Enhanced Efflux Activity Facilitates Drug Tolerance in Dormant Bacterial Cells
Yingying PuZhilun ZhaoYingxing LiJin ZouQi MaYanna ZhaoYuehua KeYun ZhuHuiyi ChenMatthew A. B. BakerHao GeYujie SunX. Sunney XieFan Bai
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Natural variations in gene expression provide a mechanism for multiple phenotypes to arise in an isogenic bacterial population. In particular, a sub-group termed persisters show high tolerance to antibiotics. Previously, their formation has been attributed to cell dormancy. Here we demonstrate that bacterial persisters, under β-lactam antibiotic treatment, show less cytoplasmic drug accumulation as a result of enhanced efflux activity. Consistently, a number of multi-drug efflux genes, particularly the central component TolC, show higher expression in persisters. Time-lapse imaging and mutagenesis studies further establish a positive correlation between tolC expression and bacterial persistence. The key role of efflux systems, among multiple biological pathways involved in persister formation, indicates that persisters implement a positive defense against antibiotics prior to a passive defense via dormancy. Finally, efflux inhibitors and antibiotics together effectively attenuate persister formation, suggesting a combination strategy to target drug tolerance.Keywords:
Efflux
Multidrug tolerance
Drug tolerance
SOS response
ABSTRACT Persisters are dormant phenotypic variants of regular cells that are tolerant to antibiotics and play an important role in recalcitrance of chronic infections to therapy. Persisters can be produced stochastically in a population untreated with antibiotics. At the same time, a deterministic component of persister formation has also been documented in a population of cells with DNA damaged by fluoroquinolone treatment. Expression of the SOS response under these conditions induces formation of persisters by increasing expression of the TisB toxin. This suggests that other stress responses may also contribute to persister formation. Of particular interest is oxidative stress that pathogens encounter during infection. Activated macrophages produce reactive oxygen and nitrogen species which induce the SoxRS and OxyR regulons. Genes controlled by these regulons deactivate the oxidants and promote repair. We examined the ability of oxidative stress induced by paraquat (PQ) to affect persister formation. Preincubation of cells with PQ produced a dramatic increase in the number of persisters surviving challenge with fluoroquinolone antibiotics. PQ did not affect killing by kanamycin or ampicillin. Persisters in a culture treated with PQ that survived a challenge with a fluoroquinolone were also highly tolerant to other antibiotics. PQ induces SoxRS, which in turn induces expression of the AcrAB-TolC multidrug-resistant (MDR) pump. Fluoroquinolones are extruded by this MDR pump, and the effect of PQ on antibiotic tolerance was largely abolished in a mutant that was defective in the pump. It appears that PQ, acting through AcrAB-TolC, reduces the concentration of fluoroquinolones in the cells. This allows a larger fraction of cells to become persisters in the presence of a fluoroquinolone. Analysis of a lexA3 mutant indeed showed a dependence of persister induction under these conditions on SOS. These findings show that induction of a classical resistance mechanism, MDR efflux, by oxidative stress leads to an increase in multidrug-tolerant persister cells.
Multidrug tolerance
Regulon
Efflux
Kanamycin
SOS response
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The ability of some bacteria within a population to tolerate antibiotic treatment is often attributed to prolonged bacterial infection 1-3 . Unlike antibiotic resistance, which generally results from genetic mutations or plasmid transfer 4,5 , antibiotic tolerance usually refers to the phenomenon that a subgroup of cells can survive high dose antibiotic treatment as a result of phenotypic heterogeneity 6,7 . Previous studies mainly associate antibiotic tolerance with cell dormancy, by hypothesizing that the lethal effects of antibiotics are disabled due to the extremely slow metabolic and proliferation rates in dormant bacteria 8,9 . However, less is known about how surviving bacteria subsequently escape from the dormant state and resuscitate, which is equally important for disease recurrence. Here we monitored the process of bacterial antibiotic tolerance and regrowth at the single-cell level, and found that each individual survival cell shows different ‘dormancy depth’, which in return regulates whether and when it can resume growth after removal of antibiotic. The persister cells are considered to be in shallow dormancy depth, while the viable but non-culturable cells (VBNC cells) are in deep dormancy depth. We further implemented time-lapse fluorescent imaging and biochemical analysis to establish that dynamic endogenous protein aggregation is an important indicator of bacterial dormancy depth. For cells to leave the dormant state and resuscitate, clearance of cellular protein aggregates and recovery of proteostasis are required. Through additional mutagenesis studies, we found the ability to recruit functional DnaK-ClpB machineries, which facilitate protein disaggregation in an ATP-dependent manner, determines the timeline (whether and when) for bacterial regrowth. Better understanding of the key factors regulating bacterial regrowth after surviving antibiotic attack could lead to new therapeutic strategies for combating bacterial antibiotic tolerance.
Multidrug tolerance
Proteostasis
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Persisters are bacterial cells that survive antibiotic treatment without acquiring resistanceconferring mutations. Upon antibiotic removal, they form a population identical to the parental one, complicating treatment of infectious diseases. Persisters are multidrug-tolerant and form in response to stresses. Persisters can form in response to fluoroquinolone (FQ) treatment through the induction of the SOS response. FQ treatment produces DNA double strand breaks (DSBs), which induce the SOS response. TisB, part of a toxin-antitoxin (TA) module, is induced by the SOS response and causes persister formation by decreasing the proton motive force and ATP levels. Some persisters also form prior to antibiotic challenge. For example, the number of persisters increases with the rise of cell density during normal growth, probably due to the stress of nutrient limitation. We wonder if non-antibiotic environmental stresses cause persister formation. In this thesis, we investigated the influence of oxidative stress and heat shock on bacterial drug tolerance, respectively. Bacterial pathogens are routinely exposed to reactive oxygen species (ROS) including hydrogen peroxide (H2O2), superoxide (O2⋅) and hydroxyl radial (OH⋅) produced by the host immune system. Bactericidal antibiotics have been reported to generate ROS. These oxidants cause damage to cellular macromolecules and induce the oxidative stress response. By pretreating a growing Escherichia coli population with superoxide-producing paraquat, we found that tolerance to FQs increased significantly. This increase in tolerance is mainly due to the induction of the efflux pump AcrAB-TolC, a part of the soxRS regulon. The efflux pump AcrABTolC decreases the intracellular concentration of FQs, reducing the amount of DSBs. Consequently, more cells can survive by repairing the damage or reducing the ATP level by thePersisters are bacterial cells that survive antibiotic treatment without acquiring resistance-conferring mutations. Upon antibiotic removal, they form a population identical to the parental one, complicating treatment of infectious diseases. Persisters are multidrug-tolerant and form in response to stresses. Persisters can form in response to fluoroquinolone (FQ) treatment through the induction of the SOS response. FQ treatment produces DNA double strand breaks (DSBs), which induce the SOS response. TisB, part of a toxin-antitoxin (TA) module, is induced by the SOS response and causes persister formation by decreasing the proton motive force and ATP levels. Some persisters also form prior to antibiotic challenge. For example, the number of persisters increases with the rise of cell density during normal growth, probably due to the stress of nutrient limitation.
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Multidrug tolerance
SOS response
Efflux
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Bacteria can survive antibiotic treatment without acquiring heritable antibiotic resistance. We investigated persistence to the fluoroquinolone ciprofloxacin in Escherichia coli. Our data show that a majority of persisters to ciprofloxacin were formed upon exposure to the antibiotic, in a manner dependent on the SOS gene network. These findings reveal an active and inducible mechanism of persister formation mediated by the SOS response, challenging the prevailing view that persisters are pre-existing and formed purely by stochastic means. SOS-induced persistence is a novel mechanism by which cells can counteract DNA damage and promote survival to fluoroquinolones. This unique survival mechanism may be an important factor influencing the outcome of antibiotic therapy in vivo.
SOS response
Persistence (discontinuity)
Multidrug tolerance
Bacterial Genetics
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Bacteria induce stress responses that protect the cell from lethal factors such as DNA-damaging agents. Bacterial populations also form persisters, dormant cells that are highly tolerant to antibiotics and play an important role in recalcitrance of biofilm infections. Stress response and dormancy appear to represent alternative strategies of cell survival. The mechanism of persister formation is unknown, but isolated persisters show increased levels of toxin/antitoxin (TA) transcripts. We have found previously that one or more components of the SOS response induce persister formation after exposure to a DNA-damaging antibiotic. The SOS response induces several TA genes in Escherichia coli. Here, we show that a knockout of a particular SOS-TA locus, tisAB/istR, had a sharply decreased level of persisters tolerant to ciprofloxacin, an antibiotic that causes DNA damage. Step-wise administration of ciprofloxacin induced persister formation in a tisAB-dependent manner, and cells producing TisB toxin were tolerant to multiple antibiotics. TisB is a membrane peptide that was shown to decrease proton motive force and ATP levels, consistent with its role in forming dormant cells. These results suggest that a DNA damage–induced toxin controls production of multidrug tolerant cells and thus provide a model of persister formation.
Multidrug tolerance
SOS response
Antitoxin
Stringent response
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Bacterial cells that stop growing but maintain viability and the capability to regrow are termed dormant and have been shown to transiently tolerate high concentrations of antimicrobials. Links between tolerance and cellular energetics as a possible explanation for the tolerance, have been investigated and have produced mixed and seemingly contradictory results. Because dormancy merely indicates growth arrest, which can be induced by various stimuli, we hypothesize that dormant cells may exist in a range of energetic states that depend on the environment. To energetically characterize different dormancies, we first induce them in a way that results in dormant populations and subsequently measure both of their main energy sources, the proton motive force magnitude and the concentration of ATP. We find that different types of dormancy exhibit characteristic energetic profiles that vary in level and dynamics. The energetic makeup was associated with survival to some antibiotics but not others. Our findings portray dormancy as a state that is rich in phenotypes with various stress survival capabilities. Because environmental conditions outside of the lab often halt or limit microbial growth, a typologization of dormant states may yield relevant insights on the survival and evolutionary strategies of these organisms.
Multidrug tolerance
Energetics
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Multidrug tolerance
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Population wide tolerance and persisters enable susceptible bacterial cells to endure hostile environments including antimicrobial exposure. The SOS response plays a significant role in generation of persister cells, population wide tolerance and shielding. The SOS pathway is an inducible DNA damage repair system that is also pivotal for bacterial adaptation, pathogenesis and diversification. In addition to two key SOS regulators, LexA and RecA, other stressors and stress responses control SOS factors. Bacteria are exposed to DNA damaging agents and other environmental and intracellular factors, including cigarette smoke, that trigger the SOS response at a number of sites within the host. The Escherichia coli TisB/IstR module is as of yet, the only known SOS regulated toxin-antitoxin module involved in persister formation. Nevertheless, the SOS response plays a key role in formation of biofilms that are highly recalcitrant to antimicrobials and can be abundant in persisters. Further the dynamic biofilm environment generates DNA damaging factors that trigger the SOS response within the biofilm, fueling bacterial adaptation and diversification. This review highlights the SOS response in relation to antimicrobial recalcetrance to antimicrobials in four clinically significant species Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Mycobacterium tuberculosis.
SOS response
Repressor lexA
Multidrug tolerance
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Bacteria produce persisters, a small subpopulation of cells that neither grow nor die in the presence of antibiotics. Persisters are tolerant against exposure to multiple antibiotics and they likely contribute to the relapse of bacterial infections after antibiotic therapy. The mechanism of persister formation is unknown, although several studies have pointed towards redundancy in persister formation mechanisms and the possible involvement of chromosomal toxin-antitoxin modules. While studying the genetic requirements for Escherichia coli persister survival after exposure to the DNA damaging antibiotic ciprofloxacin, we found that persister formation was an adaptive response to the antibiotic. Survivors to ciprofloxacin exhibited low levels of SOS induction and their survival depended largely on the SOSinducible small toxic peptide TisB. Ectopic overproduction of TisB decreased proton motive force and induced growth arrest and multidrug tolerance. Further, synthesized TisB peptide formed an anion-selective pore in an artificial lipid bilayer system. These results suggest that TisB acts as an uncoupler of oxidative phosphorylation after induction of the SOS response. These results challenge the common view of persisters as a metabolically inactive entity and show that persistence is in part an inducible response specific to a certain stress.
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Drug tolerance
Stringent response
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ABSTRACT Bacterial persistence is a transient, nonheritable physiological state that provides tolerance to bactericidal antibiotics. The stringent response, toxin-antitoxin modules, and stochastic processes, among other mechanisms, play roles in this phenomenon. How persistence is regulated is relatively ill defined. Here we show that cyclic AMP, a global regulator of carbon catabolism and other core processes, is a negative regulator of bacterial persistence in uropathogenic Escherichia coli , as measured by survival after exposure to a β-lactam antibiotic. This phenotype is regulated by a set of genes leading to an oxidative stress response and SOS-dependent DNA repair. Thus, persister cells tolerant to cell wall-acting antibiotics must cope with oxidative stress and DNA damage and these processes are regulated by cyclic AMP in uropathogenic E. coli . IMPORTANCE Bacterial persister cells are important in relapsing infections in patients treated with antibiotics and also in the emergence of antibiotic resistance. Our results show that in uropathogenic E. coli , the second messenger cyclic AMP negatively regulates persister cell formation, since in its absence much more persister cells form that are tolerant to β-lactams antibiotics. We reveal the mechanism to be decreased levels of reactive oxygen species, specifically hydroxyl radicals, and SOS-dependent DNA repair. Our findings suggest that the oxidative stress response and DNA repair are relevant pathways to target in the design of persister-specific antibiotic compounds.
Multidrug tolerance
SOS response
Stringent response
Pathogenic Escherichia coli
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