Role of the alarmone (p)ppGpp in phenotypic antibiotic tolerance

项目摘要 对抗生素耐药的病原菌的出现是一个紧迫的公共卫生问题。 耐药性通常是由一种特定的基因突变引起的，这种突变永久性地改变了生物体的 对特定抗生素的敏感性。然而，抗生素敏感性也可以短暂改变， 这是表型抗生素耐受性结果，这种现象被认为是治疗的基础 在反复细菌感染的情况下失败。抗生素耐药性是由于 细菌进入短暂的生长停滞和静止状态，在那里它们对 抑制过程如DNA复制或细胞壁合成的化合物， 避免被它们通常敏感的抗生素杀死。当寂静 在稳定期培养物中的大多数细胞的特征中，只有一小部分细胞在稳定期培养物中的特征中。 指数增长的培养物是暂时静止的。正是这些细胞-“坚持者”- 被认为是抗生素治疗复发性感染失败的原因。如何 静止只发生在一个亚群体中？一种提出的机制涉及核苷酸 第二信使（p）ppGpp，抑制广泛的生理过程，包括 转录和DNA复制导致生长停滞和静止。已经提出 细胞中（p）ppGpp量的变化可能导致生长状态的变化， 从而产生抗生素敏感性。我们最近发现，在指数增长的人口中， 的枯草芽孢杆菌表达显著更高量的（p）ppGpp合酶。中的细胞 该亚群显示出与大肠杆菌中的大多数细胞相比增加的抗生素耐受性。 不表达这种蛋白质的人群。在这项工作的一个重要扩展中，我们有 开发了一种荧光报告基因，可用于识别具有升高水平的 （p）ppGpp。我们称这些细胞为（p）ppGpphigh，它们可以通过荧光激活细胞分离 分选（FACS）。我们提出研究（p）ppGpphigh细胞的以下目的。目标1： 表征（p）ppGpphigh细胞并比较它们的生理特征，包括 蛋白质合成和DNA复制到群体的平均细胞。目标2：调查 细胞机制负责合成和降解的（p）ppGpp在 （p）ppGpphigh细胞。在这个项目中获得的知识将加深我们的 了解持留细菌的生理学，并促进代谢的鉴定 可以作为抗持久性战略的潜在目标的脆弱性。

Project Summary The emergence of pathogenic bacteria resistant to antibiotics is an urgent public health issue. Resistance typically results from a specific genetic mutation that permanently alters the organism’s sensitivity to a particular antibiotic. However, antibiotic sensitivity can also change transiently as the result of phenotypic antibiotic tolerance, a phenomenon that is thought to underly treatment failures in the case of recurrent bacterial infections. Antibiotic tolerance results from the entry of bacteria into a transient growth-arrested and quiescent state where they are less sensitive to compounds that inhibit processes such as DNA replication or cell wall synthesis and thus can escape killing by antibiotics to which they are normally sensitive. While quiescence is characteristic of most cells in a stationary phase culture, only a small fraction of the cells in a exponentially growing culture are transiently quiescent. It is these cells – “persisters” - that are thought to be responsible for the failure of antibiotic treatment in recurrent infections. How does quiescence occur in only a sub-population? One proposed mechanism involves the nucleotide second messengers (p)ppGpp that inhibit a broad range of physiological processes including transcription and DNA replication leading to growth arrest and quiescence. It has been proposed that variability in the amount of (p)ppGpp in cells could lead to variations in growth state and thereby antibiotic sensitivity. We recently found that ~1% of an exponentially growing population of Bacillus subtilis expresses substantially higher amounts of a (p)ppGpp synthase. The cells in this sub-population exhibit increased antibiotic tolerance as compared to most cells in the population that do not express this protein. In an important extension of this work, we have developed a fluorescent reporter can be used to identify individual cells with elevated levels of (p)ppGpp. We call these cells (p)ppGpphigh and they can be isolated by Fluorescent Activated Cell Sorting (FACS). We propose to investigate (p)ppGpphigh cells in the following Aims. Aim 1 is to characterize the (p)ppGpphigh cells and compare their physiological characteristics including protein synthesis and DNA replication to average cells of the population. Aim 2 is to investigate the cellular mechanisms responsible for the synthesis and degradation of (p)ppGpp in the (p)ppGpphigh cells. The knowledge gained during the course of this project will deepen our understanding of the physiology of persister bacteria and facilitate the identification of metabolic vulnerabilities that could serve as potential targets for anti-persister strategies.