A general mechanism of persister formation

持久形成的一般机制

• 批准号: 10053305
• 负责人: Kim Lewis
• 金额: $ 68.2万
• 依托单位: NORTHEASTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-11-16 至 2022-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10053305
• 关键词: Ampicillin; Animal Model; Antibiotic Resistance; Antibiotics; Bacteria; Cells; Chronic Disease; Citric Acid Cycle; Clinical; Collaborations; Cytolysis; Data; Development; Disease; Drug Tolerance; Enzymes; Escherichia coli; Etiology; Glycolysis; Goals; Growth; Immune system; Individual; Knowledge; Lactose; Link; Microbial Biofilms; Microfluidic Microchips; Microfluidics; Microscopy; Molecular; Monitor; Mothers; Nature; Noise; Oxidative Phosphorylation; Phenotype; Play; Process; Production; Reporter; Reporting; Research; Resistance; Respiration; Role; Staphylococcus aureus; Succinates; System; Testing; Time; Toxin; Variant; Work; antibiotic tolerance; antimicrobial; antitoxin; base; chemotherapy; chronic infection; design; experimental study; in vivo; instrument; mouse model; mutant; overexpression; pathogen; pathogenic bacteria; protein expression; resistance mechanism; single cell analysis; tool

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 该项目的目标是确定细菌耐药性的性质。 两种不同类型的机制使细菌能够逃避抗生素的杀灭——耐药性和耐受性； 与耐药性不同，我们对耐受性的了解是有限的。 引起抗微生物化疗耐药的慢性感染的病原体不具有耐药性。 耐受性与持久细胞有关，持久细胞是在抗生素中存活的一小部分休眠细胞。 慢性感染与生物膜有关，生物膜可以保护持续者免受免疫系统的侵害。 对持久药物耐受机制的理解将弥补知识和意愿方面的重大差距 有助于开发更好的方法来治疗慢性感染。 目前的范式主要基于对大肠杆菌的研究，认为持久性机制 细菌中的形成不保守，并且由毒素-抗毒素模块（TA）控制。 最近报道，在金黄色葡萄球菌中，TA 在持久细胞形成中没有发挥作用，而是随机减少。 稀有细胞中的 ATP 会产生休眠的持续细胞，然后我们发现 ATP 的减少与持续细胞有关。 我们还确定，虽然某些 TA 在持久性形成中发挥作用。 在大肠杆菌的特定条件下，这不是主要机制。 在这个项目中，我们将利用大肠杆菌确定细菌中持久存在形成的一般机制。 大肠杆菌，一种代表性的革兰氏阴性病原体，和金黄色葡萄球菌，一种革兰氏阳性菌，我们的初步数据。 表明能量产生成分（克雷布斯循环和糖酵解）表达的随机变化 酶 - 导致低 ATP 和持久性在这个项目中，我们将使用直接生产来表达蛋白质。 和 ATP，以建立除传统之外的能量产生成分和持久性之间的因果关系。 延时显微镜，我们将利用“母机”，即大规模并行微流体 允许同时分析数百万个单个细胞的仪器。 另一个重要的未解答的问题是持久性和临床表现之间的联系 虽然间接证据表明存在持久性，但在这个项目中，我们将确定因果关系。 设计具有减少和过度表达的持久性的病原体菌株，并将其水平与 小鼠慢性感染生物膜模型中的抗生素耐受性该项目将为研究提供新的范例。 对慢性疾病顽固性的理解，以及研究顽固性疾病的新工具。 这是微生物学家 Kim Lewis 博士之间的多位 PI 合作，他是以下研究的先驱 慢性感染的持续存在者，以及生物物理学家 Johan Paulsson 博士，他是大规模并行技术的先驱 单细胞分析。