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.

项目概要/摘要 该项目将研究先前未充分研究的称为质粒获取成本的现象， 水平基因转移（HGT）的背景。质粒转移是病原体适应的主要方式 环境压力源。这些质粒常常对细胞造成持续的负担，与持续的 质粒基因的表达量，称为质粒适应度成本。它们还引起互补， 独立的、短暂的负担，反映了质粒获得后立即发生的代谢适应。 然而，尽管适应性成本已得到充分研究，但这些短暂的机制因素 影响（称为质粒获取成本）目前尚不清楚。此信息对于 发展对病原体动态和潜在下游干预策略的基本见解。 根据我们的初步数据，我们的中心假设是收购成本是由于新陈代谢而产生的 质粒转移后立即出现失调。我们将通过两个具体目标来研究这个假设： (1) 我们将通过进行详细的研究来阐明质粒获取成本的转录基础 在标准实验室大肠杆菌中表征充分表征的 RP4 接合质粒 拉紧;我们注意到，我们的初步工作说明了购置成本的存在和程度 最初是使用这种组合完成的。具体来说，我们建立了一种新颖的实验方案，可以可靠地 量化新质粒受体的生长缺陷。在这里，我们将配对时间序列 RNA-seq 测量 通过代谢模型来阐明质粒后立即发生的代谢失调 获得;我们将使用一组合理选择的基因敲除菌株来验证这些结果 代表性质粒。总体而言，这将为观察到的采购成本建立机械解释。 (2) 我们将利用自然条件确定不同质粒获取成本的遗传决定因素 从环境和临床样本中分离出的相关质粒和菌株，以及标准品 实验室质粒。具体来说，我们将利用可用的全基因组序列进行组装 我们的合作者提供的质粒以及相应的样品，用于从两个物种中分离质粒： 大肠杆菌和肺炎克雷伯菌。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 购置成本。这将导致在自然条件下对质粒获取成本进行首次严格量化 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的 HGT 动力学的基础 人口。此外，所有质粒都将公开，这将作为宝贵的研究和参考 整个学术界的教育工具。

项目成果

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

接合质粒上的代谢基因在大肠杆菌中非常普遍，可以防止抗生素治疗。

• 发表时间: 2023-01
• 作者: Palomino, Alana;Gewurz, Danya;DeVine, Lela;Zajmi, Ujana;Moralez, Jenifer;Abu;Smith, Robert P;Lopatkin, Allison J
• 通讯作者: Lopatkin, Allison J