, suggesting a possible target for gentamicin in Leishmania.
authors.affiliation: Leishmaniasis Research Center, Kerman University of Medical Sciences, Kerman, Iran.
authors.forename: Hamid
authors.initials: H
authors.lastname: Daneshvar
authors.forename: Susan
authors.initials: S
authors.lastname: Wyllie
authors.forename: Stephen
authors.initials: S
authors.lastname: Phillips
authors.forename: Paul
authors.initials: P
authors.lastname: Hagan
authors.forename: Richard
authors.initials: R
authors.lastname: Burchmore
mesh_terms: D005786:Gene Expression Regulation / Q000187:drug effects*; D005839:Gentamicins / Q000494:pharmacology*; D006861:Hydrogen Peroxide / Q000378:metabolism / Q000494:pharmacology; D018314:Leishmania infantum / Q000235:genetics / Q000378:metabolism*; D016877:Oxidants / Q000378:metabolism / Q000494:pharmacology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D040901:Proteomics; D015800:Protozoan Proteins / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D005839:Gentamicins; D016877:Oxidants; D011500:Protein Synthesis Inhibitors; D015800:Protozoan Proteins; D006861:Hydrogen Peroxide