Investigating the contextual essentiality of the Escherichia coli efflux system

研究大肠杆菌外排系统的背景重要性

• 批准号: RGPIN-2019-04996
• 负责人: Cox, Georgina
• 金额: $ 2.7万
• 依托单位: University of Guelph
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=737998
• 关键词: Investigating; contextual; essentiality; Escherichia; coli

Efflux pumps are membrane-spanning proteins that actively transport molecules across membranes to the extracellular environment. Bacterial efflux pumps have received significant attention due to their involvement in antibiotic resistance. Despite this association, a growing body of evidence suggests that these proteins also play important roles in bacterial physiology. During metabolism, organisms produce waste metabolites that can be toxic if they reach high intracellular levels. It has been hypothesized that efflux pumps are responsible for the removal of toxic waste metabolites. Furthermore, there is also evidence that efflux pumps are involved in pH homeostasis. Using Escherichia coli as a model organism, the proposed research program is designed to provide a holistic understanding of the physiological roles of efflux-pumps in different environmental and genetic contexts, with a particular focus on their connection to metabolism and cellular homeostasis. Aim 1 of this proposal is the characterization of a conditional synthetic-sick interaction that we have observed between two efflux pumps. Deletion of two major facilitator efflux pumps causes severe growth defects when E. coli is grown in a nutrient-limited medium. We hypothesize that the loss of these efflux pumps leads to the accumulation of toxic metabolites and/or an inability of the cell to regulate cytoplasmic pH. To investigate this hypothesis we will integrate transcriptomic-metabolomic analyses and we will assess the intracellular pH of the strain. By investigating the basis of this conditional synthetic-sick interaction, we will gain insight into the essentiality of efflux pumps in bacterial physiology. Aim 2 is the first systems-level study of E. coli efflux pump essentiality in different environmental and genetic conditions. By profiling a wide variety of growth conditions in combination with extensive genetic manipulation, we will establish the contextual essentiality of the E. coli efflux family. We will characterize any conditionally essential genes and genetic interactions using the transcriptomic/metabolomic approach described in Aim 1, combined with an assessment of changes in pH tolerance and phenotypic analysis. Such studies will provide a repertoire of efflux metabolic waste substrates, bacterial stress responses induced by the loss of efflux, and a systems-level understanding of the efflux pumps involved in cellular homeostasis. Despite E. coli being a well-characterised organism, fundamental questions remain regarding the physiological roles of efflux pumps. This study is designed to address these questions and will provide the first systems-level investigation into the contextual essentiality of the E. coli efflux family. Such information will contribute to our broader understanding of bacterial physiology and will offer important insight into the origins of these proteins, which could be applicable to other bacterial species and organisms.

外排泵是一种跨膜蛋白，它主动地将分子跨膜运输到细胞外环境。细菌外排泵因其参与抗生素耐药性而受到了极大的关注。尽管存在这种关联，但越来越多的证据表明，这些蛋白质在细菌生理学中也起着重要作用。在新陈代谢过程中，生物体会产生废物代谢物，如果它们在细胞内达到很高的水平，就会有毒。据推测，外排泵负责有毒废物代谢物的清除。此外，也有证据表明外排泵参与pH稳态。利用大肠杆菌作为模式生物，该研究计划旨在全面了解外排泵在不同环境和遗传背景下的生理作用，特别关注它们与代谢和细胞稳态的联系。本提案的目的1是表征我们在两个外排泵之间观察到的条件合成病态相互作用。当大肠杆菌在营养有限的培养基中生长时，两个主要促进剂外排泵的缺失会导致严重的生长缺陷。我们假设这些外排泵的丧失导致有毒代谢物的积累和/或细胞无法调节细胞质pH。为了研究这一假设，我们将整合转录组-代谢组学分析，并评估菌株的细胞内pH。通过研究这种条件合成-疾病相互作用的基础，我们将深入了解外排泵在细菌生理学中的重要性。目的2是大肠杆菌外排泵在不同环境和遗传条件下必要性的首次系统级研究。通过分析各种各样的生长条件，结合广泛的遗传操作，我们将建立大肠杆菌外排家族的背景重要性。我们将使用Aim 1中描述的转录组学/代谢组学方法，结合对pH耐受性变化的评估和表型分析，描述任何条件必需基因和遗传相互作用。这些研究将提供一系列外排代谢废物底物，由外排丧失引起的细菌应激反应，以及对参与细胞内稳态的外排泵的系统级理解。尽管大肠杆菌是一种特征明确的有机体，但关于外排泵的生理作用的基本问题仍然存在。本研究旨在解决这些问题，并将提供大肠杆菌外排家族背景重要性的第一个系统级调查。这些信息将有助于我们更广泛地了解细菌生理学，并将为这些蛋白质的起源提供重要的见解，这可能适用于其他细菌物种和生物体。