Persister Cells Resuscitate Using Membrane Sensors that Activate Chemotaxis, Lower cAMP Levels, and Revive Ribosomes

Yamasaki, Ryota; Song, Sooyeon; Benedik, Michael J.; Wood, Thomas K.

doi:10.2139/ssrn.3399578

Cited by 24 publications

(85 citation statements)

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“…Hence, we were able to both screen for compounds that more rapidly resuscitate persister cells as well as confirm our hypotheses via single-cell microscopy. The persister cells generated in this way have been (i) confirmed eight ways (19), (ii) used to determine that persister cells wake via ribosome activation (19) and chemotaxis (20), (iii) used to show that the cells capable of resuscitation in a viable but not culturable 95 population are equivalent to persister cells (15), (iv) used to identify compounds that kill persister cells (27), and (v) used to show that the alarmone ppGpp directly creates persister cells by stimulating ribosome dimerization (16). In addition, our method to generate a high population of persister cells has been utilized by at least six independent groups (28)(29)(30)(31)(32)(33).…”

Section: Resultsmentioning

confidence: 99%

“…These proteins are related to contacting with BPOET since addition of the diluent DMSO alone and sequencing larger colonies did not identify these five proteins but instead identified TmcA, a tRNA Met cytidine acetyltransferase, which is a general factor required for translation that likely led to larger colony sizes with the diluent. Of the proteins related to BPOET, only RluD (23S rRNA pseudouridine synthase) and SrlR (represses the gut operon for glucitol metabolism) have been 120 characterized; we focused on RluD because it is related to ribosomes, and we have shown inactivating ribosomes causes persistence (23) and activating ribosomes resuscitates persister cells (16,19,20). RluD is involved in the synthesis and assembly of 70S ribosomes as well as their function based on its posttranscriptional modification of 23S rRNA to form three pseudouridine (5-ribosyl-uracil) nucelosides at positions 1911, 1915, and 1917 (34).…”

Section: Bpoet Resuscitates E Coli Persister Cells By Modifying Ribomentioning

confidence: 99%

“…In addition, persisters revive in an heterogeneous manner, by activating ribosomes; cells increase 40 their ribosome content until a threshold is reached, then they begin to elongate or divide (19). For resuscitation, the persisters sense nutrients by chemotaxis and phosphotransferase membrane proteins, reduce cAMP levels to rescue stalled ribosomes, unhybridize 100S ribosomes via HflX, and undergo chemotaxis toward fresh nutrients (20).…”

Section: Introductionmentioning

confidence: 99%

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Persister Cells Resuscitate via Ribosome Modification by 23S rRNA Pseudouridine Synthase RluD

Song

Wood

2019

Preprint

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Upon a wide range of stress conditions (e.g., nutrient, antibiotic, oxidative), a subpopulation of bacterial cells known as persisters survive by halting metabolism. These cells resuscitate rapidly to reconstitute infections once the stress is removed and nutrients are provided. However, how these dormant cells resuscitate is not understood well but involves reactivating ribosomes. By screening 10,000 5 compounds directly for stimulating Escherichia coli persister cell resuscitation, we identified that 2-{[2-(4-bromophenyl)-2-oxoethyl]thio}-3-ethyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-d]pyrimidin-4(3H)-one (BPOET) stimulates resuscitation. Critically, by screening 4,267 E. coli proteins, we determined that BPOET activates hibernating ribosomes via 23S rRNA pseudouridine synthase RluD, which increases ribosome activity. Corroborating the increased waking with RluD, production of RluD increased the 10 number of active ribosomes in persister cells. Also, inactivating the small RNA RybB which represses rluD led to faster persister resuscitation. Hence, persister cells resuscitate via activation of RluD.

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Section: Resultsmentioning

confidence: 99%

Section: Bpoet Resuscitates E Coli Persister Cells By Modifying Ribomentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Persister Cells Resuscitate via Ribosome Modification by 23S rRNA Pseudouridine Synthase RluD

Song

Wood

2019

Preprint

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“…Our previous results demonstrate that proteolytic queueing does not improve long-term survival 29 , but it is likely that the transcriptional response to proteolytic queueing and antibiotic stress improves short-term survival and recovery while other factors are required to survive for extended periods of time. Many previous works have demonstrated, ribosomes are largely inactive in persister cells [79][80][81][82] and thus translation is slow. Therefore, it is possible the transcripts we have measured are slowly building up in the cells and only being translated at low levels if at all.…”

Section: Future Directions To Expand Our Understanding Of Antibiotic mentioning

confidence: 99%

Antibiotic tolerance is associated with a broad and complex transcriptional response inE. coli

Deter

Hossain

Butzin

2020

Preprint

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In a given bacterial population, antibiotic treatment will kill a large portion of the population, while a small, tolerant subpopulation will survive. Tolerant cells disrupt the efficacy of antibiotic treatment and increase the likelihood that a population gains antibiotic resistance. When a population becomes resistant, antibiotic treatment fails, which is a major health concern. Since antibiotic tolerance often leads to resistance, we have taken a systems biology approach to examine how transcriptional networks respond to antibiotic stress so that cells can survive and recover after antibiotic treatment. We have compared gene expression with and without ampicillin in E. coli. While many previous studies have identified aspects of the antibiotic stress response at either the protein or transcriptional level, our work leverages existing knowledge of transcriptional regulation to link the dynamics of the two allowing for a more holistic approach. Here, we are the first to develop a whole-cell, transcriptional regulatory network (TRN) of antibiotic tolerant subpopulations and examine how cells respond at the transcriptional level to bactericidal concentrations of an antibiotic. Using TRN analysis, we show how certain sigma and transcription factors are affecting transcriptional regulation under antibiotic stress, and that changes in gene expression specific to ampicillin treatment can be directly linked to cell survival and recovery. The resulting network demonstrates that cells are mounting an active and coordinated response to the antibiotic that spans multiple systems and pathways. The redundancy and interconnectivity of the networks suggest that accounting for whole-cell dynamics may be crucial when modeling and studying antibiotic tolerance in the future.

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“…Anti-bacterial agents and disinfectants are effective against planktonic bacteria (bacteria suspended in liquid), however, they are not effective against biofilm-forming bacteria due to the difficulty of chemicals penetrating the biofilm. Hence, there is concern about the risk of the emergence of resistant bacteria, such as the formation of persister cells [ 15 – 18 ]. Oral pathogens that form persisters may cause recurrence of oral diseases via regrowth.…”

Section: Introductionmentioning

confidence: 99%

Rhamnolipids and surfactin inhibit the growth or formation of oral bacterial biofilm

et al. 2020

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Background Bacteria survive in various environments by forming biofilms. Bacterial biofilms often cause significant problems to medical instruments and industrial processes. Techniques to inhibit biofilm formation are essential and have wide applications. In this study, we evaluated the ability of two types of biosurfactants (rhamnolipids and surfactin) to inhibit growth and biofilm formation ability of oral pathogenic bacteria such as Aggregatibacter actinomycetemcomitans, Streptococcus mutans, and Streptococcus sanguinis. Results Rhamnolipids inhibited the growth and biofilm formation ability of all examined oral bacteria. Surfactin showed effective inhibition against S. sanguinis ATCC10556, but lower effects toward A. actinomycetemcomitans Y4 and S. mutans UA159. To corroborate these results, biofilms were observed by scanning electron microscopy (SEM) and confocal microscopy. The observations were largely in concordance with the biofilm assay results. We also attempted to determine the step in the biofilm formation process that was inhibited by biosurfactants. The results clearly demonstrated that rhamnolipids inhibit biofilm formation after the initiation process, however, they do not affect attachment or maturation. Conclusions Rhamnolipids inhibit oral bacterial growth and biofilm formation by A. actinomycetemcomitans Y4, and may serve as novel oral drug against localized invasive periodontitis.

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Persister Cells Resuscitate Using Membrane Sensors that Activate Chemotaxis, Lower cAMP Levels, and Revive Ribosomes

Cited by 24 publications

References 0 publications

Persister Cells Resuscitate via Ribosome Modification by 23S rRNA Pseudouridine Synthase RluD

Persister Cells Resuscitate via Ribosome Modification by 23S rRNA Pseudouridine Synthase RluD

Antibiotic tolerance is associated with a broad and complex transcriptional response inE. coli

Rhamnolipids and surfactin inhibit the growth or formation of oral bacterial biofilm

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