Mukhopadhyay R, Dey S, Xu N, Gage D, Lightbody J, Ouellette M, Rosen B P
Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, MI 48201, USA.
Proc Natl Acad Sci U S A. 1996 Sep 17;93(19):10383-7. 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 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.
对砷剂和锑剂耐药的利什曼原虫会排出亚砷酸盐。先前关于亚砷酸盐摄取到富含质膜的囊泡中的结果表明，转运的物质是亚砷酸盐的硫醇加合物。在本文中，我们证明对亚砷酸盐耐药的塔兰托拉利什曼原虫前鞭毛体的细胞内硫醇水平升高。对总硫醇进行高压液相色谱分析表明，一种物质的单峰升高了近40倍。通过基质辅助激光解吸/电离质谱法鉴定出该峰中的主要物质为N1,N8-双（谷胱甘肽基）亚精胺（锥虫硫醇）。亚砷酸盐的锥虫硫醇加合物可通过砷-硫醇泵有效转运。在野生型和突变体中未观察到泵活性的差异。提出了一种耐药模型，其中含Sb(V)/As(V)的化合物，包括抗利什曼原虫药物喷他脒，在细胞内被还原为Sb(III)/As(III)，与锥虫硫醇结合，并由砷-硫醇泵排出。耐药的限速步骤被认为是类金属-硫醇泵底物的形成，因此锥虫硫醇合成增加会产生耐药性。底物合成增加而非泵分子数量增加是一种新的耐药机制。
