Shan Yue, Brown Gandt Autumn, Rowe Sarah E, Deisinger Julia P, Conlon Brian P, Lewis Kim
Department of Biology, Antimicrobial Discovery Center, Northeastern University, Boston, Massachusetts, USA.
Department of Biology, Antimicrobial Discovery Center, Northeastern University, Boston, Massachusetts, USA
mBio. 2017 Feb 7;8(1):e02267-16. doi: 10.1128/mBio.02267-16.
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.
持留菌是形成对抗生素耐受的细胞亚群的休眠变体。持留菌在很大程度上导致了慢性感染对治疗的顽固性。在大肠杆菌中，一种被广泛接受的持留菌形成模型认为，ppGpp的随机积累会导致Lon蛋白酶的激活，该蛋白酶会降解抗毒素；活性毒素随后会抑制翻译，从而产生休眠的、耐药物的持留菌。我们发现，各种应激会诱导毒素-抗毒素（TA）的表达，但TA的诱导不一定会增加持留菌的数量。16S rRNA启动子rrnB P1被认为是持留菌报告基因和受ppGpp调节的毒素激活指标。通过荧光激活细胞分选（FACS），我们证实了rrnB P1-gfp低荧光细胞部分中持留菌的富集；然而，这与毒素-抗毒素无关。rrnB P1受ppGpp和ATP共同调节。我们表明，rrnB P1可以在relA/spoT缺失背景下报告持留菌，这表明rrnB P1是一个对ATP做出反应的持留菌标记。与这一发现一致，通过砷酸盐处理降低ATP水平会导致药物耐受性。降低ATP会减缓翻译，并在氟喹诺酮治疗时防止DNA双链断裂的形成。我们得出结论，ATP水平的变化通过降低抗生素靶点的活性导致持留菌的形成。
持留菌是对抗生素耐受的细胞亚群，导致慢性感染的顽固性。我们目前对持留菌形成的理解主要基于对大肠杆菌的研究。ppGpp对毒素-抗毒素系统的激活已成为一种被广泛接受的持留菌形成模型。在这项研究中，我们发现应激诱导的mRNA干扰酶型毒素的激活不一定会导致持留菌的形成。我们还发现，持留菌标记rrnB P1报告持留菌细胞是因为它检测到细胞ATP水平的下降。与此一致，降低ATP水平会降低抗生素靶点的活性，从而导致持留菌的形成。我们得出结论，ATP的随机变化是持留菌形成的主要机制。ATP的减少为持留菌的药物耐受性提供了一个令人满意的解释，因为杀菌抗生素通过破坏能量依赖靶点起作用。
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