Intracellular acidification is a hallmark of thymineless death in E. coli.
Intracellular acidification is a hallmark of thymineless death in E. coli.
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DOI：
10.1371/journal.pgen.1010456
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发表时间：
2022-10
期刊：
影响因子：
4.5
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中科院分区：
文献类型：
--
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胸苷饥饿导致细胞快速死亡。这种被称为无胸腺嘧啶死亡 (TLD) 的神秘过程是多种抗菌药物和抗肿瘤药物的潜在杀伤机制。尽管经过了数十年的调查，我们仍然缺乏对最终导致 TLD 的事件因果顺序的机械理解。在这里，我们使用了多种公正的方法来系统地确定大肠杆菌中 TLD 的遗传和调控基础。除了在先前涉及的途径中发现新基因之外，我们的研究还揭示了 TLD 中细胞内酸化的关键且先前未知的作用。我们观察到，细胞质 pH 值降低是不同遗传背景 TLD 的一个重要早期事件。此外，我们发现酸化是死亡过程中的一个因果事件，因为化学和遗传扰动会增加细胞内 pH 值，从而大大减少杀灭作用。我们还观察到接触抗生素庆大霉素后细胞内 pH 值下降，这表明细胞内酸化可能是其他抗生素杀菌作用的常见机制步骤。在大肠杆菌中发现了无胸腺嘧啶死亡，但这种杀伤现象是广泛存在的——从真核微生物到人类细胞。杀伤机制对于几种临床重要药物的作用方式至关重要，但导致细胞死亡的事件因果顺序仍知之甚少。我们采用了三种系统生物学方法来探究大肠杆菌无胸腺嘧啶死亡的潜在机制，并发现了数十种在以前未知的途径中调节存活的新基因。许多基因的功能表明 pH 稳态是一个关键因素。我们的详细研究表明，胸苷饥饿过程中会发生细胞内酸化，并且 pH 值的实验操作会对生存产生巨大影响。我们还观察到，增加细胞内 pH 值的遗传扰动会影响抗生素庆大霉素治疗后的存活率。事实上，接触庆大霉素会导致 pH 值下降，这表明酸化可能在其他抗生素的杀菌作用中发挥更广泛的作用。
Thymidine starvation causes rapid cell death. This enigmatic process known as thymineless death (TLD) is the underlying killing mechanism of diverse antimicrobial and antineoplastic drugs. Despite decades of investigation, we still lack a mechanistic understanding of the causal sequence of events that culminate in TLD. Here, we used a diverse set of unbiased approaches to systematically determine the genetic and regulatory underpinnings of TLD in Escherichia coli. In addition to discovering novel genes in previously implicated pathways, our studies revealed a critical and previously unknown role for intracellular acidification in TLD. We observed that a decrease in cytoplasmic pH is a robust early event in TLD across different genetic backgrounds. Furthermore, we show that acidification is a causal event in the death process, as chemical and genetic perturbations that increase intracellular pH substantially reduce killing. We also observe a decrease in intracellular pH in response to exposure to the antibiotic gentamicin, suggesting that intracellular acidification may be a common mechanistic step in the bactericidal effects of other antibiotics. Thymineless death was discovered in E. coli but the killing phenomenon is widespread—from eukaryotic microbes to human cells. The mechanism of killing is essential to the mode of action of several clinically important drugs, yet the causal sequence of events that lead to cell death remains poorly understood. We undertook three systems biology approaches to probe the underlying mechanisms of thymineless death in E. coli and discovered dozens of novel genes that modulate survival in previously unknown pathways. The function of many of the genes pointed to pH homeostasis as a critical factor. Our detailed studies revealed that intracellular acidification occurs during the thymidine starvation process and experimental manipulation of pH causes dramatic effects on survival. We also observed that a genetic perturbation that increases intracellular pH affects survival after treatment with the antibiotic gentamicin. Indeed, gentamicin exposure causes a decrease in pH, suggesting that acidification may play a broader role in the bactericidal effects of other antibiotics.
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