Metalloid transporters and drug resistance in Leishmania

利什曼原虫的类金属转运蛋白和耐药性

• 批准号: 7092097
• 负责人: RITA MUKHOPADHYAY
• 金额: $ 21.4万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-08 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7092097
• 关键词: Leishmania; antimony; antiprotozoal agents; drug resistance; laboratory mouse; macrophage; membrane permeability; membrane transport proteins; metal metabolism; pharmacokinetics; polymerase chain reaction; protozoal genetics; tin; tissue /cell culture; water channel; western blottings

DESCRIPTION (provided by the applicant): Leishmaniasis is a protozoan parasitic infection affecting almost 12 million people worldwide. The pentavalent antimony containing drugs, Pentostam and Glucantime are the first line of treatment against this disease. We hypothesized that the drugs are accumulated by macrophages, which reduce the Sb(V) to Sb(lll), the active form of the drug. Sb(lll) is then taken up by the intracellular amastigote form of the parasite, where it exerts its action. A complete understanding of their action requires knowledge of the pathway for these drugs. In bacteria, yeast and mammals aquaglyceroporin channels have been shown to transport As(lll) and Sb(lll). We have recently identified the genes for an aquaglyceroporin homologue, LtAQPI and LmAQPI, in Leishmania tarentolae and Leishmania major, respectively. Drug resistance has frequently been reported in field isolates, and clinical resistance is a major impediment to the treatment of this disease. Drug resistance can arise through mutation or down regulation of drug uptake system(s). When over expressed, LmAQPI sensitizes both wild type and drug resistant Leishmania promastigotes to both Sb(lll) and As(lll) in vitro. LmAQPI also re-sensitized a field resistant isolate from an Indian patient unresponsive to pentavalent antimonials. High intracellular accumulation of both Sb(lll) and As(lll) was demonstrated to be responsible for sensitivity. Specific aims include: 1) Characterization of aquaglyceroporin and Identification of additional transporters: LmAQPI will be characterized by determining its substrate specificity in oocyte or Leishmania whole cells. The channel will also be localized and residues involved in metalloid uptake will be identified. Additional homologues of LmAQPI will be identified by RT-PCR and functional complementation. 2) Generation of aquaglyceroporin knockouts: The knockouts will be created in L. major, L infantum and L. tarentolae. 3) Differential regulation ofAQPI and drug sensitivity of amastigotes in macrophages: Differential regulation of LmAQPI will be studied in vitro. Drug sensitivity of amastigotes over expressing LmAQPI will be examined in vitro and in vivo in susceptible mice. 4) Expression of AQPI in laboratory mutants and field isolates and correlation with drug uptake and resistance. Expression of LmAQPI will be monitored by western blot analysis and correlated with resistance. Drug uptake will be determined by using ICP-MS. The proposed research will greatly improve human health and advance basic research in the field of drug action and resistance in parasitic protozoa.

描述（由申请人提供）：利什曼病是一种原生动物的寄生虫感染，影响了全世界近1200万人。含有药物的五体矩阵，五骨和葡萄糖是针对该疾病的第一道治疗方法。我们假设这些药物是由巨噬细胞积累的，巨噬细胞将SB（V）降低至SB（LLL），该药物的活性形式。然后，SB（LLL）通过寄生虫的细胞内膜片形式来占用其作用。对他们的行动的完全理解需要了解这些药物的途径。在细菌中，酵母和哺乳动物的水准蛋白通道已显示为（LLL）和SB（LLL）。我们最近分别在利什曼尼亚·塔伦托拉（Leishmania Tarentolae）和利什曼尼亚（Leishmania）大满以分别确定了水甘露糖蛋白同源物，ltaqpi和lmaqpi的基因。在现场分离株中经常报道耐药性，临床耐药性是治疗该疾病的主要障碍。可以通过突变或下调药物摄取系统的耐药性。当表达过度时，LMAQPI在体外将野生型和抗药性利什曼原虫同时敏感到SB（LLL）和AS（LLL）。 LMAQPI还将来自印度患者反应迟钝的抗衡剂的抗野战力分离物重新敏感。 SB（LLL）和AS（LLL）的高细胞内积累被证明是敏感性的原因。具体目的包括：1）表征水准蛋白的表征和其他转运蛋白的识别：LMAQPI的特征是通过确定其在卵母细胞或利什曼原虫全细胞中的底物特异性来表征。该通道也将是本地化的，并且将确定参与金属摄取的残基。 LMAQPI的其他同源物将通过RT-PCR和功能互补识别。 2）产生水甘露甘露糖蛋白敲除：淘汰赛将在L. Major，L。Infantum和L. tarentolae中创建。 3）巨噬细胞中amastigotes的差异调节和药物敏感性：将在体外研究LMAQPI的差异调节。在易感小鼠的体外和体内将检查杂种片对表达LMAQPI的药物敏感性。 4）AQPI在实验室突变体和田间分离株中的表达以及与药物摄取和耐药性的相关性。 LMAQPI的表达将通过Western印迹分析监测，并与耐药性相关。药物摄取将通过使用ICP-MS确定。拟议的研究将大大改善人类健康，并在寄生原生动物的药物作用和抗药性领域提高基础研究。