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

基本信息

批准号：
10793673
负责人：
Angela Marie Mitchell
金额：
$ 7.19万
依托单位：
TEXAS A&M UNIVERSITY
依托单位国家：
美国
项目类别：
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/10793673
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

项目摘要

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.

项目概要美国每年发生近 300 万例抗生素耐药感染。这个问题尤其严重在革兰氏阴性细菌中，包围水周质的外膜（OM）充当能够排除许多抗生素的渗透性屏障。我们对肠杆菌目的 OM 感兴趣（例如，埃希氏菌、沙门氏菌、克雷伯氏菌），它们适应富含有毒分子的肠道环境，例如作为胆汁盐，需要特别强的 OM。很明显，OM 的磁导率可以是因细胞的生理状态而改变。具体来说，营养限制等压力可能会导致增强 OM 渗透屏障。阐明负责这种强化的途径将导致开发可以削弱 OM 渗透屏障的小分子的新目标。我们发现了肠杆菌共同抗原（ECA），肠杆菌目 OM 的保守成分，周质对于 OM 在压力下的不渗透性很重要。 ECA 的两种形式（磷脂连接 ECA (ECAPG) 和循环 ECA (ECACYC)) 在 OM 渗透性方面具有不同的作用；然而，他们的精确其功能仍然未知，部分原因是其生物起源的许多步骤尚不清楚。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在的抗菌目标。具体来说，该项目旨在阐明大肠杆菌 K12 中的调节和 ECA 形式的生物合成中导致抗生素耐药性的未知步骤。生化反应是这些形式的 ECA 的产生以及负责这些步骤和调节的基因是必需的这些步骤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。该假设将通过以下内容得到解决目标：确定 ECA 成为形成 ECAPG 的磷脂头基所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 生物发生中使用我们发现的抗生素敏感性抑制表型（目标 2）；并揭开我们发现的 ECA 调节的两种新途径的机制（目标 3）。这些概念上将通过高通量基因组学、遗传筛选和选择和生化技术。该项目的完成将鉴定出重要的基因和残基 ECAPG 和 ECACYC 的生物发生，代表弱化小分子开发的目标哦。此外，这将允许对 ECA 功能进行遗传分析，从而提供对肠杆菌生物学的见解。

项目成果

期刊论文数量（3）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Suppressor Mutants: History and Today's Applications.

抑制突变体：历史和当今的应用。

DOI：
发表时间：
2021-12-15
期刊：
影响因子：
0
作者：
Bautista, David E;Carr, Joseph F;Mitchell, Angela M
通讯作者：
Mitchell, Angela M

Functional diversification of gram-negative intermembrane phospholipid transporters by intrinsic substrate preference.

通过内在底物偏好实现革兰氏阴性膜间磷脂转运蛋白的功能多样化。

DOI：
发表时间：
2024-03-20
期刊：
影响因子：
0
作者：
Rai, Ashutosh K;Sawasato, Katsuhiro;Bennett, Haley C;Kozlova, Anastasiia;Sparagna, Genevieve C;Bogdanov, Mikhail;Mitchell, Angela M
通讯作者：
Mitchell, Angela M

Phosphatidylglycerol Is the Lipid Donor for Synthesis of Phospholipid-Linked Enterobacterial Common Antigen.

磷脂酰甘油是合成磷脂连接肠杆菌共同抗原的脂质供体。