Establishing a mechanistic basis for the plasmid acquisition cost

建立质粒获取成本的机制基础

• 批准号: 10291392
• 负责人: Allison Lopatkin
• 金额: $ 23.78万
• 依托单位: BARNARD COLLEGE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10291392
• 关键词: Antibiotic Resistance; Bacteria; Biochemical Pathway; Cells; Characteristics; Clinical; Code; Communities; Complement; Complex; Cost Measures; Data; Defect; Distant; Environment; Escherichia coli; Evolution; Exhibits; Foundations; Future; Gene Expression; Genes; Genetic; Genetic Determinism; Genetic Transcription; Genomics; Goals; Growth; Horizontal Gene Transfer; Hour; Human Microbiome; In Situ; Individual; Intervention; Klebsiella pneumoniae; Laboratories; Length; Measurement; Metabolic; Metabolic Pathway; Metabolism; Methods; Microbiology; Minority; Modeling; Molecular; Nature; Pathogenesis; Phenotype; Physiological; Plasmids; Play; Population; Population Dynamics; Prevalence; Protocols documentation; Public Health; Research; Role; Sampling; Series; Soil; Son of Sevenless Proteins; Testing; Time; Validation; Virulence; Water; Work; base; combat; cost; density; environmental stressor; experimental study; fitness; insight; knockout gene; microbial; microbial community; multidisciplinary; network models; novel; pathogen; plasmid DNA; reproductive success; response; success; tool; transcription factor; transcriptome sequencing; transcriptomics; undergraduate student; whole genome

Project Summary/Abstract This project will investigate the previously understudied phenomenon termed plasmid acquisition cost, in the context of horizontal gene transfer (HGT). Plasmid transfer is a dominant way that pathogens adapt to environmental stressors. These plasmids often exert a sustained burden on cells, associated with continued expression of plasmid genes, known as the plasmid fitness cost. They also induce a complementary, independent, transient burden reflective of the metabolic adaptation immediately following plasmid acquisition. However, whereas fitness costs are well-studied, the mechanistic factors underlying these transient effects, referred to as the plasmid acquisition costs, are currently unknown. This information is critical to developing fundamental insights into pathogen dynamics and potential downstream interventional strategies. Based on our preliminary data, our central hypothesis is that acquisition costs arise as a result of metabolic dysregulation immediately following plasmid transfer. We will investigate this hypothesis with two Specific Aims: (1) We will elucidate the transcriptional basis of the plasmid acquisition cost, by undertaking a detailed characterization of the well-characterized RP4 conjugative plasmid in a standard laboratory Escherichia coli strain; we note that our preliminary work illustrating the existence and extent of acquisition costs was originally done using this combination. Specifically, we established a novel experimental protocol that reliably quantifies the growth defect in new plasmid recipients. Here, we will pair time series RNA-seq measurements with metabolic modeling to elucidate the metabolic dysregulation that occurs immediately following plasmid acquisition; we will validate these results using a rationally selected set of gene knockout strains and representative plasmids. Overall, this will establish a mechanistic explanation for observed acquisition costs. (2) We will determine the genetic determinants of the acquisition cost in diverse plasmids, using naturally relevant plasmids and strains isolated from both environmental and clinical samples, as well as standard laboratory plasmids. Specifically, we will leverage available whole genome sequences with assembled plasmids from our collaborators, along with the corresponding samples, to isolate plasmids from two species: E. coli and Klebsiella pneumoniae. We will then quantify acquisition costs for each plasmid; multiple variable regression will be used to understand which plasmid characteristics are most predictive of observed acquisition costs. This will result in the first rigorous quantification of plasmid acquisition costs in naturally occurring environmental and clinical plasmids. Beyond demonstrating the generality of this phenomenon, these results will serve as the foundation for predicting and modulating HGT dynamics in more complex populations. Moreover, all plasmids will be made available, which will serve as an invaluable research and educational tool throughout the wider academic community.

项目摘要/摘要 该项目将调查先前研究的现象称为质粒的质粒成本， 水平基因转移（HGT）的上下文。质粒转移是病原体适应的主要方式 环境压力源。这些质粒通常会对细胞施加持续的负担，与持续有关 质粒基因的表达，称为质粒适应性成本。他们还诱使互补 独立的，瞬态负担反映了质粒收购后立即反映代谢适应性的。 但是，尽管健身成本得到了充分研究，但这些瞬时的机械因素 效果（称为质粒获取成本）目前尚不清楚。此信息对 发展对病原体动态和潜在下游介入策略的基本见解。 根据我们的初步数据，我们的中心假设是代谢而产生的收购成本 质粒转移后立即进行失调。我们将以两个具体的目的调查这一假设： （1）我们将通过详细说明质量获取成本的转录基础 标准实验室大肠杆菌中特征良好的RP4偶联质粒的表征 拉紧;我们注意到，我们的初步工作说明了收购成本的存在和程度是 最初是使用此组合完成的。具体而言，我们建立了一个新颖的实验协议，可靠 量化新质粒接受者的生长缺陷。在这里，我们将配对时间序列RNA-seq测量 通过代谢建模以阐明质粒后立即发生的代谢失调 获得;我们将使用合理选择的基因基因敲除菌株和 代表性质粒。总体而言，这将为观察到的获取成本建立机械解释。 （2）我们将使用自然的质粒确定采集成本的遗传决定因素 从环境和临床样品中分离出的相关质粒和菌株以及标准 实验室质粒。具体而言，我们将利用组装的整个基因组序列 我们的合作者的质粒以及相应的样品，以分离两个物种的质粒： 大肠杆菌和克雷伯氏菌肺炎。然后，我们将量化每个质粒的获取成本；多变量 回归将用于了解哪些质粒特征最能预测观察到的 获取成本。这将导致自然的第一个严格量化质粒成本的严格量化 发生环境和临床质粒。除了证明这种现象的普遍性之外， 这些结果将作为预测和调节更复杂HGT动态的基础 人群。此外，将提供所有质粒，这将是一项无价的研究， 整个更广泛的学术界的教育工具。

项目成果

Metabolic genes on conjugative plasmids are highly prevalent in Escherichia coli and can protect against antibiotic treatment.

• DOI: 10.1038/s41396-022-01329-1
• 发表时间: 2023-01
• 期刊: ISME JOURNAL
• 影响因子: 11
• 作者: Palomino, Alana;Gewurz, Danya;DeVine, Lela;Zajmi, Ujana;Moralez, Jenifer;Abu-Rumman, Fatima;Smith, Robert P.;Lopatkin, Allison J.
• 通讯作者: Lopatkin, Allison J.