Department of Biology, Texas A&M University, College Station, Texas, USA.
Institute for Integrative Genomics, Princeton Universitygrid.16750.35, Princeton, New Jersey, USA.
mBio. 2021 Dec 21;12(6):e0284621. doi: 10.1128/mBio.02846-21. Epub 2021 Nov 23.
The Gram-negative cell envelope is a complex structure delineating the cell from its environment. Recently, we found that enterobacterial common antigen (ECA) plays a role maintaining the outer membrane (OM) permeability barrier, which excludes toxic molecules including many antibiotics. ECA is a conserved carbohydrate found throughout (e.g., Salmonella, Klebsiella, and ). There are two OM forms of ECA (phosphoglyceride-linked ECA and lipopolysaccharide-linked ECA) and one periplasmic form of ECA (cyclic ECA). ECA, found in the outer leaflet of the OM, consists of a linear ECA oligomer attached to phosphoglyceride through a phosphodiester linkage. The process through which ECA is produced from polymerized ECA is unknown. Therefore, we set out to identify genes interacting genetically with ECA biosynthesis in Escherichia coli K-12 using the competition between ECA and peptidoglycan biosynthesis. Through transposon-directed insertion sequencing, we identified an interaction between and ECA biosynthesis. ElyC is an inner membrane protein previously shown to alter peptidoglycan biosynthesis rates. We found Δ was lethal specifically in strains producing ECA without other ECA forms, suggesting ECA biosynthesis impairment or dysregulation. Further characterization suggested ElyC inhibits ECA synthesis in a posttranscriptional manner. Moreover, the full impact of ElyC on ECA levels requires the presence of ECA. Our data demonstrate ECA can regulate ECA synthesis in strains wild type for . Overall, our data demonstrate ElyC and ECA act in a novel pathway that regulates the production of ECA, supporting a model in which ElyC provides feedback regulation of ECA production based on the periplasmic levels of ECA. Enterobacterial common antigen (ECA) is a conserved polysaccharide present on the surface of the outer membrane (OM) and in the periplasm of the many pathogenic bacteria belonging to , including Klebsiella pneumoniae, Salmonella enterica, and Yersinia pestis. As the OM is a permeability barrier that excludes many antibiotics, synthesis pathways for OM molecules are promising targets for antimicrobial discovery. Here, we elucidated, in E. coli K-12, a new pathway for the regulation of biosynthesis of one cell surface form of ECA, ECA. In this pathway, an inner membrane protein, ElyC, and the periplasmic form of ECA, ECA, genetically interact to inhibit the synthesis of ECA, potentially through feedback regulation based on ECA levels. This is the first insight into the pathway responsible for synthesis of ECA and represents a potential target for weakening the OM permeability barrier. Furthermore, this pathway provides a tool for experimental manipulation of ECA levels.
革兰氏阴性细胞包膜是一个复杂的结构，将细胞与其环境分隔开来。最近，我们发现肠杆菌共同抗原（ECA）在维持外膜（OM）渗透性屏障方面起着作用，该屏障排除了包括许多抗生素在内的有毒分子。ECA 是一种保守的碳水化合物，存在于多种细菌中（例如沙门氏菌、克雷伯氏菌和耶尔森氏菌）。ECA 有两种 OM 形式（磷酸甘油酯连接的 ECA 和脂多糖连接的 ECA）和一种周质形式的 ECA（环 ECA）。位于 OM 外叶的 ECA 由通过磷酸二酯键连接到磷酸甘油酯的线性 ECA 低聚物组成。ECA 从聚合的 ECA 产生的过程尚不清楚。因此，我们使用 ECA 和肽聚糖生物合成之间的竞争，通过转座子定向插入测序来确定与大肠杆菌 K-12 中 ECA 生物合成在遗传上相互作用的基因。通过转座子定向插入测序，我们发现了 和 ECA 生物合成之间的相互作用。ElyC 是一种内膜蛋白，先前已被证明可以改变肽聚糖生物合成的速度。我们发现 Δ 在仅产生 ECA 而没有其他 ECA 形式的菌株中是致命的，这表明 ECA 生物合成受损或失调。进一步的特征表明，ElyC 以转录后方式抑制 ECA 的合成。此外，ElyC 对 ECA 水平的全部影响需要 ECA 的存在。我们的数据表明 ECA 可以在野生型菌株中调节 ECA 的合成。总体而言，我们的数据表明 ElyC 和 ECA 以一种新的途径发挥作用，该途径调节 ECA 的产生，支持 ElyC 根据 ECA 的周质水平提供 ECA 产生的反馈调节的模型。肠杆菌共同抗原（ECA）是一种保守的多糖，存在于许多属于肠杆菌科的致病性细菌的外膜（OM）表面和周质中，包括肺炎克雷伯菌、沙门氏菌和鼠疫耶尔森菌。由于 OM 是一种排斥许多抗生素的渗透性屏障，因此 OM 分子的合成途径是发现抗菌药物的有希望的靶标。在这里，我们在大肠杆菌 K-12 中阐明了一种调节一种细胞表面 ECA 形式 ECA 生物合成的新途径。在该途径中，一种内膜蛋白 ElyC 和 ECA 的周质形式相互作用，抑制 ECA 的合成，可能通过基于 ECA 水平的反馈调节。这是了解负责 ECA 合成的途径的第一见解，代表了削弱 OM 渗透性屏障的潜在靶标。此外，该途径为实验操纵 ECA 水平提供了一种工具。
