Table_1_High-Throughput Proteomics Identifies Proteins With Importance to Postantibiotic Recovery in Depolarized Persister Cells.DOCX

Bacterial populations produce phenotypic variants called persisters to survive harmful conditions. Persisters are highly tolerant to antibiotics and repopulate environments after the stress has vanished. In order to resume growth, persisters have to recover from the persistent state, but the processes behind recovery remain mostly elusive. Deciphering these processes is an essential step toward understanding the persister phenomenon in its entirety. High-throughput proteomics by mass spectrometry is a valuable tool to assess persister physiology during any stage of the persister life cycle, and is expected to considerably contribute to our understanding of the recovery process. In the present study, an Escherichia coli strain, that overproduces the membrane-depolarizing toxin TisB, was established as a model for persistence by the use of high-throughput proteomics. Labeling of TisB persisters with stable isotope-containing amino acids (pulsed-SILAC) revealed an active translational response to ampicillin, including several RpoS-dependent proteins. Subsequent investigation of the persister proteome during postantibiotic recovery by label-free quantitative proteomics identified proteins with importance to the recovery process. Among them, AhpF, a component of alkyl hydroperoxide reductase, and the outer membrane porin OmpF were found to affect the persistence time of TisB persisters. Assessing the role of AhpF and OmpF in TisB-independent persisters demonstrated that the importance of a particular protein for the recovery process strongly depends on the physiological condition of a persister cell. Our study provides important insights into persister physiology and the processes behind recovery of depolarized cells.

细菌种群会产生名为persisters（持留菌）的表型变体，以在有害环境中存活。持留菌对各类抗生素具有高度耐受性，并可在胁迫解除后重新定植于环境中。若要恢复生长，持留菌必须从持留状态中脱离并复苏，但其背后的具体复苏机制迄今仍大多未被阐明。全面解析这些机制，是完整理解持留菌现象的关键一步。基于质谱分析法的高通量蛋白质组学，是评估持留菌生命周期任一阶段生理特性的有力工具，有望大幅推动我们对持留菌复苏过程的认知。

本研究借助高通量蛋白质组学技术，构建了过量表达膜去极化毒素TisB的大肠杆菌（Escherichia coli）菌株，并将其确立为持留研究的模型体系。通过稳定同位素标记氨基酸对TisB持留菌进行脉冲-SILAC（pulsed-SILAC）标记，结果揭示了其对氨苄青霉素的活跃翻译响应，其中包含多种RpoS依赖型蛋白。后续通过无标记定量蛋白质组学，对持留菌在抗生素清除后的复苏阶段的蛋白质组进行分析，鉴定出与复苏过程密切相关的关键蛋白。其中，烷基过氧化物还原酶的组分AhpF以及外膜孔蛋白OmpF被证实可影响TisB持留菌的持留时长。通过评估AhpF与OmpF在不依赖TisB的持留菌中的作用，我们证实：特定蛋白对复苏过程的重要性，强烈依赖于持留菌细胞的生理状态。本研究为持留菌的生理特性以及去极化细胞的复苏机制提供了重要的学术见解。