Abstract：
Improving the efficacy of existing antibiotics is significant for combatting antibiotic resistance that poses a major threat to human health. Carbonyl cyanide m-chlorophenylhydrazine (CCCP), a well-known protonophore for dissipating proton motive force (PMF), has been widely used to block the PMF-dependent uptake of aminoglycoside antibiotics and thus suppress aminoglycoside lethality. Here, we report that CCCP and its functional analog FCCP, but not other types of protonophores, unprecedently potentiate aminoglycosides (e.g., tobramycin and gentamicin) by 3-4 orders of magnitude killing of Escherichia coli, Staphylococcus aureus, Shigella flexneri, and Vibrio alginolyticus cells in stationary phase but not these cells in exponential phase nor other 12 bacterial species we examined. Overall, the effect of CCCP on aminoglycoside lethality undergoes a gradual transition from suppression against E. coli exponential-phase cells to potentiation against late stationary-phase cells, with the cell growth status and culture medium being crucial. Consistently, disturbance of the PMF by changing transmembrane proton gradient (ΔpH) or electric potential (ΔΨ) also potentiates tobramycin. Nevertheless, CCCP neither increases the intracellular concentration of tobramycin nor decreases the MIC of the antibiotic, thus excluding that CCCP acts as an efflux pump inhibitor to potentiate aminoglycosides. Rather, we show that the combined treatment dramatically enhances the cellular level of hydroxyl radical under both aerobic and anaerobic culturing conditions, under which the antioxidant N-acetyl cysteine fully suppresses both hydroxyl radical accumulation and cell death. Together, these findings open a new avenue to develop certain protonophores as aminoglycoside adjuvants against pathogens in stationary phase and also illustrate an essential role of hydroxyl radical in aminoglycoside lethality regardless of aerobic respiration.
摘要：
提高现有抗生素的功效对于对抗对人类健康构成重大威胁的抗生素抗性具有重要意义。羰基氰化物间氯苯肼(CCCP),一种众所周知的质子载体，用于耗散质子动力(PMF)，已被广泛用于阻断PMF依赖性的氨基糖苷类抗生素的摄取,从而抑制氨基糖苷类抗生素的致死性。这里,我们报告CCCP及其功能模拟FCCP，但不是其他类型的质子，前所未有地增强氨基糖苷类(例如，妥布霉素和庆大霉素）通过3-4个数量级杀死大肠杆菌，金黄色葡萄球菌,福氏志贺氏菌,和静止期的溶藻弧菌细胞，而不是指数期的这些细胞，也不是我们检查的其他12种细菌。总的来说,CCCP对氨基糖苷致死率的影响经历了从抑制大肠杆菌指数期细胞到增强晚期静止期细胞的逐渐转变，细胞生长状态和培养基至关重要。始终如一，通过改变跨膜质子梯度（ΔpH）或电势（ΔkW）来干扰PMF也会增强妥布霉素。然而，CCCP既不增加妥布霉素的细胞内浓度，也不降低抗生素的MIC，因此排除了CCCP作为外排泵抑制剂来增强氨基糖苷类。相反，我们表明，在好氧和厌氧培养条件下，联合处理显着提高了细胞的羟基自由基水平，抗氧化剂N-乙酰半胱氨酸完全抑制羟基自由基积累和细胞死亡。一起,这些发现为开发某些原端细胞作为氨基糖苷类佐剂在静止期对抗病原体开辟了新的途径，也说明了羟自由基在氨基糖苷类致死性中的重要作用，无论有氧呼吸如何。
