The Persistence-Inducing Toxin HokB Forms Dynamic Pores That Cause ATP Leakage

Bacterial populations harbor a small fraction of cells that display transient multidrug tolerance. These so-called persister cells are extremely difficult to eradicate and contribute to the recalcitrance of chronic infections. Several signaling pathways leading to persistence have been identified. However, it is poorly understood how the effectors of these pathways function at the molecular level. In a previous study, we reported that the conserved GTPase Obg induces persistence in Escherichia coil via transcriptional upregulation of the toxin HokB. In the present study, we demonstrate that HokB inserts in the cytoplasmic membrane where it forms pores. The pore-forming capacity of the HokB peptide is demonstrated by in vitro conductance measurements on synthetic and natural lipid bilayers, revealing an asymmetrical conductance profile. Pore formation is directly linked to persistence and results in leakage of intracellular ATP. HokB-induced persistence is strongly impeded in the presence of a channel blocker, thereby providing a direct link between pore functioning and persistence. Furthermore, the activity of HokB pores is sensitive to the membrane potential. This sensitivity presumably results from the formation of either intermediate or mature pore types depending on the membrane potential. Taken together, these results provide a detailed view on the mechanistic basis of persister formation through the effector HokB. IMPORTANCE There is increasing awareness of the clinical importance of persistence. Indeed, persistence is linked to the recalcitrance of chronic infections, and evidence is accumulating that persister cells constitute a pool of viable cells from which resistant mutants can emerge. Unfortunately, persistence is a poorly understood process at the mechanistic level. In this study, we unraveled the pore-forming activity of HokB in E. coil and discovered that these pores lead to leakage of intracellular ATP, which is correlated with the induction of persistence. Moreover, we established a link between persistence and pore activity, as the number of HokBinduced persister cells was strongly reduced using a channel blocker. The latter opens opportunities to reduce the number of persister cells in a clinical setting

Dietary Sargassum fusiforme improves memory and reduces amyloid plaque load in an Alzheimer's disease mouse model

Activation of liver X receptors (LXRs) by synthetic agonists was found to improve cognition in Alzheimer's disease (AD) mice. However, these LXR agonists induce hypertriglyceridemia and hepatic steatosis, hampering their use in the clinic. We hypothesized that phytosterols as LXR agonists enhance cognition in AD without affecting plasma and hepatic triglycerides. Phytosterols previously reported to activate LXRs were tested in a luciferase-based LXR reporter assay. Using this assay, we found that phytosterols commonly present in a Western type diet in physiological concentrations do not activate LXRs. However, a lipid extract of the 24(S)-Saringosterol-containing seaweed Sargassum fusiforme did potently activate LXR beta. Dietary supplementation of crude Sargassum fusiforme or a Sargassum fusiforme-derived lipid extract to AD mice significantly improved short-term memory and reduced hippocampal A beta plaque load by 81%. Notably, none of the side effects typically induced by full synthetic LXR agonists were observed. In contrast, administration of the synthetic LXRa activator, AZ876, did not improve cognition and resulted in the accumulation of lipid droplets in the liver. Administration of Sargassum fusiforme-derived 24(S)-Saringosterol to cultured neurons reduced the secretion of A beta 42. Moreover, conditioned medium from 24(S)-Saringosterol-treated astrocytes added to microglia increased phagocytosis of A beta. Our data show that Sargassum fusiforme improves cognition and alleviates AD pathology. This may be explained at least partly by 24(S)-Saringosterol-mediated LXR beta activation.</p

The Persistence-Inducing Toxin HokB Forms Dynamic Pores That Cause ATP Leakage

Bacterial populations harbor a small fraction of cells that display transient multidrug tolerance. These so-called persister cells are extremely difficult to eradicate and contribute to the recalcitrance of chronic infections. Several signaling pathways leading to persistence have been identified. However, it is poorly understood how the effectors of these pathways function at the molecular level. In a previous study, we reported that the conserved GTPase Obg induces persistence in Escherichia coli via transcriptional upregulation of the toxin HokB. In the present study, we demonstrate that HokB inserts in the cytoplasmic membrane where it forms pores. The pore-forming capacity of the HokB peptide is demonstrated by in vitro conductance measurements on synthetic and natural lipid bilayers, revealing an asymmetrical conductance profile. Pore formation is directly linked to persistence and results in leakage of intracellular ATP. HokB-induced persistence is strongly impeded in the presence of a channel blocker, thereby providing a direct link between pore functioning and persistence. Furthermore, the activity of HokB pores is sensitive to the membrane potential. This sensitivity presumably results from the formation of either intermediate or mature pore types depending on the membrane potential. Taken together, these results provide a detailed view on the mechanistic basis of persister formation through the effector HokB.status: Published onlin

Obg and membrane depolarization are part of a microbial bet-hedging strategy that leads to antibiotic tolerance

Within bacterial populations, a small fraction of persister cells is transiently capable of surviving exposure to lethal doses of antibiotics. As a bet-hedging strategy, persistence levels are determined both by stochastic induction and by environmental stimuli, called responsive diversification. Little is known on the mechanisms that link the low frequency of persisters to environmental signals. Our results support a central role for the conserved GTPase Obg in determining persistence in Escherichia coli in response to nutrient starvation. Obg-mediated persistence requires the stringent response alarmone (p)ppGpp and proceeds through transcriptional control of the hokB-sokB type I toxin-antitoxin module. In individual cells, increased Obg levels induce HokB expression, which in turn results in a collapse of the membrane potential, leading to dormancy. Obg also controls persistence in Pseudomonas aeruginosa and thus constitutes a conserved regulator of antibiotic tolerance. Combined, our findings signify an important step towards unravelling shared genetic mechanisms underlying persistence.publisher: Elsevier articletitle: Obg and Membrane Depolarization Are Part of a Microbial Bet-Hedging Strategy that Leads to Antibiotic Tolerance journaltitle: Molecular Cell articlelink: http://dx.doi.org/10.1016/j.molcel.2015.05.011 content_type: article copyright: Copyright © 2015 Elsevier Inc. All rights reserved.status: publishe

Obg and membrane depolarization are part of a microbial bet-hedging strategy that leads to antibiotic tolerance

Within bacterial populations, a small fraction of per-sister cells is transiently capable of surviving exposure to lethal doses of antibiotics. As a bet-hedging strategy, persistence levels are determined both by stochastic induction and by environmental stimuli called responsive diversification. Little is known about the mechanisms that link the low frequency of per-sisters to environmental signals. Our results support a central role for the conserved GTPase Obg in determining persistence in Escherichia coli in response to nutrient starvation. Obg-mediated persistence requires the stringent response alarmone (p) ppGpp and proceeds through transcriptional control of the hokB-sokB type I toxin-antitoxin module. In individual cells, increased Obg levels induce HokB expression, which in turn results in a collapse of the membrane potential, leading to dormancy. Obg also controls persistence in Pseudomonas aeruginosa and thus constitutes a conserved regulator of antibiotic tolerance. Combined, our findings signify an important step toward unraveling shared genetic mechanisms underlying persistence