Persister cells resuscitate via ribosome modification by 23S rRNA pseudouridine synthase RluD

Abstract

Summary Upon a wide range of stress conditions (e.g. nutrient, antibiotic, oxidative), a subpopulation of bacterial cells known as persisters survives 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 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 4267 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 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.

A Primary Physiological Role of Toxin/Antitoxin Systems Is Phage Inhibition

“…In contrast to work showing chromosomal TA systems like MqsR/MqsA affect the stress response, the impact of TA systems on persistence is not convincing, primarily because the fold changes in most persister experiments with individual TA systems are usually small (less than 10-fold) and strains with multiple TA systems deleted do not show consistent phenotypes. Persistence is an extreme stress response that occurs when a subpopulation of cells becomes dormant due to ribosome dimerization as a direct result of increased ppGpp levels ( Kim and Wood, 2016 ; Kim et al, 2018 ; Song and Wood, 2020a , b ; Yamasaki et al, 2020 ). Specifically, inactivation of 10 TA systems did not affect E. coli persistence for several groups ( Harms et al, 2017 ; Goormaghtigh et al, 2018 ; Svenningsen et al, 2019 ).…”

Are we really studying persister cells?

“…Building on the research of Hobby and Bigger, persister cell formation is an elegantly regulated response to environmental conditions (Kwan et al ., 2013). Critically, by increasing the fraction of persister cells so that they may be studied directly, it has been discovered that inactivation of ribosomes via dimerization is the likely general mechanism leading to persistence (Kim et al ., 2018a; Kim et al ., 2018b; Wood et al ., 2019; Wood and Song, 2020; Yamasaki et al ., 2020; Song and Wood, 2020a, 2020b). Therefore, actively growing, even exponentially growing cells, can become persistent, if challenged with stress and a threshold level of ribosomes dimerize (Kwan et al ., 2013; Wood and Song, 2020).…”

Evidence of defined temporal expression patterns that lead a gram-negative cell out of dormancy

“…Like classic dormant cysts and spores, the addition of fresh nutrients has been observed to immediately resuscitate persister cells [58]. Not unlike R. centenum cysts that prioritize ribosome synthesis early in germination, the ribosome content of persister cells are known to play a role in persister cell awakening with cells that have higher levels of ribosomes waking up first [61,62]. This phenomenon has been attributed to the mandatory need for protein production for successful persister cell awakening [61,62].…”

“…Not unlike R. centenum cysts that prioritize ribosome synthesis early in germination, the ribosome content of persister cells are known to play a role in persister cell awakening with cells that have higher levels of ribosomes waking up first [61,62]. This phenomenon has been attributed to the mandatory need for protein production for successful persister cell awakening [61,62]. One long-term goal of this study, and others, is to determine if there are commonalities in processes by which different species both become dormant and also awaken from dormancy.…”

“…Cyst germination was imaged every 5 minutes the with recorded images stitched into a movie. (AVI) S1 Table. A spread sheet listing 1667 genes that undergo differential expression between the 0th-hour sample, and every other time point, was then calculated using the DE-Seq2 package in R [62]. A gene was considered as a Differentially Expressed Gene (DEG) when the log 2 fold change was � ± 1.5 with a false discovery rate adjusted p-value of <0.05.…”