Spanka Daniel-Timon, Konzer Anne, Edelmann Daniel, Berghoff Bork A
Institute for Microbiology and Molecular Biology, Justus Liebig University Giessen, Giessen, Germany.
Biomolecular Mass Spectrometry, Max-Planck-Institute for Heart and Lung Research, Bad Nauheim, Germany.
Front Microbiol. 2019 Mar 6;10:378. doi: 10.3389/fmicb.2019.00378. eCollection 2019.
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 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.
细菌群体产生称为持留菌的表型变体以在有害条件下存活。持留菌对抗生素具有高度耐受性，并在应激消失后重新在环境中繁殖。为了恢复生长，持留菌必须从持留状态中恢复过来，但恢复背后的过程大多仍不清楚。破解这些过程是全面理解持留菌现象的关键一步。通过质谱进行的高通量蛋白质组学是评估持留菌生命周期任何阶段持留菌生理学的宝贵工具，有望极大地促进我们对恢复过程的理解。在本研究中，通过使用高通量蛋白质组学，建立了一种过量产生膜去极化毒素TisB的菌株作为持留菌模型。用含稳定同位素的氨基酸对TisB持留菌进行标记（脉冲式稳定同位素标记氨基酸定量法）揭示了对氨苄青霉素的活跃翻译反应，包括几种依赖RpoS的蛋白质。随后通过无标记定量蛋白质组学对抗生素后恢复期间的持留菌蛋白质组进行研究，确定了对恢复过程重要的蛋白质。其中，烷基过氧化氢还原酶的一个组分AhpF和外膜孔蛋白OmpF被发现会影响TisB持留菌的持留时间。评估AhpF和OmpF在不依赖TisB的持留菌中的作用表明，特定蛋白质对恢复过程的重要性很大程度上取决于持留菌细胞的生理状态。我们的研究为持留菌生理学以及去极化细胞恢复背后的过程提供了重要见解。
