Trypanothione Overproduction and Resistance to Antimonials and Arsenicals in Leishmania

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 93; no. 19; pp. 10383 - 10387
• Main Authors: Mukhopadhyay, Rita, Dey, Saibal, Xu, Naxing, Gage, Douglas, Lightbody, James, Ouellette, Marc, Rosen, Barry P.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 17.09.1996; National Acad Sciences; National Academy of Sciences
• Subjects: Adducts; Animals; Antimony Sodium Gluconate - toxicity; Antiprotozoal Agents - toxicity; Arsenic compounds; Arsenic Poisoning; Arsenites; Cell lines; Chromatography, High Pressure Liquid; Cysteine - metabolism; Drug Resistance - genetics; Glutathione - analogs & derivatives; Glutathione - biosynthesis; Glutathione - metabolism; Infections; Leishmania - drug effects; Leishmania - metabolism; Leishmania tarentolae; Mass spectroscopy; Medical research; Microorganisms; Models, Biological; Mutation; Parasites; Parasitism; Promastigotes; Pumps; Sodium; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Spermidine - analogs & derivatives; Spermidine - biosynthesis; Thiols
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.93.19.10383

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 N$^{1}$,N$^{8}$-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 ratelimiting 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.