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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.

摘要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伤-耐药性和耐受性赋予的能力。与抵抗不同，我们对宽容的了解是有限的。巧合的是，大多数导致对抗菌化学疗法无效的慢性感染的病原体不是耐药性的。耐受性与持久性有关，持久性是一小部分休眠细胞，在抗生素中存活。许多慢性感染与生物膜有关，生物膜可保护持久感染者免受免疫系统的侵害。一个对持续耐药机制的理解将缩小知识和意志上的重大差距，有助于开发更好的治疗慢性感染的方法。目前的研究范式主要是基于E.认为，持久的机制，形成在细菌中不保守，并且由毒素-抗毒素模块（TA）控制。但我们最近报道，在S.金黄色葡萄球菌中，TA在持久性形成中不起作用。相反，稀有细胞中的ATP会产生休眠的持留细胞。然后我们发现ATP的减少与持续性有关， E.大肠杆菌也是。我们还确定，虽然一些TA在持续形成中发挥作用， E.大肠杆菌，这不是主要的机制。在这个项目中，我们将使用E. 大肠杆菌（代表性革兰氏阴性病原体）和S.金黄色葡萄球菌，一种革兰氏阳性菌种。我们的初步数据表明能量产生组分-克雷布斯循环和糖酵解表达随机变化酶-导致低ATP和持久性。在这个项目中，我们将使用直接报告基因进行蛋白质表达和ATP，以建立能源生产组件和持久性之间的因果关系。除了传统的时间推移显微镜，我们将利用“母机”，一个大规模并行微流体一种可以同时分析数百万个细胞的仪器。另一个重要的未回答的问题是坚持者和临床表现之间的联系，疾病虽然间接证据指向持续存在，但因果关系尚未确定。在这个项目中，我们将设计具有减少的病原体菌株;和过表达的持续生产，并将其水平与小鼠慢性感染的生物膜模型中的抗生素耐受性。该项目将提供一个新的范例，对慢性病顽固性的理解，以及研究顽固性疾病的新工具。这是一个多PI之间的合作博士金刘易斯，微生物学家谁开创了研究，慢性感染的坚持者，以及约翰·保尔森博士，一位开创了大规模平行单细胞分析