Biogenesis of cyclic and phospholipid-linked enterobacterial common antigen

环状和磷脂连接的肠细菌共同抗原的生物发生

• 批准号: 10293347
• 负责人: Angela Marie Mitchell
• 金额: $ 36.81万
• 依托单位: TEXAS A&M UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-09 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10293347
• 关键词: Acute; Address; Anabolism; Antibiotic Resistance; Antibiotics; Antigens; Biochemical; Biochemical Reaction; Biogenesis; Biology; Carbohydrates; Cell Membrane Permeability; Cells; Cellular Structures; Cessation of life; Defect; Development; Enteral; Environment; Escherichia; Escherichia coli; Escherichia coli K12; Feedback; Genes; Genetic; Genetic Determinism; Genetic Screening; Genetic Transcription; Genomics; Goals; Gram-Negative Bacteria; Head; Hydrophobicity; Infection; Investigation; Klebsiella; Knowledge; Length; Link; Lipids; Maintenance; Membrane; Mutation; Nature; Nutrient; Pathogenicity; Pathway interactions; Peptidoglycan; Periodicity; Permeability; Phenotype; Phospholipids; Physiological; Play; Production; Reaction; Regulation; Regulatory Pathway; Role; Salmonella; Stress; Structure; Surface; Techniques; Transcription Regulation Pathway; United States; Work; Yersinia; antibiotic resistant infections; antimicrobial; aqueous; base; bile salts; cell envelope; economic cost; enterobacterial common antigen; experience; genetic analysis; genetic selection; improved; innovation; insight; interest; member; novel; periplasm; response; small molecule

PROJECT SUMMARY Nearly 3 million antibiotic resistant infections occur per year in the United States. This problem is especially acute in gram-negative bacteria, where the outer membrane (OM) which surrounds the aqueous periplasm acts as a permeability barrier capable of excluding many antibiotics. We are interested in the OM of Enterobacterales (e.g., Escherichia, Salmonella, Klebsiella), which are adapted to an enteric environment rich in toxic molecules, such as bile salts, necessitating an especially strong OM. It has become clear that the permeability of the OM can be altered by the physiological state of the cell. Specifically, stresses such as nutrient limitation can result in strengthening of the OM permeability barrier. Elucidation of the pathways responsible for this strengthening will lead to new targets for the development of small molecules that can weaken the OM permeability barrier. We have found enterobacterial common antigen (ECA), a conserved component of the Enterobacterales OM and periplasm, to be important for OM impermeability under stress. Two forms of ECA (phospholipid-linked ECA (ECAPG), and cyclic ECA (ECACYC)) have different roles related to OM permeability; however, their precise functions remain unknown, in part, because many steps in their biogenesis are poorly understood. Our long-term goal is to understand the biogenesis of ECA to facilitate functional studies and identify potential antimicrobial targets. Specifically, this project aims to elucidate, in Escherichia coli K12, the regulation of and unknown steps in biogenesis of the forms of ECA contributing to antibiotic resistance. Biochemical reactions are required for these forms of ECA to be produced and yet the genes responsible for these steps and the regulation of these steps are largely unknown. The central hypothesis is that ECAPG and ECACYC can be differentiate through their unique biosynthetic genes and regulatory roles. This hypothesis will be addressed with the following aims: identify the genes and substrate necessary for ECA to become a phospholipid head group forming ECAPG using genetic interactions with other biosynthesis pathways (Aim 1); elucidate factors and mechanisms involved in ECACYC biogenesis using an antibiotic sensitivity suppression phenotype we discovered (Aim 2); and uncover the mechanisms of the two novel pathways of ECA regulation we discovered (Aim 3). These conceptually innovative aims will be approached through a blend of high-throughput genomics, genetic screens and selections, and biochemical techniques. Completion of this project will identify genes and residues important for biogenesis of ECAPG and ECACYC, which represent targets for development of small molecules weakening the OM. In addition, this will allow genetic analyses of ECA function, providing insights into Enterobacterales biology.

项目总结 美国每年发生近300万例抗药性感染。这个问题尤其严重。 在革兰氏阴性细菌中，包裹房水周质的外膜(OM)起到了 可阻挡多种抗生素的渗透性屏障。我们对肠杆菌属的OM感兴趣(例如， 大肠杆菌、沙门氏菌、克雷伯氏菌)，它们适应于富含有毒分子的肠道环境，如 作为胆盐，需要特别强烈的OM。很明显，OM的渗透性可以是 被细胞的生理状态改变的。具体地说，诸如营养限制之类的压力会导致 OM渗透性屏障的加固。阐明导致这种强化意志的途径 为开发能够削弱OM渗透性屏障的小分子开辟了新的靶点。我们 发现了肠杆菌共同抗原(ECA)，这是肠杆菌属OM和 周质，对于OM在胁迫下的防渗性是重要的。两种形式的ECA(磷脂连接的ECA (ECAPG)和循环ECA(ECACYC)具有与OM通透性相关的不同作用；然而，它们的精确 功能仍然未知，部分原因是人们对其生物发生的许多步骤知之甚少。 我们的长期目标是了解ECA的生物发生，以促进功能研究和确定潜在的 抗菌靶标。具体地说，这个项目的目的是阐明在大肠杆菌K12中，和 导致抗生素耐药性的ECA形式的生物发生中未知的步骤。生化反应是 产生这些形式的ECA所需的，但负责这些步骤和调节的基因 这些步骤中有哪些在很大程度上是未知的。中心假设是ECAPG和ECACyC是可以区分的 通过它们独特的生物合成基因和调节作用。这一假设将通过以下几个方面得到解决 目的：确定ECA成为形成ECAPG的磷脂头基所需的基因和底物 利用基因与其他生物合成途径的相互作用(目标1)；阐明相关因素和机制 在使用我们发现的抗生素敏感性抑制表型的ECACyC生物发生中(目标2)；并发现 我们发现的ECA调节的两个新途径的机制(目标3)。从概念上讲， 创新的目标将通过高通量基因组学、基因筛选和 选择和生化技术。该项目的完成将确定重要的基因和残基 ECAPG和ECACyC的生物发生，它们代表了小分子的发展靶点，削弱了 奥姆。此外，这将允许对ECA功能的遗传分析，提供对肠杆菌生物学的洞察。