The ability of some bacteria within a population to tolerate antibiotic treatment is often attributed to prolonged bacterial infection. Unlike antibiotic resistance, which generally results from genetic mutations or plasmid transfer, antibiotic tolerance usually refers to the phenomenon that a subgroup of cells can survive high dose antibiotic treatment as a result of phenotypic heterogeneity. Previous studies mainly associate antibiotic tolerance with cell dormancy, by hypothesizing that the lethal effects of antibiotics are disabled due to the extremely slow metabolic and proliferation rates in dormant bacteria. However, less is known about how surviving bacteria subsequently escape from the dormant state and resuscitate, which is equally important for disease recurrence. Here we monitored the process of bacterial antibiotic tolerance and regrowth at the single-cell level, and found that each individual survival cell shows different dormancy depth, which in return regulates whether and when it can resume growth after removal of antibiotic. The persister cells are considered to be in shallow dormancy depth, while the viable but non-culturable cells (VBNC cells) are in deep dormancy depth. We further implemented time-lapse fluorescent imaging and biochemical analysis to establish that dynamic endogenous protein aggregation is an important indicator of bacterial dormancy depth. For cells to leave the dormant state and resuscitate, clearance of cellular protein aggregates and recovery of proteostasis are required. Through additional mutagenesis studies, we found the ability to recruit functional DnaK-ClpB machineries, which facilitate protein disaggregation in an ATP-dependent manner, determines the timeline (whether and when) for bacterial regrowth. Better understanding of the key factors regulating bacterial regrowth after surviving antibiotic attack could lead to new therapeutic strategies for combating bacterial antibiotic tolerance.

Dynamic protein aggregation regulates bacterial dormancy depth critical for antibiotic tolerance