The dynamic transition of persistence towards the VBNC state during stationary phase is driven by protein aggregation

Abstract

Decades of research into bacterial persistence has been unable to fully characterize this antibiotic-tolerant phenotype, thereby hampering the development of therapies effective against chronic infections. Although some active persister mechanisms have been identified, the prevailing view is that cells become persistent because they enter a dormant state. We therefore characterized starvation-induced dormancy in Escherichia coli. Our findings indicate that dormancy develops gradually; persistence strongly increases during stationary phase and decreases again as persisters enter the viable but nonculturable (VBNC) state. Importantly, we show that dormancy development is tightly associated with progressive protein aggregation, which occurs concomitantly with ATP depletion during starvation. Persisters contain protein aggregates in an early developmental stage while VBNC cells carry more mature aggregates. Finally, we show that at least one persister protein, ObgE, works by triggering aggregation and thereby changing the dynamics of persistence and dormancy development. These findings provide evidence for a genetically-controlled, gradual development of persisters and VBNC cells through protein aggregation.

Protein Aggregation as a Bacterial Strategy to Survive Antibiotic Treatment

“…This hypothesis is supported by the observation that in Escherichia coli persisters and VBNC cells more often contain aggregates than nondormant cells (Pu et al, 2019;Yu et al, 2019;Cesar et al, 2020) and that protein aggregation in these dormant cells occurs more intensely (Dewachter et al, 2021). Moreover, the intensity of aggregation, measured by expression of IbpA-msfGFP and therefore the amount of proteins that are aggregated, appears to be correlated to dormancy depth at the single-cell level; shallowly dormant persisters carry low intensity aggregates, while deeper dormant VBNC cells contain more intense aggregates (Dewachter et al, 2021). However, not all cells with protein aggregates are dormant (Dewachter et al, 2021), which suggests that a certain level or threshold of aggregation is needed in the cells to shift to the dormant state.…”

“…Recently, experimental support for this hypothesis has emerged suggesting that both persistence and the VBNC state are linked to protein aggregation and that progressive aggregation can drive the development from persistence to the VBNC state (Figure 1) (Pu et al, 2019;Dewachter et al, 2021). Indeed, previous work also demonstrated a link between aggregation and persistence (Leszczynska et al, 2013;Mordukhova and Pan, 2014;Pu et al, 2019;Yu et al, 2019;Dewachter et al, 2021;Huemer et al, 2021). In this review, we elaborate on the steadily growing number of studies linking protein aggregation and persistence.…”

“…Due to their lower level of organization, amorphous aggregates are not related to these new functionalities (Bednarska et al, 2013). Another beneficial effect of aggregates is their ability to protect the cell against stress (Leszczynska et al, 2013;Mordukhova and Pan, 2014;Govers et al, 2018;Pu et al, 2019;Yu et al, 2019;Dewachter et al, 2021;Huemer et al, 2021). It is not known yet if this increased stress tolerance is a general property or if it is linked to a specific type and/or composition of aggregates.…”

“…Some examples of stresses that induce both dormant phenotypes are nutrient (Betts et al, 2002;Mishra et al, 2012), oxidative (Wu et al, 2012;Li et al, 2014), osmotic (Roth et al, 1988;Murakami et al, 2005), acid (Cunningham et al, 2009;Hong W. et al, 2012), and temperature stress (Oliver et al, 1991;Cardoso et al, 2010). Additionally, both persistence and the VBNC state are linked to the general stress response (Boaretti et al, 2003;Murakami et al, 2005), toxin-antitoxin modules (Moyed and Bertrand, 1983;Korch and Hill, 2006), and protein aggregation (Leszczynska et al, 2013;Mordukhova and Pan, 2014;Pu et al, 2019;Yu et al, 2019;Cesar et al, 2020;Dewachter et al, 2021;Huemer et al, 2021). Persisters and VBNC cells thus share many similarities.…”

“…Persisters and VBNC cells thus share many similarities. Therefore, it is hypothesized that they represent different stages of the same dormancy program with different dormancy depths; persisters and VBNC cells reside in a shallow and deep dormant state, respectively (Li et al, 2014;Ayrapetyan et al, 2015;Kim et al, 2018;Pu et al, 2019;Dewachter et al, 2021).…”