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Growth arrest by HipA induction causes intracellular accumulation of amino acids, indicating that HipA does not trigger ppGpp synthesis by causing amino acid depletion. Light gray and dark gray bars represent log2 amino acid concentration ratios of HipA-arrested cells to growing cells, as measured in cells 1 and 2 h after HipA induction by addition of aTc. Black bars indicate ratios (HipA-arrested cells/growing cells) of flux through indicated amino acid pools determined using kinetic flux profiling. A value of 0 indicates no change. For concentration measurements, error bars represent the standard deviations of the results of three biological replicates. Error bars in flux bars represent uncertainty in flux determination, as described in Materials and Methods. Asterisks indicate amino acid fluxes for which values could not be determined.
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Persistence is a phenomenon whereby a subpopulation of bacterial cells enters a transient growth-arrested state that confers
antibiotic tolerance. While entrance into persistence has been linked to the activities of toxin proteins, the molecular mechanisms
by which toxins induce growth arrest and the persistent state remain unclear. Here, we show t...
Citations
... Analogous to antibiotic persistence, it is intuitive that the dormancy of stressed and starved bacteria in vivo might impair phage therapy. Previous work indeed showed that the productivity of phage infections is positively correlated with host growth rate and that fully growth-arrested cells are refractory to phage replication [21][22][23][24][25] . ...
... Our study confirms previous notion that bacteriophages are generally unable to directly replicate on deep-dormant bacteria 24,25 , but presents a new P. aeruginosa phage named Paride with the unique ability to kill deep-dormant bacteria by direct lytic replication ( Figs. 1 and 2b). Notably, no E. coli phage with this ability could be isolated despite considerable efforts. ...
Bacteriophages are ubiquitous viral predators that have primarily been studied using fast-growing laboratory cultures of their bacterial hosts. However, microbial life in nature is mostly in a slow- or non-growing, dormant state. Here, we show that diverse phages can infect deep-dormant bacteria and suspend their replication until the host resuscitates (“hibernation”). However, a newly isolated Pseudomonas aeruginosa phage, named Paride, can directly replicate and induce the lysis of deep-dormant hosts. While non-growing bacteria are notoriously tolerant to antibiotic drugs, the combination with Paride enables the carbapenem meropenem to eradicate deep-dormant cultures in vitro and to reduce a resilient bacterial infection of a tissue cage implant in mice. Our work might inspire new treatments for persistent bacterial infections and, more broadly, highlights two viral strategies to infect dormant bacteria (hibernation and direct replication) that will guide future studies on phage-host interactions.
... However, various stresses such as oxidative and acid stresses activate the transcription of rpoS and increase the formation of persister cells in E. coli [34]. Many studies have demonstrated a clear relationship between the stringent response and SOS response with persister formation [37][38][39]. ...
Bacterial persister cells are quiescent, slow-growing or growth-arrested phenotypic variants of normal bacterial cells that are transiently tolerant to antibiotics. It seems that persister cells are the main cause of the recurrence of various chronic infections. Stress response (RpoS-mediated), toxin-antitoxin (TA) systems, inhibition of ATP production, reactive oxygen species (ROS), efflux pumps, bacterial SOS response, cell-to-cell communication and stringent response (ppGpp- mediated) are the primary potential mechanisms for persistence cell formation. However, eradicating persistent cells is challenging as the specific molecular mechanisms that initiate their formation remain fuzzy and unknown. Here we reviewed and summarized the current understanding of how bacterial persister cells are formed, controlled, and destroyed.
... The overproduction of RelE or HipA causes an increase in the persister population. HipA inhibits translation by the phosphorylation of EF-Tu [115], stimulates the RelA-dependent synthesis of (p)ppGpp [116], and phosphorylates glutamyl-tRNA synthetase (GltX), which becomes inactivated by phosphorylation by HipA [117]. RelE cleaves mRNA at the ribosomal A site with high codon specificity [118]. ...
Despite continuing progress in medical and surgical procedures, staphylococci remain the major Gram-positive bacterial pathogens that cause a wide spectrum of diseases, especially in patients requiring the utilization of indwelling catheters and prosthetic devices implanted temporarily or for prolonged periods of time. Within the genus, if Staphylococcus aureus and S. epidermidis are prevalent species responsible for infections, several coagulase-negative species which are normal components of our microflora also constitute opportunistic pathogens that are able to infect patients. In such a clinical context, staphylococci producing biofilms show an increased resistance to antimicrobials and host immune defenses. Although the biochemical composition of the biofilm matrix has been extensively studied, the regulation of biofilm formation and the factors contributing to its stability and release are currently still being discovered. This review presents and discusses the composition and some regulation elements of biofilm development and describes its clinical importance. Finally, we summarize the numerous and various recent studies that address attempts to destroy an already-formed biofilm within the clinical context as a potential therapeutic strategy to avoid the removal of infected implant material, a critical event for patient convenience and health care costs.
... The biological processes that govern the acquisition of tolerance and/or persistence are influenced by different factors such as genetic background, nutrient availability and other environmental conditions [4]. For instance, mutations in hipA, hipB and metG trigger the stringent response, which inhibits bacterial growth and results in increased tolerance to β-lactams and fluoroquinolones [5][6][7][8]. ...
... (p)ppGpp is associated with tolerance and persistence towards β-lactams and fluoroquinolones [6][7][8][20][21][22][23][24][25]. If the cell is at a non-growing state due to high levels of (p) ppGpp there are less active molecular targets available for interacting with the antibiotics and thus the bacteria become tolerant. ...
... Glyphosate induces the stringent response [40], which is characterised by cell growth arrest, a necessary step in the development of tolerance or persistence [51]. In addition, (p)ppGpp has been directly implicated with both phenomena [6][7][8][20][21][22][23][24][25]. Therefore it is possible that the strong effect of glyphosate on tolerance and persistence towards the three tested antibiotics is associated with the accumulation of (p)ppGpp. ...
Glyphosate is a herbicide widely used in food production that blocks the synthesis of aromatic amino acids in plants and in microorganisms and also induces the accumulation of the alarmone (p)ppGpp. The purpose of this study was to investigate whether glyphosate affects the resistance, tolerance or persistence of bacteria towards three different classes of antibiotics and the possible role of (p)ppGpp in this activity. Glyphosate did not affect the minimum inhibitory concentration of the tested antibiotics, but enhanced bacterial tolerance and/or persistence towards them. The upshift in ciprofloxacin and kanamycin tolerance was partially dependent on the presence of relA that promotes (p)ppGpp accumulation in response to glyphosate. Conversely, the strong increase in ampicillin tolerance caused by glyphosate was independent of relA. We conclude that by inducing aromatic amino acid starvation glyphosate contributes to the temporary increase in E. coli tolerance or persistence, but does not affect antibiotic resistance.
... Although arrest of cell growth and dormancy are the most common mechanisms underlying persister cell formation, a large body of evidence indicates persisters can be heterogeneous, demonstrating diverse physiological activities. Persister cells may have active electron transport chains 21,22 , produce ATP 23,24 , and futilely synthesize and degrade RNAs 25 . Even in apparent dormancy, persister cells must sustain a minimum adenylate energy charge 26 and must synthesize and repair cellular components that are denatured or degraded during antibiotic treatment 12,26 . ...
Bacterial persister cells are temporarily tolerant to bactericidal antibiotics but are not necessarily dormant and may exhibit physiological activities leading to cell damage. Based on the link between fluoroquinolone-mediated SOS responses and persister cell recovery, we screened chemicals that target fluoroquinolone persisters. Metabolic inhibitors (e.g., phenothiazines) combined with ofloxacin (OFX) perturbed persister levels in metabolically active cell populations. When metabolically stimulated, intrinsically tolerant stationary phase cells also became OFX-sensitive in the presence of phenothiazines. The effects of phenothiazines on cell metabolism and physiology are highly pleiotropic: at sublethal concentrations, phenothiazines reduce cellular metabolic, transcriptional, and translational activities; impair cell repair and recovery mechanisms; transiently perturb membrane integrity; and disrupt proton motive force by dissipating the proton concentration gradient across the cell membrane. Screening a subset of mutant strains lacking membrane-bound proteins revealed the pleiotropic effects of phenothiazines potentially rely on their ability to inhibit a wide range of critical metabolic proteins. Altogether, our study further highlights the complex roles of metabolism in persister cell formation, survival and recovery, and suggests metabolic inhibitors such as phenothiazines can be selectively detrimental to persister cells.
... Previous work suggested that the productivity of phage infections is positively correlated with host growth rate and that fully growth-arrested cells are refractory to phage replication 12,[23][24][25][26] . The resource limitation during bacterial dormancy therefore seems to be a major barrier to phage infection, and this phenomenon is weaponized against these viruses by a wide variety of bacterial immunity systems that shut down cellular physiology upon phage infection to restrict viral spread 27,28 . ...
Bacteriophages are fierce viral predators with no regard for pathogenicity or antibiotic resistance of their bacterial hosts. Despite early recognition of their therapeutic potential and the current escalation of bacterial multidrug resistance, phages have so far failed to become a regular treatment option in clinical practice. One reason is the occasional discrepancy between poor performance of selected phages in vivo despite high potency in vitro. Similar resilience of supposedly drug-sensitive bacterial infections to antibiotic treatment has been linked to persistence of dormant cells inside patients. Given the abundance of non-growing bacteria also in the environment, we wondered whether some phages can infect and kill these antibiotic-tolerant cells. As shown previously, most phages failed to replicate on dormant hosts and instead entered a state of hibernation or pseudolysogeny. However, we isolated a new Pseudomonas aeruginosa phage named Paride with the exciting ability to directly kill dormant, antibiotic-tolerant hosts by lytic replication, causing sterilization of deep-dormant cultures in synergy with the β-lactam meropenem. Intriguingly, efficient replication of Paride on dormant hosts depends on the same bacterial stress responses that also drive antibiotic tolerance. We therefore suggest that Paride hijacks weak spots in the dormant physiology of antibiotic-tolerant bacteria that could be exploited as Achilles′ heels for the development of new treatments targeting resilient bacterial infections.
... Overexpression of persistence-inducing toxins (tisB, hokB, hipA) increased the survival against BAC by more than 10-fold (Fig. 1f). The function of HipA and HokB requires (p)ppGpp, a global alarmone that controls the starvation-induced stringent response [42][43][44][45] . Consistent with this, a mutant that lacks both (p) ppGpp-synthesizing enzymes, relA and spoT, showed 20-fold decreased survival (Fig. 1f). ...
Biocides used as disinfectants are important to prevent the transmission of pathogens, especially during the current antibiotic resistance crisis. This crisis is exacerbated by phenotypically tolerant persister subpopulations that can survive transient antibiotic treatment and facilitate resistance evolution. Here, we show that E. coli displays persistence against a widely used disinfectant, benzalkonium chloride (BAC). Periodic, persister-mediated failure of disinfection rapidly selects for BAC tolerance, which is associated with reduced cell surface charge and mutations in the lpxM locus, encoding an enzyme for lipid A biosynthesis. Moreover, the fitness cost incurred by BAC tolerance turns into a fitness benefit in the presence of antibiotics, suggesting a selective advantage of BAC-tolerant mutants in antibiotic environments. Our findings highlight the links between persistence to disinfectants and resistance evolution to antimicrobials.
... Notably, there is evidence that self-digestion mediates the metabolism of persister cells, particularly those formed throughout the stationary phase (Figure 1). In fact, numerous independent studies have shown that persisters can harbor ETC activities [55,234], catabolize certain substrates to generate proton motive force (PMF) [234][235][236], produce energy molecules [237,238], and drive the futile production and degradation of RNA, leading to energy generation and dissipation [239]. Persister cells must also be able to repair antibiotic-induced damage to survive [16,17], and most repair mechanisms (e.g., DNA repair) are strongly ATPdependent [240][241][242][243]. Further, a number of independent groups have shown that deletion of enzymes associated with the TCA cycle and ETC (e.g., sdhA, sucA, mdh) drastically reduce persister levels, indicating the importance of energy metabolism in persister cell formation and/or survival [21, 55,244]. ...
... (iii) Persister metabolism is a controversial topic, reflecting the complexity and diversity of persister cell formation, survival, and resuscitation mechanisms, as well as the influence of culture conditions [31, 61,299]. Although persisters are mostly non-growing cells [33, [300][301][302], and their metabolism is generally lower than that of exponentially growing cells [13,37,102,103,233], these phenotypes might be at a metabolic steady state, providing energy molecules necessary for their survival [237,239]. Although it is well established that autophagy plays a crucial role in the metabolism of drug-tolerant cancer cells, it remains to be determined whether this is also true for bacterial persisters. ...
Cellular self-digestion is an evolutionarily conserved process occurring in prokaryotic cells that enables survival under stressful conditions by recycling essential energy molecules. Self-digestion, which is triggered by extracellular stress conditions, such as nutrient depletion and overpopulation, induces degradation of intracellular components. This self-inflicted damage renders the bacterium less fit to produce building blocks and resume growth upon exposure to fresh nutrients. However, self-digestion may also provide temporary protection from antibiotics until the self-digestion-mediated damage is repaired. In fact, many persistence mechanisms identified to date may be directly or indirectly related to self-digestion, as these processes are also mediated by many degradative enzymes, including proteases and ribonucleases (RNases). In this review article, we will discuss the potential roles of self-digestion in bacterial persistence.
... TA systems have been suggested to promote adaptation to various stresses (3). The ectopic expression of many toxins causes cells to enter a growth-arrested state in which they are stress and antibiotic tolerant (5)(6)(7). Additionally, the transcription of many TA systems increases in response to a range of stresses (8). However, there are few cases of strong, reproducible deletion phenotypes for TA systems, and recent work has demonstrated that toxins may not be activated even if their transcription is induced by a stress condition (9,10). ...
Toxin-antitoxin (TA) systems are widespread genetic modules found in almost all bacteria that can regulate their growth and may play prominent roles in phage defense. Escherichia coli encodes 11 TA systems in which the toxin is a known or predicted endoribonuclease. The targets and cleavage specificities of these endoribonucleases have remained largely uncharacterized, precluding an understanding of how each impacts cell growth and an assessment of whether they have distinct or overlapping targets.
... TA systems have been suggested to promote adaptation to various stresses 3 . The ectopic expression of many toxins causes cells to enter a growth-arrested state in which they are stressand antibiotic-tolerant [5][6][7] . Additionally, the transcription of many TA systems increases in response to a range of stresses 8 . ...
Toxin-antitoxin systems are widely distributed genetic modules typically featuring toxins that can inhibit bacterial growth and antitoxins that can reverse inhibition. Although Escherichia coli encodes 11 toxins with known or putative endoribonuclease activity, the target of most of these toxins remain poorly characterized. Using a new RNA-seq pipeline that enables the mapping and quantification of RNA cleavage with single-nucleotide resolution, we characterize the targets and specificities of 9 endoribonuclease toxins from E. coli . We find that these toxins use low-information cleavage motifs to cut a significant proportion of mRNAs in E. coli , but not tRNAs or the rRNAs from mature ribosomes. However, all the toxins, including those that are ribosome-dependent and cleave only translated RNA, inhibit ribosome biogenesis. This inhibition likely results from the cleavage of ribosomal protein transcripts, which disrupts the stoichiometry and biogenesis of new ribosomes and causes the accumulation of aberrant ribosome precursors. Collectively, our results provide a comprehensive, global analysis of endoribonuclease-based toxin-antitoxin systems in E. coli and support the conclusion that, despite their diversity, each disrupts translation and ribosome biogenesis.
Importance
Toxin-antitoxin systems are widespread genetic modules found in almost all bacteria that can regulate their growth and may play prominent roles in phage defense. Escherichia coli encodes 11 TA systems in which the toxin is a known or predicted endoribonuclease. The targets and cleavage specificities of these endoribonucleases have remained largely uncharacerized, precluding an understanding of how each impacts cell growth and an assessment of whether they have distinct or overlapping targets. Using a new and broadly applicable RNA-seq pipeline, we present a global analysis of 9 endoribonulease toxins from E. coli . We find that each uses a relatively low-information cleavage motif to cut a large proportion of mRNAs in E. coli , but not tRNAs or mature rRNAs. Notably, although the precise set of targets varies, each toxin efficiently disrupts ribosome biogenesis, primarily by cleaving the mRNAs of ribosomal proteins. In sum, the analyses presented provide new, comprehensive insights into the cleavage specificities and targets of almost all endoribonuclease toxins in E. coli . Despite different specificities, our work reveals a striking commonality in function as each toxin disrupts ribosome biogenesis and translation.
