Biochemistry Division, CSIR-Central Drug Research Institute, Jankipuram Extension, Sitapur Road, Lucknow 226031, India.
Global Biotechnology Resource Center, 145 Rosewood Drive, Streamwood, IL 60107, USA.
Acta Trop. 2019 Nov;199:105158. doi: 10.1016/j.actatropica.2019.105158. Epub 2019 Sep 3.
Visceral leishmaniasis (VL) is a neglected tropical disease caused by protozoan Leishmania donovani parasite which may be fatal if left untreated. While drug-sensitive parasites are able to live and multiply within the host macrophages, they develop resistance to drugs used against them for survival and multiplication in the infected patients undergoing routine treatment. Development of new agents devoid of such drug resistance potential is achievable by identifying new drug targets in the parasite. One such target is the key regulator of intracellular vesicular trafficking protein, RabGTPase which belongs to the Ras GTPase superfamily. We recently elucidated whole genome sequence (WGS) of L. donovani (clinical Indian isolate; BHU 1220, GenBank: AVPQ00000000.1) and identified Ldrab6 gene. We now provide experimental evidence for this gene's ability to impart drug-resistant phenotype to wild-type (sensitive) Leishmania upon transfection. trans-Dibenzalacetone (DBA), a synthetic analog of curcumin, was used to determine its antileishmanial activity in wild-type parasites and parasites transfected with Ldrab6 gene. Dose-response study showed that DBA had no effect on transfected parasites at 20 µg/mL dose, whereas wild-type promastigotes showed 50% inhibition (IC) at the same dose. This indicates the development of resistant mechanism in the transfected parasites due to enhancement of the copy number of Ldrab6 gene in L. donovani parasites. Flow cytometric analysis revealed elevated level of thiols in transfectants when compared to wild-type parasites treated with DBA. To assess the functional activity of multidrug resistance-associated protein (MRP) pump in transfectants, the accumulation of calcein, a known MRP pump substrate and probenecid, a known MRP pump regulator, were analyzed. The results indicate that Ldrab6 gene in Leishmania conferred resistance by the well-established mechanism of drug-thiol conjugation and sequestration by ABC transporter multidrug resistance-protein A (MRPA). Accordingly, Leishmania parasites transfected with Ldrab6 gene can be used as an experimental cell line for the screening of new lead molecules for their propensity to develop drug resistance.
内脏利什曼病（VL）是一种被忽视的热带病，由原生动物利什曼原虫寄生虫引起，如果不治疗可能致命。虽然对药物敏感的寄生虫能够在宿主巨噬细胞内生存和繁殖，但它们会产生抗药性，以在接受常规治疗的感染患者中生存和繁殖。通过在寄生虫中识别新的药物靶点，可以开发出没有这种耐药潜力的新药物。其中一个靶点是细胞内囊泡运输蛋白的关键调节剂 RabGTPase，它属于 Ras GTPase 超家族。我们最近阐明了利什曼原虫（临床印度分离株；BHU 1220，GenBank：APVQ00000000.1）的全基因组序列（WGS），并鉴定了 Ldrab6 基因。我们现在提供实验证据证明，该基因能够赋予野生型（敏感型）利什曼原虫转染后的耐药表型。反式二苯甲酰丙酮（DBA），姜黄素的合成类似物，用于确定其在野生型寄生虫和转染 Ldrab6 基因的寄生虫中的抗利什曼原虫活性。剂量反应研究表明，DBA 在 20μg/mL 剂量下对转染的寄生虫没有影响，而相同剂量的野生型前鞭毛体显示出 50%的抑制（IC）。这表明由于 L. donovani 寄生虫中 Ldrab6 基因的拷贝数增加，转染的寄生虫中出现了耐药机制。与用 DBA 处理的野生型寄生虫相比，流式细胞术分析显示转染子中的巯基水平升高。为了评估转染子中多药耐药相关蛋白（MRP）泵的功能活性，分析了 calcein 的积累，calcein 是一种已知的 MRP 泵底物和丙磺舒，一种已知的 MRP 泵调节剂。结果表明，利什曼原虫中的 Ldrab6 基因通过药物-巯基缀合和 ABC 转运蛋白多药耐药蛋白 A（MRPA）的隔离的既定机制赋予了耐药性。因此，转染 Ldrab6 基因的利什曼原虫可以用作实验细胞系，用于筛选具有耐药性发展倾向的新先导分子。
