Systems biological interrogation of bacterial persistence

摘要 抗生素持久性仍然是有效清除慢性细菌的最具挑战性的障碍之一 感染。尽管自最初发现以来已经有几十年了，我们仍然对它缺乏基本的了解 分子网络如何实现持久性的定义特征：表型异质性和耐受性 致死水平的抗生素暴露。许多关于持久性的工作都是现象学的， 机械论的研究，到目前为止，只集中在几个候选通路上，未能提供 对这种复杂的表型有足够的了解。在这里，我们提出了一种无偏见的系统生物学方法 来描述坚持不懈的遗传和监管基础。其主要目标是精确定义 通常只占种群的一小部分(&lt；10-4)的持久细胞的细胞状态。我们会 利用我们之前开发的大肠杆菌超持久性遗传和化学诱导模型来触发 并通过使用我们最近开发的 单细胞RNA测序技术(PETRI-SEQ)。这将使我们能够定义过程的动态 在单细胞分辨率下，并识别高度特异的持久标记，我们可以用来纯化它们到接近- 同质性。然后，我们将通过使用我们最近优化的 体内蛋白质-DNA和RNA-RNA相互作用的技术。结合单细胞RNA图谱和全球 监管互动将使我们能够生成关键监管事件的因果图形模型 潜伏在持久层之下。我们将利用我们最近开发的CRISPR干扰技术(CAME)来 系统地确定所有必需和非必需基因的敲除扰动如何影响 持久度的定量参数，包括持久率和杀伤率。最后，通过将 监管和遗传地图的持久性，我们将识别和验证最关键的漏洞节点 其靶向将消除持久病毒的形成和生存。这些观测的巨大规模将 揭示关于姊妹代、基因调控和 生理学，到目前为止。这是一个关键的基础，我们可以在此基础上制定合理的战略，以减少 持久者的临床负担。最后，这里独特的概念和技术方法将作为一种 探索其他复杂细菌现象的遗传和调控基础的蓝图，如生物膜， 其中表型的异质性是一个决定性的标志。

Summary Antibiotic persistence remains one of the most challenging barriers to effective clearance of chronic bacterial infections. Despite the intervening decades since its original discovery, we still lack a basic understanding of how molecular networks implement the defining features of persistence: phenotypic heterogeneity and tolerance to lethal levels of antibiotic exposure. Much of the work on persistence has been phenomenological and the mechanistic studies which, so far, have only focused on a few candidate pathways, have failed to provide an adequate understanding of this complex phenotype. Here, we propose an unbiased systems biology approach to characterize the genetic and regulatory underpinnings of persistence. A primary aim is to precisely define the cellular state of persister cells which typically make up only a small (< 10-4) fraction of the population. We will utilize our previously developed genetic and chemically induced models of E. coli hyper-persistence to trigger persister cells, and to follow the trajectory of their phenotypic diversification by using our recently developed single-cell RNA sequencing technology (PETRI-seq). This will enable us to define the dynamics of the process at single-cell resolution, and identify highly specific persister markers, which we can use to purify them to near- homogeneity. We will then profile the global gene regulatory state of persisters by using our recently optimized technologies for in vivo Protein-DNA and RNA-RNA interactions. Combining the single-cell RNA atlas and global regulatory interactions will enable us to generate causal graphical models of pivotal regulatory events that underlie persister formation. We will utilize our recently developed CRISPR-interference technology (CALM) to systematically determine how knock-down perturbations to all essential and non-essential genes affect quantitative parameters of persistence, including persister-fraction and kill-rates. Finally, by combining the regulatory and genetic maps of persistence, we will identify and validate the most critical vulnerability nodes whose targeting will eliminate persister formation and survival. The massive scale of these observations will reveal the most comprehensive and unbiased global view of persister generation, gene regulation, and physiology, to date. This is a critical foundation upon which we can devise rational strategies for reducing the clinical burden of persisters. Finally, the unique conceptual and technological approaches here will serve as a blueprint for exploring the genetic and regulatory basis of other complex bacterial phenomena, such as biofilms, where phenotypic heterogeneity is a defining hallmark.