HokB Monomerization and Membrane Repolarization Control Persister Awakening.

Every bacterial population harbors a small subpopulation of so-called persisters that are transiently antibiotic tolerant. These persisters are associated with the recalcitrance of chronic infections because they can recolonize the host after antibiotic removal. Although several effectors have been described to induce persistence, persister cell awakening is poorly understood. We previously reported that the toxin HokB induces persistence via pore formation, resulting in membrane depolarization and ATP leakage. We now delineate mechanisms responsible for the awakening of HokB-induced persisters. We show that HokB dimerization by the oxidoreductase DsbA is essential for pore formation and peptide stability. Pores are disassembled via DsbC-mediated monomerization, which targets HokB for DegQ-mediated degradation. Finally, pore disassembly allows membrane repolarization by the electron transport chain, supporting cells to resume growth. These results provide a detailed view of both the formation and awakening of HokB-induced persister cells.

Toxin Induction or Inhibition of Transcription or Translation Posttreatment Increases Persistence to Fluoroquinolones.

Toxin-antitoxin modules are widespread in prokaryotes, and the capacity of toxin accumulation to increase the tolerances of bacteria to antibiotics has been well documented. The conventional model for this functionality implies that an overabundance of toxin arrests bacterial growth, which inhibits processes targeted by antibiotics and thereby limits their corruption and the lethal damage that would ensue. Implicit in this model is that toxins exert their influence on antibiotic lethality before and/or during treatment, even though they are also present and functional after treatment concludes. Given recent evidence establishing that the period following antibiotic treatment (recovery) is important for the survival of nongrowing bacterial populations treated with fluoroquinolones (FQs), we assayed to what extent toxins influence bacterial survival during the recovery period. With both LdrD and MazF, toxins of type I and II systems, respectively, controlling accumulation to occur only after FQ treatment of nongrowing cultures resulted in significant increases in persisters. Further genetic investigation revealed important roles for homologous recombination and nucleotide excision repair machinery. Focusing on the wild type, we did not observe any SOS-induced toxin functioning in this manner; however, an analogous phenomenon was observed for wild-type Escherichia coli as well as uropathogenic E. coli (UPEC) when transcription or translation was inhibited during the post-FQ recovery period. Collectively, these data reveal the capacity of toxins to thwart FQ killing even after the treatment has concluded and show that FQ treatment of nongrowing bacteria can be rendered largely ineffective if bacteria cannot readily resume translation and growth at the conclusion of treatment. IMPORTANCE Overabundances of toxins have been shown to increase the antibiotic tolerances of bacteria. Largely, these effects have been attributed to the abilities of toxins to inhibit bacterial growth before and during antibiotic exposure. In this study, we assessed to what extent toxins can influence bacterial survival following antibiotic treatment, rather than before or during. Using two mechanistically distinct toxins, we show that their accumulations after antibiotic exposure have the capacity to increase the abundances of fluoroquinolone persisters from nongrowing populations. Further, we show with wild-type and uropathogenic E. coli that chemical inhibition of growth, not just that induced by toxins, produces analogous results. These observations reveal another dimension of how toxins influence antibiotic tolerance and highlight the importance of postantibiotic physiology on bacterial survival.

Modulation of Toxin-Antitoxin System Rnl AB Type II in Phage-Resistant Gammaproteobacteria Surviving Photodynamic Treatment.

Type II toxin-antitoxin (TA) systems are the particular type of TA modules which take part in different kinds of cellular actions, such as biofilm formation, persistence, stress endurance, defense of the bacterial cell against multiple phage attacks, plasmid maintenance, and programmed cell death in favor of bacterial population. Although several bioinformatics and Pet lab studies have already been conducted to understand the functionality of already discovered TA systems, still, more work in this area is required. Rnl AB type II TA module, which is composed of RnlA toxin and RnlB antitoxin, is a newly discovered type II TA module which takes part in the defense mechanism against T4 bacteriophage attack in Escherichia coli K-12 strain MH1 that has not been widely studied in other bacteria. Because of the significant role of class Gammaproteobacteriacea in a diverse range of health problems, we chose here to focus on this class to survey the presence of the Rnl AB TA module. For better categorization and description of the distribution of this module in this class of bacteria, the corresponding phylogenetic trees are illustrated here. Neighbor-joining and the maximum parsimony methods were used in this study to take a look at the distribution of domains present in RnlA and RnlB proteins, among members of Gammaproteobacteria. Also, the possible roles of photodynamic therapy (PDT) in providing a substrate for better phage therapy are herein discussed.

Novel Inhibitors of Toxin HipA Reduce Multidrug Tolerant Persisters.

Persisters are a small fraction of drug-tolerant bacteria without any genotype variations. Their existence in many life-threatening infectious diseases presents a major challenge to antibiotic therapy. Persistence is highly related to toxin-antitoxin modules. HipA (high persistence A) was the first toxin found to contribute to Escherichia coli persistence. In this study, we used structure-based virtual screening for HipA inhibitors discovery and identified several novel inhibitors of HipA that remarkably reduced E. coli persistence. The most potent one decreased the persister fraction by more than five-fold with an in vitro K D of 270 ± 90 nM and an ex vivo EC50 of 46 ± 2 and 28 ± 1 µM for ampicillin and kanamycin screening, respectively. These findings demonstrated that inhibition of toxin can reduce bacterial persistence independent of the antibiotics used and provided a framework for persistence treatment by interfering with the toxin-antitoxin modules.