Bacterial persisters: molecular mechanisms and therapeutic development

Abstract

Persisters refer to genetically drug susceptible quiescent (non-growing or slow growing) bacteria that survive in stress environments such as antibiotic exposure, acidic and starvation conditions. These cells can regrow after stress removal and remain susceptible to the same stress. Persisters are underlying the problems of treating chronic and persistent infections and relapse infections after treatment, drug resistance development, and biofilm infections, and pose significant challenges for effective treatments. Understanding the characteristics and the exact mechanisms of persister formation, especially the key molecules that affect the formation and survival of the persisters is critical to more effective treatment of chronic and persistent infections. Currently, genes related to persister formation and survival are being discovered and confirmed, but the mechanisms by which bacteria form persisters are very complex, and there are still many unanswered questions. This article comprehensively summarizes the historical background of bacterial persisters, details their complex characteristics and their relationship with antibiotic tolerant and resistant bacteria, systematically elucidates the interplay between various bacterial biological processes and the formation of persister cells, as well as consolidates the diverse anti-persister compounds and treatments. We hope to provide theoretical background for in-depth research on mechanisms of persisters and suggest new ideas for choosing strategies for more effective treatment of persistent infections.

Fig. 1

Milestone events in persister research. Since the initial discovery of bacterial persistence phenomenon in 1942, significant findings have progressively unveiled the clinical implications of bacterial persisters in human diseases. Created with BioRender.com

Fig. 2

Distinguishing characteristics of persister cells from resistant and tolerant bacteria in in vitro models. According to the definition reported in the literature, when exposed to antibiotics, the homogeneous population of sensitive (green), persister (red), “tolerant,” (yellow) or resistant bacteria (purple) exhibit different scenarios (a) and antibiotic killing kinetics (b). Following the addition of a bactericidal antibiotic, sensitive bacteria could be completely killed and its kill curve is a decreasing straight line. Even after the antibiotic is removed, the bacteria cannot revive. Persister refers to a small fraction within the bacterial population that, when exposed to antibiotics, the bulk growing bacteria are killed rapidly, while the persisters are still alive. Once the antibiotic is removed, persisters can resuscitate and resume growth. “Tolerant bacteria” is a whole population of bacteria with persister-like tolerance, which are killed more slowly than normal growing bacteria and are capable of regrowth upon antibiotic removal. Resistant cells, unaffected by antibiotics, grow in the presence of the antibiotic and exhibit an ascending straight-line without being killed, indicating their survival and proliferation despite antibiotic exposure. Created with BioRender.com

Fig. 3

The coexistence and dynamic interconversions of sensitive bacteria, persisters/tolerant bacteria and resistant bacteria during host infections. In the host environment, the bacterial population is heterogeneous and significantly more complex than that in vitro. The metabolic activities of bacteria within the persister population are not uniform; there are bacteria with slow metabolism known as shallow persisters and those with metabolic dormancy known as deep persisters. From an evolutionary perspective, persistence under certain conditions can lead to resistance development, via mutations or transfer of resistance genes. Created with BioRender.com

Fig. 4

Mechanisms of persister formation via TA modules. The anti-toxins of type II TA modules are proteins that are usually degraded by the protease ClpP or Lon in response to (p)ppGpp signaling. These toxins mediate bacteria to enter a persistence state by disrupting replication and translation processes, such as interfering with DNA gyrase, acting as mRNA endonucleases, inactivating glutamyl-tRNA synthetase (GltX) and acetylate-tRNA. The anti-toxins of type I TA modules are antisense RNAs which are activated by the “SOS” response and (p)ppGpp signaling. These toxins are usually small proteins that insert and form pores in bacterial membranes, causing loss of proton motive force (PMF) and ATP production. Created with BioRender.com

Fig. 5

Mechanisms of persister formation via energy metabolism. Persisters have lower metabolic activity than non-persisters and this reduced energy metabolic activity could be mediated through (a) G3P metabolism, (b) aerobic respiration, (c) tricarboxylic acid (TCA) cycle and (d) methylcitrate cycle (MCC), which may allow persisters to enter a dormant state and survive in adverse conditions. For example, reduced ATP levels decrease the activity of ATP-dependent antibiotic targets, while decreased proton motive force (PMF) restricts the entrance of antibiotics such as aminoglycosides and thus enhances the tolerance ability. Created with BioRender.com

Fig. 6

Mechanisms of persister formation via SOS response, stringent response and quorum sensing. The SOS response is triggered by DNA damage, facilitating genetic repair to enhance cell survival under stress. It also contributes to the activation of type I TA module TisB/IstR as well. The stringent response is a global response to nutrition deprivation or limitation (such as carbon, amino acid nitrogen and phosphate), where (p)ppGpp is an important messenger molecule. This response induces bacterial dormancy through downstream pathways involving TA modules and ribosomes. Quorum sensing is a bacterial communication process that coordinates behavior based on population density. Bacteria release signaling molecules (AHL, CSP, indole etc.), which can enhance efflux pumps or disrupt metabolism. Created with BioRender.com

Fig. 7

Proposed drug combination strategies for treating persistent infections. (1) Combination of drugs killing both metabolically active bacteria and persister cells per Yin-Yang model 24. (2) Compounds sensitizing persisters or inhibiting persister formation combined with traditional antibiotics. (3) Utilizing mechanistically diverse drugs to potentially eliminate the persister population. (4) Combining antibacterial agents and immunomodulators to modulate host immunity. Created with BioRender.com