Brian W. Kwan

Bacterial Persister Cell Formation and Dormancy

ABSTRACT Bacterial cells may escape the effects of antibiotics without undergoing genetic change; these cells are known as persisters. Unlike resistant cells that grow in the presence of antibiotics, persister cells do not grow in the presence of antibiotics. These persister cells are a small fraction of exponentially growing cells (due to carryover from the inoculum) but become a significant fraction in the stationary phase and in biofilms (up to 1%). Critically, persister cells may be a major cause of chronic infections. The mechanism of persister cell formation is not well understood, and even the metabolic state of these cells is debated. Here, we review studies relevant to the formation of persister cells and their metabolic state and conclude that the best model for persister cells is still dormancy, with the latest mechanistic studies shedding light on how cells reach this dormant state.

Arrested Protein Synthesis Increases Persister-Like Cell Formation

ABSTRACT Biofilms are associated with a wide variety of bacterial infections and pose a serious problem in clinical medicine due to their inherent resilience to antibiotic treatment. Within biofilms, persister cells comprise a small bacterial subpopulation that exhibits multidrug tolerance to antibiotics without undergoing genetic change. The low frequency of persister cell formation makes it difficult to isolate and study persisters, and bacterial persistence is often attributed to a quiescent metabolic state induced by toxins that are regulated through toxin-antitoxin systems. Here we mimic toxins via chemical pretreatments to induce high levels of persistence (10 to 100%) from an initial population of 0.01%. Pretreatment of Escherichia coli with (i) rifampin, which halts transcription, (ii) tetracycline, which halts translation, and (iii) carbonyl cyanide m-chlorophenylhydrazone, which halts ATP synthesis, all increased persistence dramatically. Using these compounds, we demonstrate that bacterial persistence results from halted protein synthesis and from environmental cues.

Quorum sensing enhancement of the stress response promotes resistance to quorum quenching and prevents social cheating

Quorum sensing (QS) coordinates the expression of virulence factors and allows bacteria to counteract the immune response, partly by increasing their tolerance to the oxidative stress generated by immune cells. Despite the recognized role of QS in enhancing the oxidative stress response, the consequences of this relationship for the bacterial ecology remain unexplored. Here we demonstrate that QS increases resistance also to osmotic, thermal and heavy metal stress. Furthermore a QS-deficient lasR rhlR mutant is unable to exert a robust response against H2O2 as it has less induction of catalase and NADPH-producing dehydrogenases. Phenotypic microarrays revealed that the mutant is very sensitive to several toxic compounds. As the anti-oxidative enzymes are private goods not shared by the population, only the individuals that produce them benefit from their action. Based on this premise, we show that in mixed populations of wild-type and the mexR mutant (resistant to the QS inhibitor furanone C-30), treatment with C-30 and H2O2 increases the proportion of mexR mutants; hence, oxidative stress selects resistance to QS compounds. In addition, oxidative stress alone strongly selects for strains with active QS systems that are able to exert a robust anti oxidative response and thereby decreases the proportion of QS cheaters in cultures that are otherwise prone to invasion by cheats. As in natural environments stress is omnipresent, it is likely that this QS enhancement of stress tolerance allows cells to counteract QS inhibition and invasions by social cheaters, therefore having a broad impact in bacterial ecology.

Combatting bacterial infections by killing persister cells with mitomycin C

Persister cells are a multi-drug tolerant subpopulation of bacteria that contribute to chronic and recalcitrant clinical infections such as cystic fibrosis and tuberculosis. Persisters are metabolically dormant, so they are highly tolerant to all traditional antibiotics which are mainly effective against actively growing cells. Here, we show that the FDA-approved anti-cancer drug mitomycin C (MMC) eradicates persister cells through a growth-independent mechanism. MMC is passively transported and bioreductively activated, leading to spontaneous cross-linking of DNA, which we verify in both active and dormant cells. We find MMC effectively eradicates cells grown in numerous different growth states (e.g. planktonic cultures and highly robust biofilm cultures) in both rich and minimal media. Additionally, MMC is a potent bactericide for a broad range of bacterial persisters, including commensal Escherichia coli K-12 as well as pathogenic species of E. coli, Staphylococcus aureus and Pseudomonas aeruginosa. We also demonstrate the efficacy of MMC in an animal model and a wound model, substantiating the clinical applicability of MMC against bacterial infections. Therefore, MMC is the first broad-spectrum compound capable of eliminating persister cells, meriting investigation as a new approach for the treatment of recalcitrant infections.

Persistence Increases in the Absence of the Alarmone Guanosine Tetraphosphate by Reducing Cell Growth.

Most bacterial cells are stressed, and as a result, some become tolerant to antibiotics by entering a dormant state known as persistence. The key intracellular metabolite that has been linked to this persister state is guanosine tetraphosphate (ppGpp), the alarmone that was first linked to nutrient stress. In Escherichia coli, ppGpp redirects protein production during nutrient stress by interacting with RNA polymerase directly and by inhibiting several proteins. Consistently, increased levels of ppGpp lead to increased persistence; but, the mechanism by which elevated ppGpp translates into persistence has not been determined. Hence, we explored persistence in the absence of ppGpp so that the underlying mechanism of persister cell formation could be explored. We found that persister cells still form, although at lower levels, in the absence of ppGpp. Additionally, the toxin/antitoxin systems that we investigated (MqsR, MazF, GhoT, and YafQ) remain able to increase persistence dramatically in the absence of ppGpp. By overproducing each E. coli protein from the 4287 plasmid vectors of the ASKA library and selecting for increased persistence in the absence of ppGpp (via a relA spoT mutant), we identified five new proteins, YihS, PntA, YqjE, FocA, and Zur, that increase persistence simply by reducing cell growth.

Phosphodiesterase DosP increases persistence by reducing cAMP which reduces the signal indole

Persisters are bacteria that are highly tolerant to antibiotics due to their dormant state and are of clinical significance owing to their role in infections. Given that the population of persisters increases in biofilms and that cyclic diguanylate (c‐di‐GMP) is an intracellular signal that increases biofilm formation, we sought to determine whether c‐di‐GMP has a role in bacterial persistence. By examining the effect of 30 genes from Escherichia coli, including diguanylate cyclases that synthesize c‐di‐GMP and phosphodiesterases that breakdown c‐di‐GMP, we determined that DosP (direct oxygen sensing phosphodiesterase) increases persistence by over a thousand fold. Using both transcriptomic and proteomic approaches, we determined that DosP increases persistence by decreasing tryptophanase activity and thus indole. Corroborating this effect, addition of indole reduced persistence. Despite the role of DosP as a c‐di‐GMP phosphodiesterase, the decrease in tryptophanase activity was found to be a result of cyclic adenosine monophosphate (cAMP) phosphodiesterase activity. Corroborating this result, the reduction of cAMP via CpdA, a cAMP‐specific phosphodiesterase, increased persistence and reduced indole levels similarly to DosP. Therefore, phosphodiesterase DosP increases persistence by reducing the interkingdom signal indole via reduction of the global regulator cAMP. Biotechnol. Bioeng. 2015;112: 588–600.

The MqsR/MqsA toxin/antitoxin system protects Escherichia coli during bile acid stress.

Toxin/antitoxin (TA) systems are ubiquitous within bacterial genomes, and the mechanisms of many TA systems are well characterized. As such, several roles for TA systems have been proposed, such as phage inhibition, gene regulation and persister cell formation. However, the significance of these roles is nebulous due to the subtle influence from individual TA systems. For example, a single TA system has only a minor contribution to persister cell formation. Hence, there is a lack of defining physiological roles for individual TA systems. In this study, phenotype assays were used to determine that the MqsR/MqsA type II TA system of Escherichia coli is important for cell growth and tolerance during stress from the bile salt deoxycholate. Using transcriptomics and purified MqsR, we determined that endoribonuclease toxin MqsR degrades YgiS mRNA, which encodes a periplasmic protein that promotes deoxycholate uptake and reduces tolerance to deoxycholate exposure. The importance of reducing YgiS mRNA by MqsR is evidenced by improved growth, reduced cell death and reduced membrane damage when cells without ygiS are stressed with deoxycholate. Therefore, we propose that MqsR/MqsA is physiologically important for E. coli to thrive in the gallbladder and upper intestinal tract, where high bile concentrations are prominent.

de novo Synthesis of a Bacterial Toxin/Antitoxin System

The prevalence of toxin/antitoxin (TA) systems in almost all genomes suggests they evolve rapidly. Here we show that an antitoxin from a type V system (GhoS, an endoribonuclease specific for the mRNA of the toxin GhoT) can be converted into a novel toxin (ArT) simply by adding two mutations. In contrast to GhoS, which increases growth, the new toxin ArT decreases growth dramatically in Escherichia coli. Transmission electron microscopy analysis revealed that the nucleoid in ArT-producing cells is concentrated and appears hollow. Whole-transcriptome profiling revealed ArT cleaves 50 additional transcripts, which shows that the endoribonuclease activity of GhoS has been broadened as it was converted to ArT. Furthermore, we evolved an antitoxin for the new toxin ArT from two unrelated antitoxin templates, the protein-based antitoxin MqsA and RNA-based antitoxin ToxI, and showed that the evolved MqsA and ToxI variants are able to counteract the toxicity of ArT. In addition, the de novo TA system was found to increase persistence, a phenotype commonly associated with TA systems. Therefore, toxins and antitoxins from disparate systems can be interconverted.

Toxin MqsR Cleaves Single-Stranded mRNA with Various 5 Ends

Toxin/antitoxin (TA) systems are the means by which bacterial cells become persistent; that is, those cells that are tolerant to multiple environmental stresses such as antibiotics by becoming metabolically dormant. These persister cells are responsible for recalcitrant infections. Once toxins are activated by the inactivation of antitoxins (e.g., stress‐triggered Lon degradation of the antitoxin), many toxins reduce metabolism by inhibiting translation (e.g., cleaving mRNA, reducing ATP). The MqsR/MqsA TA system of Escherichia coli cleaves mRNA to help the cell withstand oxidative and bile acid stress. Here, we investigated the role of secondary structure and 5′ mRNA processing on MqsR degradation of mRNA and found that MqsR cleaves only single‐stranded RNA at 5′‐GCU sites and that MqsR is equally active against RNA with 5′‐triphosphate, 5′‐monophosphate, and 5′‐hydroxyl groups.