A general mechanism of persister formation

摘要 该项目的目标是确定细菌耐药的性质。 两种不同类型的机制允许细菌通过抗药性和耐受性来逃避死亡 由姊妹细胞赋予的。与抵抗不同，我们对耐受性的了解是有限的。矛盾的是，大多数人 导致慢性感染的病原体对抗菌素化疗不耐药。 耐受性与持久者有关，持久者是一小部分在抗生素中存活的休眠细胞。许多 慢性感染与生物膜有关，生物膜保护持久者免受免疫系统的攻击。一个 对持久性药物耐受机制的理解将弥合知识和意愿方面的重大差距 有助于开发更好的治疗慢性感染的方法。 目前的范例，主要是基于对大肠杆菌的研究，认为持久剂的机制 细菌之间的形成不是保守的，由毒素-抗毒素模块(TA)控制。然而，我们 最近报道，在金黄色葡萄球菌中，Tas在持续体的形成中没有作用。相反，是随机减少的 稀有细胞中的三磷酸腺苷会产生休眠的持续体。然后我们发现，ATP的减少与持久力有关 在大肠杆菌中也能形成。我们还证实，虽然一些TA在植物体内的持续体形成中起作用 在大肠杆菌的特定条件下，这并不是主要的机制。 在这个项目中，我们将利用E。 革兰氏阴性杆菌和革兰氏阳性菌金黄色葡萄球菌。我们的初步数据 表明能量产生组分-Krebs循环和糖酵解表达的随机变化 酶--导致低ATP和持久力。在这个项目中，我们将使用直接记者进行蛋白质表达 和三磷酸腺苷，以建立能源生产组件和持久者之间的因果关系。除了传统的 时间推移显微镜，我们将利用“母机”，一种大规模并行的微流体 允许同时分析数百万个单个细胞的仪器。 另一个重要的悬而未决的问题是持久者和临床表现之间的联系。 疾病。虽然间接证据指向了持久者，但因果关系尚未确定。在这个项目中，我们将 设计持久菌产量减少和过表达的病原菌，并将它们的水平与 小鼠慢性感染生物膜模型中的抗生素耐受性。该项目将提供一种新的范例 对慢性病顽固性的理解，以及研究顽固者的新工具。 这是金·刘易斯博士之间的多PI合作，他是微生物学家，他是研究 坚持慢性感染，以及生物物理学家约翰·保尔森博士，他开创了大规模平行 单细胞分析。

Abstract The goal of the project is to determine the nature of bacterial drug tolerance. Two different types of mechanisms allow bacteria to evade killing by antibiotics – resistance; and tolerance conferred by persister cells. Unlike resistance, our knowledge of tolerance is limited. Paradoxically, most pathogens that cause chronic infections recalcitrant to antimicrobial chemotherapy are not drug resistant. Tolerance has been linked to persisters, a small subpopulation of dormant cells that survive antibiotics. Many chronic infections are associated with biofilms, which protect persisters from the immune system. An understanding of the mechanism of persister drug tolerance will close a significant gap in knowledge and will contribute to the development of better approaches to treat chronic infections. The current paradigm, based primarily on the study of E. coli, holds that mechanisms of persister formation are not conserved among bacteria, and are governed by toxin-antitoxin modules (TA). However, we recently reported that in S. aureus, TAs play no role in persister formation. Rather, a stochastic decrease in ATP in rare cells produces dormant persisters. We then found that a decrease in ATP is linked to persister formation in E. coli as well. We also established that while some TAs play a role in persister formation under specific conditions in E. coli, this is not the main mechanism. In this project, we will determine the general mechanism by which persisters form in bacteria using E. coli, a representative Gram negative pathogen, and S. aureus, a Gram positive species,. Our preliminary data indicate that stochastic variation in expression of energy producing components - Krebs cycle and glycolytic enzymes - leads to low ATP and persisters. In this project, we will use direct reporters for protein expression and ATP to establish causality between energy producing components and persisters. Apart from conventional time-lapse microscopy, we will take advantage of the “mother machine”, a massively parallel microfluidics instrument that allows simultaneous analysis of millions of individual cells. Another important unanswered question is the link between persisters and the clinical manifestation of disease. While indirect evidence points to persisters, causality is yet to be established. In this project, we will design pathogen strains with diminished; and overexpressed production of persisters, and link their levels to antibiotic tolerance in biofilm models of murine chronic infection. This project will provide a new paradigm for the understanding of recalcitrance of chronic diseases, and new tools for the study of persisters. This is a multi-PI collaboration between Dr. Kim Lewis, a microbiologist who pioneered the studies of persisters in chronic infections, and Dr. Johan Paulsson, a biophysicist who pioneered massively parallel single-cell analysis.