ATP-Dependent Persister Formation in Escherichia coli.
ATP-Dependent Persister Formation in Escherichia coli.
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DOI：
10.1128/mbio.02267-16
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发表时间：
2017-02-07
期刊：
影响因子：
6.4
通讯作者：
Lewis K
中科院分区：
文献类型：
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作者：
Shan Y;Brown Gandt A;Rowe SE;Deisinger JP;Conlon BP;Lewis K
持续存在是休眠变体，形成对抗生素具有耐受性的细胞亚群。慢性感染不接受治疗的主要原因是持续存在的人。在大肠杆菌中，一种广泛接受的持久细胞形成模型认为，ppGpp 的随机积累会导致 Lon 蛋白酶的激活，从而降解抗毒素。然后活性毒素抑制翻译，导致休眠的、耐药的持续存在。我们发现各种应激都会诱导毒素-抗毒素（TA）表达，但诱导 TA 并不一定会增加持久性。 16S rRNA 启动子 rrnB P1 被认为是持久报告基因和受 ppGpp 调节的毒素激活的指标。使用荧光激活细胞分选 (FACS)，我们确认了 rrnB P1-gfp 暗细胞部分中持续细胞的富集；然而，这与毒素-抗毒素无关。 rrnB P1 受 ppGpp 和 ATP 共同调节。我们发现 rrnB P1 可以在 relA/spoT 缺失背景中报告持久细胞，这表明 rrnB P1 是响应 ATP 的持久细胞标记。与这一发现一致，通过砷酸盐治疗降低 ATP 水平会导致药物耐受。降低 ATP 会减慢翻译速度并防止氟喹诺酮治疗后 DNA 双链断裂的形成。我们得出的结论是，ATP 水平的变化通过降低抗生素靶标的活性导致持久性形成。持续细胞是抗生素耐受细胞的亚群，导致慢性感染的顽固性。我们目前对持久细胞形成的理解主要基于对大肠杆菌的研究。 ppGpp 激活毒素-抗毒素系统已成为广泛接受的持久细胞形成模型。在这项研究中，我们发现应激诱导的 mRNA 干扰酶型毒素激活并不一定会导致持久细胞的形成。我们还发现持久细胞标记 rrnB P1 报告持久细胞，因为它检测到细胞 ATP 水平下降。与此一致的是，降低 ATP 水平会降低抗生素靶点活性，从而导致持久性形成。我们得出结论，ATP 的随机变化是持久细胞形成的主要机制。 ATP 的减少为持久者的药物耐受性提供了令人满意的解释，因为杀菌抗生素通过破坏能量依赖性目标来发挥作用。
Persisters are dormant variants that form a subpopulation of cells tolerant to antibiotics. Persisters are largely responsible for the recalcitrance of chronic infections to therapy. In Escherichia coli, one widely accepted model of persister formation holds that stochastic accumulation of ppGpp causes activation of the Lon protease that degrades antitoxins; active toxins then inhibit translation, resulting in dormant, drug-tolerant persisters. We found that various stresses induce toxin-antitoxin (TA) expression but that induction of TAs does not necessarily increase persisters. The 16S rRNA promoter rrnB P1 was proposed to be a persister reporter and an indicator of toxin activation regulated by ppGpp. Using fluorescence-activated cell sorting (FACS), we confirmed the enrichment for persisters in the fraction of rrnB P1-gfp dim cells; however, this is independent of toxin-antitoxins. rrnB P1 is coregulated by ppGpp and ATP. We show that rrnB P1 can report persisters in a relA/spoT deletion background, suggesting that rrnB P1 is a persister marker responding to ATP. Consistent with this finding, decreasing the level of ATP by arsenate treatment causes drug tolerance. Lowering ATP slows translation and prevents the formation of DNA double-strand breaks upon fluoroquinolone treatment. We conclude that variation in ATP levels leads to persister formation by decreasing the activity of antibiotic targets. Persisters are a subpopulation of antibiotic-tolerant cells responsible for the recalcitrance of chronic infections. Our current understanding of persister formation is primarily based on studies of E. coli. The activation of toxin-antitoxin systems by ppGpp has become a widely accepted model for persister formation. In this study, we found that stress-induced activation of mRNA interferase-type toxins does not necessarily cause persister formation. We also found that the persister marker rrnB P1 reports persister cells because it detects a drop in cellular ATP levels. Consistent with this, lowering the ATP level decreases antibiotic target activity and, thus, leads to persister formation. We conclude that stochastic variation in ATP is the main mechanism of persister formation. A decrease in ATP provides a satisfactory explanation for the drug tolerance of persisters, since bactericidal antibiotics act by corrupting energy-dependent targets.