Efflux pumps and the emergence of antibiotic resistance in single cells

外排泵和单细胞抗生素耐药性的出现

• 批准号: 9505088
• 负责人: Mary Dunlop
• 金额: $ 24.72万
• 依托单位: BOSTON UNIVERSITY (CHARLES RIVER CAMPUS)
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-02-01 至 2020-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9505088
• 关键词: Address; Antibiotic Resistance; Antibiotic Therapy; Antibiotics; Area; Bacteria; Cell Cycle; Cell Division Process; Cell division; Cells; Clinical; Computer Simulation; Data; Dependence; Development; Dose; Drug Efflux; Drug Tolerance; Drug resistance; Escherichia coli; Evolution; Fluoroquinolones; Frequencies; Genes; Individual; Knowledge; Lead; Length; Light; Link; Measurement; Measures; Mediating; Microscopy; Modeling; Multi-Drug Resistance; Mutagenesis; Mutate; Mutation; Nature; Pharmaceutical Preparations; Pharmacotherapy; Play; Population; Public Health; Pump; Research; Resistance; Role; Schedule; Testing; Tetracyclines; Time; Work; antibiotic efflux; base; beta-Lactams; clinically relevant; cost; efflux pump; experimental study; improved; inhibitor/antagonist; innovation; mathematical model; microorganism; mutant; novel; optogenetics; pathogen; pathogenic bacteria; repaired; resistance frequency; resistance mechanism; resistance mutation; therapy design; treatment strategy

Project Summary / Abstract Antibiotic resistance is a major problem in clinical settings as bacteria continuously evolve to circumvent drugs. The majority of our understanding on antibiotic resistance comes from studies on the genetic changes that cause it, however bacteria can also transiently survive antibiotic exposure even without permanent genetic changes. A key question is whether transient resistance can serve as a stepping stone to permanent drug resistance. To address this, we focus here on multi-drug efflux pumps, which are a canonical example of a transient resistance mechanism. Expression of multi-drug efflux pumps, such as AcrAB-TolC and its homologs, allows cells to export a broad range of antibiotics including b-lactams, fluoroquinolones, tetracyclines, and many others. Although pumps enable survival in antibiotics, they are costly to express so cells commonly turn them on only temporarily. Two recent studies have shown that in Escherichia coli AcrAB-TolC expression varies from cell to cell within a population and that pump levels and drug export rates vary within populations as a result. Importantly, our preliminary data show a link between single-cell efflux pump expression and mutation rate. Based on these results, our central hypothesis is that the AcrAB-TolC pump provides transient resistance and elevated mutation rates that differ between cells in a population, allowing some cells to survive antibiotics longer while increasing mutagenesis to promote the emergence of resistant mutants. We will test this hypothesis using an approach that integrates single-cell time-lapse microscopy, optogenetically-controlled efflux pumps, and mathematical modeling to measure the duration and time dependence of efflux-mediated antibiotic survival and mutation. Our approach centers around two Aims: (1) Quantify single-cell relationship between AcrAB-TolC expression and duration of transient resistance. (2) Determine how variability in AcrAB- TolC impacts expression of mutation-related genes and the emergence of permanent resistance. This research is significant because multi-drug efflux pumps are ubiquitous in pathogenic bacteria and are often implicated in the initial stages of drug resistance. In addition to measuring the role of efflux pumps in the evolution of antibiotic resistance, this work is likely to be generally relevant for understanding and eliminating nucleation points for drug resistance. The work is innovative because of the dynamic, single-cell approach to quantifying the emergence of antibiotic resistance and in addition proposes a novel role for efflux pumps in elevating mutation rates.

项目总结/摘要 抗生素耐药性是临床环境中的主要问题，因为细菌不断进化以规避药物。 我们对抗生素耐药性的大部分了解来自对遗传变化的研究， 然而，细菌也可以短暂地生存抗生素暴露，即使没有永久的遗传 变化一个关键的问题是短暂的耐药是否可以作为永久性药物的垫脚石 阻力为了解决这个问题，我们在这里集中在多药物外排泵，这是一个典型的例子， 瞬态阻力机制多药外排泵如AcrAB-TolC及其同源物的表达， 允许细胞输出广泛的抗生素，包括β-内酰胺类、氟喹诺酮类、四环素类和 其他许多人.尽管泵能够在抗生素中存活，但它们的表达成本很高，因此细胞通常会转向 他们只是暂时的。最近的两项研究表明，在大肠杆菌中AcrAB-TolC的表达 并且泵水平和药物输出速率在群体内变化 结果导致了严重的后果。重要的是，我们的初步数据显示了单细胞外排泵表达与细胞凋亡之间的联系。 突变率基于这些结果，我们的中心假设是AcrAB-TolC泵提供了瞬时的 耐药性和突变率升高，使群体中的细胞之间存在差异，使一些细胞能够存活 抗生素的作用时间较长，同时增加诱变，以促进耐药突变体的出现。我们将测试 这一假设使用了一种方法，该方法集成了单细胞延时显微镜，光遗传学控制， 外排泵和数学建模，以测量外排介导的 抗生素存活和突变。我们的方法围绕两个目标：（1）量化单细胞关系 AcrAB-TolC表达与短暂耐药持续时间之间的关系。(2)确定AcrAB的变异性- TolC影响突变相关基因的表达和永久抗性的出现。本研究 是重要的，因为多药外排泵在病原菌中普遍存在， 抗药性的最初阶段除了测量外排泵在进化中的作用外， 抗生素耐药性，这项工作可能是普遍相关的理解和消除核 耐药性的分数。这项工作是创新的，因为动态的，单细胞的方法来量化 抗生素耐药性的出现，并提出了一个新的作用外排泵在提高 突变率