Department of Microbiology and Immunology, Otago School of Medical Sciences, University of Otago, Dunedin 9054, New Zealand.
Department of Molecular Cell Biology, Amsterdam Institute for Molecules, Medicines and Systems, VU University Amsterdam, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands.
J Antimicrob Chemother. 2015 Jul;70(7):2028-37. doi: 10.1093/jac/dkv054. Epub 2015 Mar 8.
It is not fully understood why inhibiting ATP synthesis in Mycobacterium species leads to death in non-replicating cells. We investigated the bactericidal mode of action of the anti-tubercular F1Fo-ATP synthase inhibitor bedaquiline (Sirturo™) in order to further understand the lethality of ATP synthase inhibition.
Mycobacterium smegmatis strains were used for all the experiments. Growth and survival during a bedaquiline challenge were performed in multiple media types. A time-course microarray was performed during initial bedaquiline challenge in minimal medium. Oxygen consumption and proton-motive force measurements were performed on whole cells and inverted membrane vesicles, respectively.
A killing of 3 log10 cfu/mL was achieved 4-fold more quickly in minimal medium (a glycerol carbon source) versus rich medium (LB with Tween 80) during bedaquiline challenge. Assessing the accelerated killing condition, we identified a transcriptional remodelling of metabolism that was consistent with respiratory dysfunction but inconsistent with ATP depletion. In glycerol-energized cell suspensions, bedaquiline caused an immediate 2.3-fold increase in oxygen consumption. Bedaquiline collapsed the transmembrane pH gradient, but not the membrane potential, in a dose-dependent manner. Both these effects were dependent on binding to the F1Fo-ATP synthase.
Challenge with bedaquiline results in an electroneutral uncoupling of respiration-driven ATP synthesis. This may be a determinant of the bactericidal effects of bedaquiline, while ATP depletion may be a determinant of its delayed onset of killing. We propose that bedaquiline binds to and perturbs the a-c subunit interface of the Fo, leading to futile proton cycling, which is known to be lethal to mycobacteria.
目前尚不完全清楚为什么抑制分枝杆菌属中的 ATP 合成会导致非复制细胞死亡。我们研究了抗结核 F1Fo-ATP 合酶抑制剂贝达喹啉（Sirturo™）的杀菌作用机制，以便进一步了解 ATP 合酶抑制的致死性。
所有实验均使用耻垢分枝杆菌菌株。在多种培养基类型中进行贝达喹啉挑战期间的生长和存活。在最小培养基中进行初步贝达喹啉挑战时进行了时间过程微阵列。在整个细胞和倒置膜囊泡上分别进行了氧消耗和质子动力测量。
在最小培养基（甘油碳源）中，贝达喹啉挑战时对数减少 3 个 CFU/mL 的杀菌速度比在富含培养基（含吐温 80 的 LB）中快 4 倍。在评估加速杀菌条件时，我们发现代谢的转录重塑与呼吸功能障碍一致，但与 ATP 耗竭不一致。在甘油供能的细胞悬浮液中，贝达喹啉会立即引起氧消耗增加 2.3 倍。贝达喹啉以剂量依赖性方式使跨膜 pH 梯度，但不使膜电位崩溃。这两种作用都依赖于与 F1Fo-ATP 合酶的结合。
贝达喹啉的挑战导致呼吸驱动的 ATP 合成的电中性解偶联。这可能是贝达喹啉杀菌作用的决定因素，而 ATP 耗竭可能是其延迟杀菌作用的决定因素。我们提出贝达喹啉结合并扰乱 Fo 的 a-c 亚基界面，导致无效质子循环，这已知对分枝杆菌具有致死性。
