Biogenesis of cyclic and phospholipid-linked enterobacterial common antigen

项目摘要 在美国，每年有近300万例抗生素耐药性感染。这一问题尤为突出 在革兰氏阴性菌中，包围水周质的外膜（OM）起着 渗透屏障能够排除许多抗生素。我们感兴趣的是肠球菌目的OM（例如， 大肠杆菌属、沙门氏菌属、克雷伯氏菌属），其适应于富含毒性分子的肠道环境，例如 作为胆汁盐，需要特别强的OM。很明显，OM的渗透性可以是 被细胞的生理状态所改变。具体来说，营养限制等压力可能导致 加强OM渗透屏障。阐明负责这种强化的途径将 导致新的目标，为发展的小分子，可以削弱OM渗透屏障。我们 已经发现肠杆菌共同抗原（ECA），一种肠杆菌目OM的保守组分， 周质，是重要的OM不渗透性在胁迫下。两种形式的ECA（磷脂连接的ECA） （ECAPG）和环状ECA（ECACYC））具有与OM渗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的生物起源，以促进功能研究并确定潜在的 抗菌目标。具体而言，本项目旨在阐明大肠杆菌K12中， ECA形式的生物发生中的未知步骤导致抗生素耐药性。生化反应是 需要这些形式的ECA被生产，但基因负责这些步骤和调控， 这些步骤在很大程度上是未知的。中心假设是ECAPG和ECACYC可以区分 通过其独特的生物合成基因和调节作用。这个假设将通过以下内容来解决 目的：鉴定ECA成为形成ECAPG的磷脂头基所必需的基因和底物 利用与其他生物合成途径的遗传相互作用（目标1）;阐明相关因素和机制 在ECACYC生物发生中，我们发现了抗生素敏感性抑制表型（目的2）;并揭示了 我们发现的两种新的ECA调节途径的机制（目的3）。这些概念 创新的目标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的重要性， ECAPG和ECACYC的生物发生，它们代表了小分子发育的靶点， OM。此外，这将允许ECA功能的遗传分析，提供对Enterobacterales生物学的见解。

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.