The Dynamic Transition of Persistence toward the Viable but Nonculturable State during Stationary Phase Is Driven by Protein Aggregation

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 mecha-nisms 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 develop-ment is tightly associated with progressive protein aggregation, which occurs concomi-tantly 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, even at endogenous levels, and thereby changing the dynamics of persistence and dor-mancy development. These findings provide evidence for a genetically controlled, grad-ual development of persisters and VBNC cells through protein aggregation. IMPORTANCE While persistence and the viable but nonculturable (VBNC) state are cur-rently investigated in isolation, our results strongly indicate that these phenotypes rep-resent different stages of the same dormancy program and that they should therefore be studied within the same conceptual framework. Moreover, we show here for the first time that the dynamics of protein aggregation perfectly match the onset and fur-ther development of bacterial dormancy and that different dormant phenotypes are linked to different stages of protein aggregation. Our results thereby strongly hint at a causal relationship between both. Because many conditions known to trigger persist-ence are also known to influence aggregation, it is tempting to speculate that a variety of different persister pathways converge at the level of protein aggregation. If so, aggregation could emerge as a general principle that underlies the development of persistence which could be exploited for the design of antipersister therapies.

Automated summary

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, even at endogenous levels, 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. IMPORTANCE While persistence and the viable but nonculturable (VBNC) state are currently investigated in isolation, our results strongly indicate that these phenotypes represent different stages of the same dormancy program and that they should therefore be studied within the same conceptual framework. Moreover, we show here for the first time that the dynamics of protein aggregation perfectly match the onset and further development of bacterial dormancy and that different dormant phenotypes are linked to different stages of protein aggregation. Our results thereby strongly hint at a causal relationship between both. Because many conditions known to trigger persistence are also known to influence aggregation, it is tempting to speculate that a variety of different persister pathways converge at the level of protein aggregation. If so, aggregation could emerge as a general principle that underlies the development of persistence which could be exploited for the design of antipersister therapies.