ATP-Dependent Dynamic Protein Aggregation Regulates Bacterial Dormancy Depth Critical for Antibiotic Tolerance

Abstract

Graphical Abstract Highlights d The degree of drug-tolerant cells being dormant can be measured by ''dormancy depth'' d Cellular dark foci, proved to be protein aggresomes, indicate dormancy depth d Depletion of intracellular ATP is the major force driving aggresomes formation d DnaK is vital in the disaggregation of aggresomes when a dormant cell resuscitates In this work, Pu et al. introduced a concept of ''dormancy depth'' that provides a unifying framework for understanding both persisters and viable but non-culturable cells. Subsequent mechanistic investigations revealed how ATP-dependent dynamic protein aggregation regulates cellular dormancy and resuscitation, the fine control of which facilitates bacterial drug tolerance. SUMMARYCell dormancy is a widespread mechanism used by bacteria to evade environmental threats, including antibiotics. Here we monitored 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 the lag time for cell resuscitation after removal of antibiotic. We further established that protein aggresome-a collection of endogenous protein aggregates-is an important indicator of bacterial dormancy depth, whose formation is promoted by decreased cellular ATP level. For cells to leave the dormant state and resuscitate, clearance of protein aggresome and recovery of proteostasis are required. We revealed that the ability to recruit functional DnaK-ClpB machineries, which facilitate protein disaggregation in an ATP-dependent manner, determines the lag time 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.

ppGpp Ribosome Dimerization Model for Bacterial Persister Formation and Resuscitation

“…By inhibiting the activity of ribosomes, we have developed means to convert E. coli cells into a 20 population that consists solely of persister cells (6) so that insights may be made about persister cell resuscitation. Using this approach, which has been verified by six independent labs to produce bona fide persister cells (7)(8)(9)(10)(11)(12), we have shown that persister cells resuscitate rapidly primarily in response to external signals (13), such as fresh nutrients, rather than stochastically (14). Critically, persister cell resuscitation is heterogeneous and depends on the number of active ribosomes; once a threshold of active ribosomes is reached, persister cells elongate and divide (13).…”

Persister cells resuscitate via ribosome modification by 23S rRNA pseudouridine synthase RluD

“…Furthermore, our method has been used to (i) determine that persister cells wake via ribosome activation (Kim et al, 2018b) and (ii) by activating chemotaxis (Yamasaki et al, 2019), (iii) show that the cells capable of resuscitation in a viable but not culturable population are equivalent to persister cells (Kim et al, 2018a), (iv) identify compounds that kill persister cells and (v) reveal that the alarmone ppGpp directly creates persister cells by stimulating ribosome dimerization . In addition, our method to generate a high population of persister cells has been utilized by at least six independent groups (Grassi et al, 2017;Cui et al, 2018;Narayanaswamy et al, 2018;Sulaiman et al, 2018;Tkhilaishvili et al, 2018;Pu et al, 2019).…”

Persister Cells Resuscitate via Ribosome Modification by 23S rRNA Pseudouridine Synthase RluD

“…The persister cells generated in this way have been (i) confirmed eight ways (19), (ii) used to determine that persister cells wake via ribosome activation (19) and chemotaxis (20), (iii) used to show that the cells capable of resuscitation in a viable but not culturable 95 population are equivalent to persister cells (15), (iv) used to identify compounds that kill persister cells (27), and (v) used to show that the alarmone ppGpp directly creates persister cells by stimulating ribosome dimerization (16). In addition, our method to generate a high population of persister cells has been utilized by at least six independent groups (28)(29)(30)(31)(32)(33).…”