Lactobacillus Persisters Formation and Resuscitation.

Lactobacillus is a commonly used probiotic, and many researchers have focused on its stress response to improve its functionality and survival. However, studies on persister cells, dormant cells that aid bacteria in surviving general stress, have focused on pathogenic bacteria that cause infection, not Lactobacillus. Thus, understanding Lactobacillus persister cells will provide essential clues for understanding how Lactobacillus survives and maintains its function under various environmental conditions. We treated Lactobacillus strains with various antibiotics to determine the conditions required for persister formation using kill curves and transmission electron microscopy. In addition, we observed the resuscitation patterns of persister cells using single-cell analysis. Our results show that Lactobacillus creates a small population of persister cells (0.0001-1% of the bacterial population) in response to beta-lactam antibiotics such as ampicillin and amoxicillin. Moreover, only around 0.5-1% of persister cells are heterogeneously resuscitated by adding fresh media; the characteristics are typical of persister cells. This study provides a method for forming and verifying the persistence of Lactobacillus and demonstrates that antibiotic-induced Lactobacillus persister cells show characteristics of dormancy, sensitivity of antibiotics, same as exponential cells, multi-drug tolerance, and resuscitation, which are characteristics of general persister cells. This study suggests that the mechanisms of formation and resuscitation may vary depending on the characteristics, such as the membrane structure of the bacterial species.

Single-cell analysis reveals that cryptic prophage protease LfgB protects Escherichia coli during oxidative stress by cleaving antitoxin MqsA.

Although toxin/antitoxin (TA) systems are ubiquitous, beyond phage inhibition and mobile element stabilization, their role in host metabolism is obscure. One of the best-characterized TA systems is MqsR/MqsA of Escherichia coli, which has been linked previously to protecting gastrointestinal species during the stress it encounters from the bile salt deoxycholate as it colonizes humans. However, some recent whole-population studies have challenged the role of toxins such as MqsR in bacterial physiology since the mqsRA locus is induced over a hundred-fold during stress, but a phenotype was not found upon its deletion. Here, we investigate further the role of MqsR/MqsA by utilizing single cells and demonstrate that upon oxidative stress, the TA system MqsR/MqsA has a heterogeneous effect on the transcriptome of single cells. Furthermore, we discovered that MqsR activation leads to induction of the poorly characterized yfjXY ypjJ yfjZF operon of cryptic prophage CP4-57. Moreover, deletion of yfjY makes the cells sensitive to H2O2, acid, and heat stress, and this phenotype was complemented. Hence, we recommend yfjY be renamed to lfgB (less fatality gene B). Critically, MqsA represses lfgB by binding the operon promoter, and LfgB is a protease that degrades MqsA to derepress rpoS and facilitate the stress response. Therefore, the MqsR/MqsA TA system facilitates the stress response through cryptic phage protease LfgB.IMPORTANCEThe roles of toxin/antitoxin systems in cell physiology are few and include phage inhibition and stabilization of genetic elements; yet, to date, there are no single-transcriptome studies for toxin/antitoxin systems and few insights for prokaryotes from this novel technique. Therefore, our results with this technique are important since we discover and characterize a cryptic prophage protease that is regulated by the MqsR/MqsA toxin/antitoxin system in order to regulate the host response to oxidative stress.

Toxin/antitoxin systems induce persistence and work in concert with restriction/modification systems to inhibit phage.

IMPORTANCE: To date, there are no reports of phage infection-inducing persistence. Therefore, our results are important since we show for the first time that a phage-defense system, the MqsRAC toxin/antitoxin system, allows the host to survive infection by forming persister cells, rather than inducing cell suicide. Moreover, we demonstrate that the MqsRAC system works in concert with restriction/modification systems. These results imply that if phage therapy is to be successful, anti-persister compounds need to be administered along with phages.

CRISPR-Cas Controls Cryptic Prophages.

The bacterial archetypal adaptive immune system, CRISPR-Cas, is thought to be repressed in the best-studied bacterium, Escherichia coli K-12. We show here that the E. coli CRISPR-Cas system is active and serves to inhibit its nine defective (i.e., cryptic) prophages. Specifically, compared to the wild-type strain, reducing the amounts of specific interfering RNAs (crRNA) decreases growth by 40%, increases cell death by 700%, and prevents persister cell resuscitation. Similar results were obtained by inactivating CRISPR-Cas by deleting the entire 13 spacer region (CRISPR array); hence, CRISPR-Cas serves to inhibit the remaining deleterious effects of these cryptic prophages, most likely through CRISPR array-derived crRNA binding to cryptic prophage mRNA rather than through cleavage of cryptic prophage DNA, i.e., self-targeting. Consistently, four of the 13 E. coli spacers contain complementary regions to the mRNA sequences of seven cryptic prophages, and inactivation of CRISPR-Cas increases the level of mRNA for lysis protein YdfD of cryptic prophage Qin and lysis protein RzoD of cryptic prophage DLP-12. In addition, lysis is clearly seen via transmission electron microscopy when the whole CRISPR-Cas array is deleted, and eliminating spacer #12, which encodes crRNA with complementary regions for DLP-12 (including rzoD), Rac, Qin (including ydfD), and CP4-57 cryptic prophages, also results in growth inhibition and cell lysis. Therefore, we report the novel results that (i) CRISPR-Cas is active in E. coli and (ii) CRISPR-Cas is used to tame cryptic prophages, likely through RNAi, i.e., unlike with active lysogens, active CRISPR-Cas and cryptic prophages may stably co-exist.

Escherichia coli cryptic prophages sense nutrients to influence persister cell resuscitation.

Cryptic prophages are not genomic junk but instead enable cells to combat myriad stresses as an active stress response. How these phage fossils affect persister cell resuscitation has, however, not been explored. Persister cells form as a result of stresses such as starvation, antibiotics and oxidative conditions, and resuscitation of these persister cells likely causes recurring infections such as those associated with tuberculosis, cystic fibrosis and Lyme disease. Deletion of each of the nine Escherichia coli cryptic prophages has no effect on persister cell formation. Strikingly, elimination of each cryptic prophage results in an increase in persister cell resuscitation with a dramatic increase in resuscitation upon deleting all nine prophages. This increased resuscitation includes eliminating the need for a carbon source and is due to activation of the phosphate import system resulting from inactivating the transcriptional regulator AlpA of the CP4-57 cryptic prophage. Deletion of alpA increases persister resuscitation, and AlpA represses phosphate regulator PhoR. Both phosphate regulators PhoP and PhoB stimulate resuscitation. This suggests a novel cellular stress mechanism controlled by cryptic prophages: regulation of phosphate uptake which controls the exit of the cell from dormancy and prevents premature resuscitation in the absence of nutrients.

'Viable but non-culturable cells' are dead.

Most bacteria lead lives of quiet desperation, so they sleep. By sleeping, bacteria survive ubiquitous stress, such as antibiotics, and can resuscitate to reconstitute infections. As for other nearly universal and highly regulated processes such as biofilm formation, in persistence, a small population of cells have an elegantly-regulated pathway to become dormant. By inactivating their ribosomes, persister cells sleep through stress and resuscitate once (i) the stress is removed, (ii) nutrients are presented and (iii) ribosome content reaches a threshold. During stress, cells often become spheroid and die, becoming hollow, membrane-enclosed vessels. How cellular content is lost is unclear, but it is obvious that these 'cell shells' are dead; i.e., 'There's no there there'. Critically, due to their intact membranes, the shells appear with membrane-impenetrant stains as 'viable' particles. Unfortunately, the microbiology field of 'viable but non-culturable cells' (VBNCs), though important for demonstrating the existence of dormant bacteria as a result of myriad stress states, has often mistaken these non-viable shells as viable particles that mysteriously may be reborn, when an appropriate incantation is made. We argue here, based on experimental data, that if resuscitation occurs, it is the persister (always-viable) cell population that revives, rather than the cell husks, which are dead.

The Primary Physiological Roles of Autoinducer 2 in Escherichia coli Are Chemotaxis and Biofilm Formation.

Autoinducer 2 (AI-2) is a ubiquitous metabolite but, instead of acting as a "universal signal," relatively few phenotypes have been associated with it, and many scientists believe AI-2 is often a metabolic byproduct rather than a signal. Here, the aim is to present evidence that AI-2 influences both biofilm formation and motility (swarming and chemotaxis), using Escherichia coli as the model system, to establish AI-2 as a true signal with an important physiological role in this bacterium. In addition, AI-2 signaling is compared to the other primary signal of E. coli, indole, and it is shown that they have opposite effects on biofilm formation and virulence.

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

Toxin/antitoxin (TA) systems are present in most prokaryote genomes. Toxins are almost exclusively proteins that reduce metabolism (but do not cause cell death), and antitoxins are either RNA or proteins that counteract the toxin or the RNA that encodes it. Although TA systems clearly stabilize mobile genetic elements, after four decades of research, the physiological roles of chromosomal TA systems are less clear. For example, recent reports have challenged the notion of TA systems as stress-response elements, including a role in creating the dormant state known as persistence. Here, we present evidence that a primary physiological role of chromosomally encoded TA systems is phage inhibition, a role that is also played by some plasmid-based TA systems. This includes results that show some CRISPR-Cas system elements are derived from TA systems and that some CRISPR-Cas systems mimic the host growth inhibition invoked by TA systems to inhibit phage propagation.

Combatting Persister Cells With Substituted Indoles.

Given that a subpopulation of most bacterial cells becomes dormant due to stress, and that the resting cells of pathogens can revive and reconstitute infections, it is imperative to find methods to treat dormant cells to eradicate infections. The dormant bacteria that are not spores or cysts are known as persister cells. Remarkably, in contrast to the original report that incorrectly indicated indole increases persistence, a large number of indole-related compounds have been found in the last few years that kill persister cells. Hence, in this review, along with a summary of recent results related to persister cell formation and resuscitation, we focus on the ability of indole and substituted indoles to combat the persister cells of both pathogens and non-pathogens.

Toxin/Antitoxin System Paradigms: Toxins Bound to Antitoxins Are Not Likely Activated by Preferential Antitoxin Degradation.

Periodically, a scientific field should examine its early premises. For ubiquitous toxin/antitoxin (TA) systems, several initial paradigms require adjustment based on accumulated data. For example, it is now clear that under physiological conditions, there is little evidence that toxins of TA systems cause cell death and little evidence that TA systems cause persistence. Instead, TA systems are utilized to reduce metabolism during stress, inhibit phages, stabilize genetic elements, and influence biofilm formation (bacterial cells attached via an extracellular matrix). In this essay, it is argued that toxins bound to antitoxins are not likely to become activated by preferential antitoxin degradation but instead, de novo toxin synthesis in the absence of stoichiometric amounts of antitoxin activates toxins.

ppGpp ribosome dimerization model for bacterial persister formation and resuscitation.

Stress is ubiquitous for bacteria and can convert a subpopulation of cells into a dormant state known as persistence, in which cells are tolerant to antimicrobials. These cells revive rapidly when the stress is removed and are likely the cause of many recurring infections such as those associated with tuberculosis, cystic fibrosis, and Lyme disease. However, how persister cells are formed is not understood well. Here we propose the ppGpp ribosome dimerization persister (PRDP) model in which the alarmone guanosine pentaphosphate/tetraphosphate (henceforth ppGpp) generates persister cells directly by inactivating ribosomes via the ribosome modulation factor (RMF), the hibernation promoting factor (Hpf), and the ribosome-associated inhibitor (RaiA). We demonstrate that persister cells contain a large fraction of 100S ribosomes, that inactivation of RMF, HpF, and RaiA reduces persistence and increases single-cell persister resuscitation and that ppGpp has no effect on single-cell persister resuscitation. Hence, a direct connection between ppGpp and persistence is shown along with evidence of the importance of ribosome dimerization in persistence and for active ribosomes during resuscitation.

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

Persistence, the stress-tolerant state, is arguably the most vital phenotype since nearly all cells experience nutrient stress, which causes a sub-population to become dormant. However, how persister cells wake to reconstitute infections is not understood well. Here, using single-cell observations, we determined that Escherichia coli persister cells resuscitate primarily when presented with specific carbon sources, rather than spontaneously. In addition, we found that the mechanism of persister cell waking is through sensing nutrients by chemotaxis and phosphotransferase membrane proteins. Furthermore, nutrient transport reduces the level of secondary messenger cAMP through enzyme IIA; this reduction in cAMP levels leads to ribosome resuscitation and rescue. Resuscitating cells also immediately commence chemotaxis toward nutrients, although flagellar motion is not required for waking. Hence, persister cells wake by perceiving nutrients via membrane receptors that relay the signal to ribosomes via the secondary messenger cAMP, and persisters wake and utilize chemotaxis to acquire nutrients.

Forming and waking dormant cells: The ppGpp ribosome dimerization persister model.

Procaryotes starve and face myriad stresses. The bulk population actively resists the stress, but a small population weathers the stress by entering a resting stage known as persistence. No mutations occur, and so persisters behave like wild-type cells upon removal of the stress and regrowth; hence, persisters are phenotypic variants. In contrast, resistant bacteria have mutations that allow cells to grow in the presence of antibiotics, and tolerant cells survive antibiotics better than actively-growing cells due to their slow growth (such as that of the stationary phase). In this review, we focus on the latest developments in studies related to the formation and resuscitation of persister cells and propose the guanosine pentaphosphate/tetraphosphate (henceforth ppGpp) ribosome dimerization persister (PRDP) model for entering and exiting the persister state.

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

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.

Comment on Metabolomics for a Millenniums-Old Crop: Tea Plant (Camellia sinensis).

Metabolomics is the study of metabolite profiles at the system level. Since its introduction in the early 2000s, metabolomics has greatly contributed to the understanding of the distribution of metabolites in organisms under various physiological conditions. In this comment, we show our research on the temporal development of metabolomics in general and in agricultural, food, and nutritional sciences. According to our investigation, metabolomics develops in a sigmoid kinetics. On the basis of the analysis, we made a prediction on the future of the metabolomics study, which may benefit the research community in the field.

Identification of a potent indigoid persister antimicrobial by screening dormant cells.

The subpopulation of bacterial cells that survive myriad stress conditions (e.g., nutrient deprivation and antimicrobials) by ceasing metabolism, revive by activating ribosomes. These resuscitated cells can reconstitute infections; hence, it is imperative to discover compounds which eradicate persister cells. By screening 10,000 compounds directly for persister cell killing, we identified 5-nitro-3-phenyl-1H-indol-2-yl-methylamine hydrochloride (NPIMA) kills Escherichia coli persister cells more effectively than the best indigoid found to date, 5-iodoindole, and better than the DNA-crosslinker cisplatin. In addition, NPIMA eradicated Pseudomonas aeruginosa persister cells in a manner comparable to cisplatin. NPIMA also eradicated Staphylococcus aureus persister cells but was less effective than cisplatin. Critically, NPIMA kills Gram-positive and Gram-negative bacteria by damaging membranes and causing lysis as demonstrated by microscopy and release of extracellular DNA and protein. Furthermore, NPIMA was effective in reducing P. aeruginosa and S. aureus cell numbers in a wound model, and no resistance was found after 1 week. Hence, we identified a potent indigoid that kills persister cells by damaging their membranes.