Vacuolar ATPase depletion affects mitochondrial ATPase function, kinetoplast dependency, and drug sensitivity in trypanosomes
Vacuolar ATPase depletion affects mitochondrial ATPase function, kinetoplast dependency, and drug sensitivity in trypanosomes
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DOI：
10.1073/pnas.1505411112
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发表时间：
2015-07-21
影响因子：
11.1
通讯作者：
Horn, David
中科院分区：
文献类型：
--
作者：
Baker, Nicola;Hamilton, Graham;Horn, David
动质体寄生虫在人类和动物中引起致命的疾病。动基体本身含有线粒体基因组，由一个巨大的、复杂的DNA网络组成，也是一个重要的药物靶点。例如，Isometamidium是一种关键的兽药，在非洲锥虫的动基体中积累。观察到动基体独立性和异脒抗性，其中两个扇区F1 Fo-ATP合酶的F-1-γ-亚基中的某些突变允许线粒体膜电位的F-0-独立产生。为了进一步探索动基体生物学和耐药性，我们在非洲锥虫中筛选了一个基因组规模的RNA干扰文库，以了解异美他啶耐药机制。我们的筛选确定了14个V-ATP酶亚基和所有4个adaptin-3亚基，暗示了抗性中的酸性隔室缺陷; V-ATP酶酸化溶酶体和相关细胞器，而adaptin-3负责这些细胞器之间的运输。与耗尽的V-ATP酶或adaptin-3亚基的独立菌株的isometamidium耐药，和V-ATP酶的化学抑制phenocopies这种效果。虽然药物在动基体中的积累在V-ATP酶亚基耗尽后继续，但在这些细胞和耗尽适应素-3或内质网膜复合物亚基的细胞中，也在我们的筛选中鉴定出了吖啶诱导的动基体损失。与动质体可分配性一致，V-ATP酶缺陷细胞具有寡霉素抗性，表明ATP合酶解偶联和绕过正常的F-O-A6亚基要求;该亚基是唯一的动质体编码产物，最终需要在血流形式的锥虫中的活力。因此，我们描述了30个基因和3个蛋白质复合物与动质体依赖的增长。影响这些基因的突变可以解释运动障碍和多药耐药性的自然病例。我们的研究结果还揭示了潜在的保守通信之间的区室化的两个部门的旋转ATP酶。
Kinetoplastid parasites cause lethal diseases in humans and animals. The kinetoplast itself contains themitochondrial genome, comprising a huge, complex DNA network that is also an important drug target. Isometamidium, for example, is a key veterinary drug that accumulates in the kinetoplast in African trypanosomes. Kinetoplast independence and isometamidium resistance are observed where certain mutations in the F-1-gamma-subunit of the two-sector F1Fo-ATP synthase allow for F-o-independent generation of a mitochondrial membrane potential. To further explore kinetoplast biology and drug resistance, we screened a genome-scale RNA interference library in African trypanosomes for isometamidium resistance mechanisms. Our screen identified 14 V-ATPase subunits and all 4 adaptin-3 subunits, implicating acidic compartment defects in resistance; V-ATPase acidifies lysosomes and related organelles, whereas adaptin-3 is responsible for trafficking among these organelles. Independent strains with depleted V-ATPase or adaptin-3 subunits were isometamidium resistant, and chemical inhibition of the V-ATPase phenocopied this effect. While drug accumulation in the kinetoplast continued after V-ATPase subunit depletion, acriflavine-induced kinetoplast loss was specifically tolerated in these cells and in cells depleted for adaptin-3 or endoplasmic reticulum membrane complex subunits, also identified in our screen. Consistent with kinetoplast dispensability, V-ATPase defective cells were oligomycin resistant, suggesting ATP synthase uncoupling and bypass of the normal F-o-A6-subunit requirement; this subunit is the only kinetoplast-encoded product ultimately required for viability in bloodstream-form trypanosomes. Thus, we describe 30 genes and 3 protein complexes associated with kinetoplast-dependent growth. Mutations affecting these genes could explain natural cases of dyskinetoplasty and multidrug resistance. Our results also reveal potentially conserved communication between the compartmentalized two-sector rotary ATPases.