Abstract
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.
Highlights
The rise of antibiotic resistance among pathogens is a major threat to the human health care system (Lewis, 2013), and gains ever-expanding attention
Among the 24 proteins with increased abundance during recovery, we identified a component of the alkyl hydroperoxide reductase (AhpF) and an outer membrane porin (OmpF)
E. coli strains were grown in lysogeny broth (LB) or M9 minimal medium at 37◦C with continuous shaking at 180 rpm
Summary
The rise of antibiotic resistance among pathogens is a major threat to the human health care system (Lewis, 2013), and gains ever-expanding attention. Bacteria have developed alternative strategies to survive an antibiotic challenge. Bigger realized very early after the introduction of penicillin that a small subpopulation of otherwise susceptible Staphylococcus aureus cultures survived a penicillin treatment for several days. He termed these surviving cells “persisters” (Bigger, 1944). Persisters are transiently drug-tolerant phenotypic variants within
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