Role of Efflux Pumps and Intracellular Thiols in Natural Antimony Resistant Isolates of Leishmania donovani

BackgroundIn view of the recent upsurge in the phenomenon of therapeutic failure, drug resistance in Leishmania, developed under natural field conditions, has become a great concern yet little understood. Accordingly, the study of determinants of antimony resistance is urgently warranted. Efflux transporters have been reported in Leishmania but their role in clinical resistance is still unknown. The present study was designed to elucidate the mechanism of natural antimony resistance in L. donovani field isolates by analyzing the functionality of efflux pump(s) and expression profiles of known genes involved in transport and thiol based redox metabolism Methodology/Principal FindingsWe selected 7 clinical isolates (2 sensitive and 5 resistant) in addition to laboratory sensitive reference and SbIII resistant mutant strains for the present study. Functional characterization using flow cytometry identified efflux pumps that transported substrates of both P-gp and MRPA and were inhibited by the calmodulin antagonist trifluoperazine. For the first time, verapamil sensitive efflux pumps for rhodamine 123 were observed in L. donovani that were differentially active in resistant isolates. RT-PCR confirmed the over-expression of MRPA in isolates with high resistance index only. Resistant isolates also exhibited consistent down regulation of AQP1 and elevated intracellular thiol levels which were accompanied with increased expression of ODC and TR genes. Interestingly, γ-GCS is not implicated in clinical resistance in L. donovani isolates. Conclusions/SignificanceHere we demonstrate for the first time, the role of P-gp type plasma membrane efflux transporter(s) in antimony resistance in L. donovani field isolates. Further, decreased levels of AQP1 and elevated thiols levels have emerged as biomarkers for clinical resistance.背景 鉴于近年来治疗失败现象愈发频发，自然环境中产生的利什曼原虫（Leishmania）耐药性已成为备受关注却鲜有阐明的难题，因此亟需开展锑剂耐药决定因素的相关研究。目前虽已有利什曼原虫外排转运蛋白（efflux transporters）的相关报道，但其在临床耐药中的作用仍未明确。本研究旨在通过分析外排泵的功能特性，以及参与转运过程与巯基氧化还原代谢（thiol based redox metabolism）的已知基因的表达谱，阐明杜氏利什曼原虫（L. donovani）野外分离株的天然锑剂耐药机制。方法/主要结果 本研究共纳入7株临床分离株（2株敏感株、5株耐药株），同时设置实验室敏感参考株与三价锑（SbIII）耐药突变株作为对照。通过流式细胞术进行功能鉴定，结果发现可转运P-糖蛋白（P-gp）与多药耐药相关蛋白A（MRPA）底物的外排泵，且该类外排泵可被钙调蛋白拮抗剂三氟拉嗪（trifluoperazine）抑制。本研究首次在杜氏利什曼原虫中观察到对罗丹明123（rhodamine 123）具有底物特异性的维拉帕米（verapamil）敏感型外排泵，且该类外排泵在耐药分离株中活性存在差异。逆转录聚合酶链反应（RT-PCR）结果证实，仅在高耐药指数的分离株中存在MRPA的过表达现象。耐药分离株同时表现出水通道蛋白1（AQP1）的持续下调，以及细胞内巯基水平升高，后者伴随鸟氨酸脱羧酶（ODC）与硫氧还蛋白还原酶（TR）基因表达的上调。值得注意的是，γ-谷氨酰半胱氨酸合成酶（γ-GCS）并未参与杜氏利什曼原虫临床分离株的耐药过程。结论与意义 本研究首次证实了P-糖蛋白型质膜外排转运蛋白在杜氏利什曼原虫野外分离株的锑剂耐药过程中的作用。此外，水通道蛋白1（AQP1）表达下调与细胞内巯基水平升高可作为临床耐药的生物标志物。