title: Trypanothione overproduction and resistance to antimonials and arsenicals in Leishmania.
abstract: Leishmania resistant to arsenicals and antimonials extrude arsenite. Previous results of arsenite uptake into plasma membrane-enriched vesicles suggested that the transported species is a thiol adduct of arsenite. In this paper, we demonstrate that promastigotes of arsenite-resistant Leishmania tarentolae have increased levels of intracellular thiols. High-pressure liquid chromatography of the total thiols showed that a single peak of material was elevated almost 40-fold. The major species in this peak was identified by matrix-assisted laser desorption/ionization mass spectrometry as N1,N8-bis-(glutathionyl)spermidine (trypanothione). The trypanothione adduct of arsenite was effectively transported by the As-thiol pump. No difference in pump activity was observed in wild type and mutants. A model for drug resistance is proposed in which Sb(V)/As(V)-containing compounds, including the antileishmanial drug Pentostam, are reduced intracellularly to Sb(III)/As(III), conjugated to trypanothione, and extruded by the As-thiol pump. The rate-limiting step in resistance is proposed to be formation of the metalloid-thiol pump substrates, so that increased synthesis of trypanothione produces resistance. Increased synthesis of the substrate rather than an increase in the number of pump molecules is a novel mechanism for drug resistance.
authors.affiliation: Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, MI 48201, USA.
authors.forename: R
authors.initials: R
authors.lastname: Mukhopadhyay
authors.forename: S
authors.initials: S
authors.lastname: Dey
authors.forename: N
authors.initials: N
authors.lastname: Xu
authors.forename: D
authors.initials: D
authors.lastname: Gage
authors.forename: J
authors.initials: J
authors.lastname: Lightbody
authors.forename: M
authors.initials: M
authors.lastname: Ouellette
authors.forename: B P
authors.initials: BP
authors.lastname: Rosen
mesh_terms: D000818:Animals; D000967:Antimony Sodium Gluconate / Q000633:toxicity*; D000981:Antiprotozoal Agents / Q000633:toxicity*; D020261:Arsenic Poisoning; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000378:metabolism; D004351:Drug Resistance / Q000235:genetics; D005978:Glutathione / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000378:metabolism; D007891:Leishmania / Q000187:drug effects / Q000378:metabolism*; D008954:Models, Biological; D009154:Mutation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013095:Spermidine / Q000031:analogs & derivatives* / Q000096:biosynthesis
chemical_list: D000981:Antiprotozoal Agents; C044809:trypanothione; D005978:Glutathione; D003545:Cysteine; D013095:Spermidine; D000967:Antimony Sodium Gluconate