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

项目摘要 对抗生素产生耐药性的病原菌的出现是一个紧迫的公共卫生问题。 抗药性通常是由一种特定的基因突变引起的，这种突变永久地改变了有机体的 对一种特定抗生素的敏感性。然而，抗生素敏感性也可能发生短暂的变化，如 表型抗生素耐药的结果，一种被认为治疗不足的现象 在反复细菌感染的情况下失败。抗生素耐药是由于进入 细菌进入一种短暂的生长停滞和静止状态，在这种状态下它们对 抑制DNA复制或细胞壁合成等过程的化合物，因此可以 避免被抗生素杀死，而抗生素通常是敏感的。而静止则是 大多数细胞处于静止期培养的特点，只有一小部分细胞在 呈指数级增长的文化暂时停滞不前。正是这些细胞--“持久者”-- 被认为是反复感染时抗生素治疗失败的原因。何以 静止期只出现在亚群中吗？一种被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停滞。已经有人提议 细胞中(P)ppGpp含量的变化可能会导致生长状态和 因此对抗生素的敏感性。我们最近发现，在呈指数级增长的人口中，约1%的人 枯草芽孢杆菌表达大量的(P)ppGpp合成酶。中的单元格 与大多数细胞相比，这一亚群表现出更高的抗生素耐受性 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 开发的一种荧光报告器可以用来识别单个细胞，这些细胞的水平升高 (P)ppGpp。我们称这些细胞为(P)ppGpphigh，它们可以通过荧光激活的细胞分离出来 分类(FACS)。我们建议研究(P)ppGpphigh细胞以达到以下目的。目标1是 表征(P)ppGpphigh细胞并比较它们的生理特性，包括 蛋白质合成和DNA复制到群体的平均细胞。目标2是调查 (P)ppGpp合成和降解的细胞机制 (P)ppGpphigh细胞。在这个项目的过程中获得的知识将加深我们的 了解宿存细菌的生理学并促进代谢的鉴定 可能成为反持久策略的潜在目标的漏洞。

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.