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: 10621314
• 负责人: Angela Marie Mitchell
• 金额: $ 36.7万
• 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/10621314
• 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; Cyclization; Defect; Development; Enteral; Environment; Escherichia; Escherichia coli; Escherichia coli K12; Exclusion; 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; Surface; Techniques; Transcription Regulation Pathway; United States; Work; Yersinia; antibiotic resistant infections; antimicrobial; aqueous; bile salts; cell envelope; economic cost; enterobacterial common antigen; experience; genetic analysis; genetic selection; improved; innovation; insight; interest; member; novel; periplasm; posttranscriptional; 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.

项目摘要 在美国，每年发生近300万种抗生素感染。这个问题尤其严重 在革兰氏阴性细菌中，围绕着含水的外膜的外膜（OM）充当 能够排除许多抗生素的渗透性屏障。我们对肠杆菌的OM感兴趣（例如 Escherichia，沙门氏菌，克雷伯氏菌），适用于富含有毒分子的肠环境， 作为胆汁盐，需要特别强大的OM。很明显，OM的渗透性可能是 通过细胞的生理状态改变。具体而言，诸如营养限制之类的压力可能导致 加强OM渗透性屏障。阐明负责这种加强的途径将 导致新的靶标，用于开发可以削弱OM渗透性屏障的小分子。我们 已经发现肠杆菌普通抗原（ECA），肠杆菌的保守成分OM和 周期，对于在压力下对OM不渗透很重要。两种形式的ECA（磷脂连接的ECA） （ECAPG）和环状ECA（ECACACACC）具有与OM渗透性有关的不同作用；但是，它们的精确度 部分功能仍然未知，部分原因是其生物发生的许多步骤对待很少了解。 我们的长期目标是了解ECA的生物发生，以促进功能研究并确定潜力 抗菌靶。具体而言，该项目旨在在大肠杆菌K12中阐明和 ECA形式生物发生的未知步骤，导致抗生素耐药性。生化反应是 生产这些形式的ECA所必需 这些步骤在很大程度上是未知的。中心假设是ECAPG和ECACACAC可以区分 通过其独特的生物合成基因和调节作用。该假设将通过以下来解决 目的：确定ECA成为形成ECAPG的磷脂头组所需的基因和底物 使用与其他生物合成途径的遗传相互作用（AIM 1）；阐明涉及的因素和机制 在Ecacyc生物发生中，我们发现了我们发现的抗生素敏感性抑制表型（AIM 2）；并发现 我们发现了ECA调节的两种新型途径的机制（AIM 3）。从概念上讲 将通过高通量基因组学，遗传筛选和 选择和生化技术。该项目的完成将确定基因和残基对 ECAPG和ECACACAC的生物发生，代表了弱分子发展的靶标 om。此外，这将允许对ECA功能的遗传分析，从而提供对肠杆菌生物学的见解。