Real-time monitoring of peptidoglycan synthesis by membrane-reconstituted penicillin-binding proteins.
Real-time monitoring of peptidoglycan synthesis by membrane-reconstituted penicillin-binding proteins.
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DOI：
10.7554/elife.61525
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发表时间：
2021-02-24
期刊：
影响因子：
7.7
通讯作者：
Vollmer W
中科院分区：
文献类型：
--
作者：
Hernández-Rocamora VM;Baranova N;Peters K;Breukink E;Loose M;Vollmer W
肽聚糖是细菌细胞被膜的重要组成部分，其围绕细胞质膜以保护细胞免受渗透裂解。重要的抗生素如β-内酰胺和糖肽靶向肽聚糖生物合成。A类青霉素结合蛋白（PBP）是一种双功能的膜结合肽聚糖酶，它通过转肽作用将聚糖链固定并连接相邻的茎肽。这些酶在其生理膜环境中如何工作还知之甚少。在这里，我们开发了一种新的Förster共振能量转移为基础的测定遵循真实的时间的反应A类PBPs重组脂质体或支持的脂质双层，并应用此测定与PBP 1B同系物大肠杆菌，铜绿假单胞菌，鲍曼不动杆菌在存在或不存在其同源脂蛋白激活剂。我们的试验将允许解开在脂质双层环境中肽聚糖合成的机制，并可进一步开发用于高通量筛选新的抗菌剂。
Peptidoglycan is an essential component of the bacterial cell envelope that surrounds the cytoplasmic membrane to protect the cell from osmotic lysis. Important antibiotics such as β-lactams and glycopeptides target peptidoglycan biosynthesis. Class A penicillin-binding proteins (PBPs) are bifunctional membrane-bound peptidoglycan synthases that polymerize glycan chains and connect adjacent stem peptides by transpeptidation. How these enzymes work in their physiological membrane environment is poorly understood. Here, we developed a novel Förster resonance energy transfer-based assay to follow in real time both reactions of class A PBPs reconstituted in liposomes or supported lipid bilayers and applied this assay with PBP1B homologues from Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii in the presence or absence of their cognate lipoprotein activator. Our assay will allow unravelling the mechanisms of peptidoglycan synthesis in a lipid-bilayer environment and can be further developed to be used for high-throughput screening for new antimicrobials.