Efflux pumps and the emergence of antibiotic resistance in single cells

项目摘要/摘要 随着细菌不断进化以规避药物，抗生素耐药性是临床环境中的一个主要问题。 我们对抗生素耐药性的大部分了解来自于对基因变化的研究 然而，即使没有永久的基因，细菌也可以在接触抗生素的情况下暂时存活下来 改变。一个关键的问题是，短暂的耐药性是否可以成为永久药物的垫脚石 抵抗。为了解决这一问题，我们在这里重点介绍多药外排泵，这是一个典型的 暂态电阻机制。AcrAB-TolC及其同系物等多药外排泵的表达 允许细胞输出多种抗生素，包括β-内酰胺类、氟喹诺酮类、四环素类和 还有很多其他人。尽管泵能够在抗生素中存活，但表达它们的成本很高，因此细胞通常会转向 他们只是暂时戴上。最近的两项研究表明，AcrAB-TolC在大肠杆菌中表达 在一个群体中不同的细胞不同，泵浦水平和药物输出率在群体中也不同 结果。重要的是，我们的初步数据显示，单细胞外排泵的表达与 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 群体中不同细胞之间的抗药性和高突变率，使一些细胞得以存活 抗生素的使用时间延长，同时增加诱变作用，促进耐药突变株的出现。我们将测试 这一假说使用了一种集成了单细胞时间推移显微镜的方法，光基因控制 外排泵，以及测量外排介导的持续时间和时间相关性的数学模型 抗生素的存活和突变。我们的方法围绕两个目标：(1)量化单细胞关系 AcrAB-TolC表达与短暂性抗性持续时间之间的关系。(2)确定AcrAB的可变性如何- TolC影响突变相关基因的表达和永久耐药的出现。这项研究 之所以意义重大，是因为多种药物外排泵在病原菌中普遍存在，并经常与 耐药的最初阶段。除了测量外排泵在疾病演变中的作用 抗生素耐药性，这项工作很可能对理解和消除成核具有普遍意义 耐药分数。这项工作具有创新性，因为它采用了动态的单细胞量化方法 抗生素耐药性的出现，并提出了外排泵在提高 突变率。

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.