Metalloid transporters and drug resistance in Leishmania

利什曼原虫的类金属转运蛋白和耐药性

基本信息

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
项目类别：
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

项目摘要

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寄生虫感染，影响全世界近 1200 万人。含五价锑的药物 Pentostam 和 Glucantime 是治疗这种疾病的一线药物。我们假设药物由巨噬细胞积累，巨噬细胞将 Sb(V) 还原为 Sb(III)（药物的活性形式）。然后，Sb(III) 被寄生虫的细胞内无鞭毛体形式吸收，并在那里发挥作用。要完全了解它们的作用，需要了解这些药物的作用途径。在细菌、酵母和哺乳动物中，水甘油孔蛋白通道已被证明可转运 As(III) 和 Sb(III)。我们最近在塔伦托利什曼原虫和硕大利什曼原虫中分别鉴定了水甘油孔蛋白同源物 LtAQPI 和 LmAQPI 的基因。野外分离株经常报道耐药性，临床耐药性是治疗该疾病的主要障碍。耐药性可以通过药物摄取系统的突变或下调而产生。当过度表达时，LmAQPI 在体外使野生型和耐药利什曼原虫前鞭毛体对 Sb(III) 和 As(III) 敏感。 LmAQPI 还使来自印度患者的对五价锑无反应的抗场分离株重新变得敏感。 Sb(III) 和 As(III) 的细胞内高积累被证明是敏感性的原因。具体目标包括： 1) 水甘油孔蛋白的表征和其他转运蛋白的鉴定：LmAQPI 将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。该通道也将被定位，并且与类金属吸收相关的残留物将被识别。 LmAQPI 的其他同源物将通过 RT-PCR 和功能互补来鉴定。 2)水甘油孔蛋白敲除的产生：将在L.major、Linfantum和L.tarentolae中产生敲除。 3)巨噬细胞中AQPI的差异调节和无鞭毛体的药物敏感性：将在体外研究LmAQPI的差异调节。将在易感小鼠体内和体外检查过表达 LmAQPI 的无鞭毛体的药物敏感性。 4) AQPI在实验室突变体和野外分离株中的表达及其与药物摄取和耐药性的相关性。 LmAQPI 的表达将通过蛋白质印迹分析进行监测并与抗性相关。将使用 ICP-MS 测定药物摄取。拟议的研究将极大地改善人类健康并推进寄生原生动物药物作用和耐药性领域的基础研究。