A general mechanism of persister formation

持久形成的一般机制

基本信息

批准号：
10291419
负责人：
Kim Lewis
金额：
$ 68.2万
依托单位：
NORTHEASTERN UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-11-16 至 2023-10-31
项目状态：
已结题

项目摘要

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.

抽象的该项目的目的是确定细菌药物耐受性的性质。两种不同类型的机制允许细菌逃避抗生素杀死 - 耐药性；和宽容由持久细胞会议。与抵抗不同，我们对宽容的了解是有限的。矛盾的是，大多数引起慢性感染抗菌化疗的慢性感染的病原体不是抗药性的。耐受性与持久性有关，这是对抗生素存活的休眠细胞的少量亚群。许多慢性感染与生物膜有关，该生物膜可保护持久性免受免疫系统的影响。一个理解持久药物耐受性机制将缩小知识的显着差距，并将有助于开发更好的治疗慢性感染方法。当前的范式主要基于大肠杆菌的研究，持续的机制在细菌中不保守形成，并且由毒素 - 抗毒素模块（TA）控制。但是，我们最近报道说，在金黄色葡萄球菌中，TA在持续形成中没有任何作用。而是随机减少稀有细胞中的ATP会产生休眠的持久性。然后，我们发现ATP的减少与迫害有关大肠杆菌也形成。我们还确定，尽管某些TA在持续形成中发挥了作用大肠杆菌中的特定条件，这不是主要机制。在这个项目中，我们将确定使用E的细菌中持续形成的一般机制。大肠杆菌，一种代表性的革兰氏阴性病原体和金黄色链球菌，革兰氏阳性物种。我们的初步数据表明能量产生成分表达的随机变化 - 克雷布斯循环和糖酵解酶 - 导致ATP和持久性低。在这个项目中，我们将使用直接记者进行蛋白质表达和ATP以在能源生产组成部分和持久性之间建立因果关系。除了传统延时显微镜，我们将利用“母机”，这是一种大规模平行的微流体允许简单分析数百万个单个单元的仪器。另一个重要的未解决的问题是持久者与临床表现之间的联系疾病。尽管间接证据表明持续存在，但尚未建立偶然性。在这个项目中，我们将设计病原体菌株的减少；并过表达言论的产生，并将其级别与鼠慢性感染的生物膜模型中的抗生素耐受性。该项目将为对慢性疾病的顽固性的理解以及用于研究言论的新工具。这是微生物学家金·刘易斯（Kim Lewis）博士之间的多个PI合作，他开创了研究慢性感染中的持久性，生物物理学家约翰·保尔森（Johan Paulsson）单细胞分析。