Persister‐promoting bacterial toxin TisB produces anion‐selective pores in planar lipid bilayers

Gurnev, Philip A.; Ortenberg, Ron; Dörr, Tobias; Lewis, Kim; Bezrukov, Sergey M.

doi:10.1016/j.febslet.2012.06.021

Cited by 92 publications

(100 citation statements)

References 23 publications

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“…In some cases, a pore-forming function has been indicated (38,39), but in most others the mechanism of action is unclear and the phenotypic effects do not necessarily require pore formation (22,40). In the Bacillus subtilis bsrG/SR4 system, pore formation has been explicitly ruled out (41).…”

Section: Discussionmentioning

confidence: 99%

Examination of Enterococcus faecalis Toxin-Antitoxin System Toxin Fst Function Utilizing a Pheromone-Inducible Expression Vector with Tight Repression and Broad Dynamic Range

et al. 2017

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Tools for regulated gene expression in Enterococcus faecalis are extremely limited. In this report, we describe the construction of an expression vector for E. faecalis, designated pCIE, utilizing the P Q pheromone-responsive promoter of plasmid pCF10. We demonstrate that this promoter is tightly repressed, responds to nanogram quantities of the peptide pheromone, and has a large dynamic range. To demonstrate its utility, the promoter was used to control expression of the toxic peptides of two par family toxin-antitoxin (TA) loci present in E. faecalis, par pAD1 of the pAD1 plasmid and par EF0409 located on the E. faecalis chromosome. The results demonstrated differences in the modes of regulation of toxin expression and in the effects of toxins of these two related systems. We anticipate that this vector will be useful for further investigation of par TA system function as well as the regulated expression of other genes in E. faecalis. IMPORTANCE E. faecalis is an important nosocomial pathogen and a model organism for examination of the genetics and physiology of Gram-positive cocci. While numerous genetic tools have been generated for the manipulation of this organism, vectors for the regulated expression of cloned genes remain limited by high background expression and the use of inducers with undesirable effects on the cell. Here we demonstrate that the P Q pheromone-responsive promoter is repressed tightly enough to allow cloning of TA system toxins and evaluate their effects at very low induction levels. This tool will allow us to more fully examine TA system function in E. faecalis and to further elucidate its potential roles in cell physiology.KEYWORDS Enterococcus, expression vector, pheromone, toxin-antitoxin systems E nterococci have emerged over the past few decades as major nosocomial pathogens. They are responsible for 15% of health care-associated urinary tract infections (1), cause 5 to 15% of all infectious endocarditis cases (2), and are currently the second leading cause of health care-associated bacteremia (1). Treatment is complicated by intrinsic resistance to a variety of antibiotics and acquired resistance to high-level aminoglycosides, vancomycin, and other antibiotics (3). A variety of mobile genetic elements (MGE) facilitate the transfer of antibiotic resistance determinants (4). Enterococcus faecalis and Enterococcus faecium cause the majority of infections, with E. faecalis generally being more pathogenic and E. faecium displaying greater antibiotic resistance, particularly to vancomycin. E. faecalis has also emerged as a model organism for

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Section: Discussionmentioning

confidence: 99%

Examination of Enterococcus faecalis Toxin-Antitoxin System Toxin Fst Function Utilizing a Pheromone-Inducible Expression Vector with Tight Repression and Broad Dynamic Range

et al. 2017

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“…Under these conditions, most of the persisters are formed in a TisB-dependent manner. The TisB protein is a typical antimicrobial peptide (13,39), which acts by forming anion channels in the membrane (15). A decrease in proton motive force (PMF) and ATP then shuts down the antibiotic targets, leading to dormancy and drug tolerance.…”

Section: Discussionmentioning

confidence: 99%

Role of Oxidative Stress in Persister Tolerance

Vulić

Keren

et al. 2012

Antimicrob Agents Chemother

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ABSTRACTPersisters 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 alexA3mutant 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.

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“…Therefore, the surviving cells could resume growth and become vulnerable again, yet there is no significant loss of viability for an extended period of time. The induction of the SOS-controlled TisB toxin dissipates the proton motive force (Gurnev et al 2012) and depletes the cellular ATP pool (Unoson and Wagner 2008), possibly preventing further damage by effectively shutting down the cell after the repair of the original lesions. However, this mechanism would operate in only a fraction of cases as the survival is TisB dependent only at high concentrations of FQ.…”

Section: Discussionmentioning

confidence: 99%

“…The SOS-regulated tisAB/istR TA locus contributes to tolerance to ciprofloxacin (Dörr et al 2010). When overproduced, the membrane peptide TisB decreases proton motive force and intracellular ATP levels (Gurnev et al 2012). During SOS induction, tisB is overexpressed, which may induce stasis and prevent killing by the antibiotic.…”

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confidence: 99%

Tolerance ofEscherichia colito Fluoroquinolone Antibiotics Depends on Specific Components of the SOS Response Pathway

2013

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Bacteria exposed to bactericidal fluoroquinolone (FQ) antibiotics can survive without becoming genetically resistant. Survival of these phenotypically resistant cells, commonly called "persisters," depends on the SOS gene network. We have examined mutants in all known SOS-regulated genes to identify functions essential for tolerance in Escherichia coli. The absence of DinG and UvrD helicases and the Holliday junction processing enzymes RuvA and RuvB leads to a decrease in survival. Analysis of the respective mutants indicates that, in addition to repair of double-strand breaks, tolerance depends on the repair of collapsed replication forks and stalled transcription complexes. Mutation in recF results in increased survival, which identifies RecAF recombination as a poisoning mechanism not previously linked to FQ lethality. DinG acts upstream of SOS promoting its induction, whereas RuvAB participates in repair only. UvrD directly promotes all repair processes initiated by FQ-induced damage and prevents RecAF-dependent misrepair, making it one of the crucial SOS functions required for tolerance. IN the face of a potentially lethal stress, the presence of phenotypic variants of genetically identical bacterial cells can make a difference between survival of the lineage and extermination. A clonal population of cells of a strain genetically susceptible to an antibiotic can harbor tolerant phenocopies that will survive the antibiotic challenge. These cells are termed "persisters" and are of special interest because of their ability to influence the outcome of antibiotic treatments (Lewis 2010;Mulcahy et al. 2010). Toxins from chromosomally encoded toxin-antitoxin (TA) modules have been shown to play a role in persister formation under certain condition (Keren et al. 2004;Dörr et al. 2010;Gerdes and Maisonneuve 2012) To date, screening of E. coli mutant libraries has yielded no single mutants that completely lack persisters (Spoering 2006;Hansen et al. 2008). These studies have shown that there are many pathways that lead to persister formation (Lewis 2010). We have shown that tolerance to fluoroquinolone (FQ) antibiotics depends on a functional SOS response (Dörr et al. 2009). The SOS-regulated tisAB/istR TA locus contributes to tolerance to ciprofloxacin (Dörr et al. 2010). When overproduced, the membrane peptide TisB decreases proton motive force and intracellular ATP levels (Gurnev et al. 2012). During SOS induction, tisB is overexpressed, which may induce stasis and prevent killing by the antibiotic. However, it influences tolerance only at high ciprofloxacin concentration [1003 minimal inhibitory concentration (MIC)]; therefore, it is not the only mechanism of FQ tolerance.Fluoroquinolones target gyrase and topoisomerase IV (Drlica and Zhao 1997). Upon FQ binding, these enzymes become endonucleases introducing double-strand breaks (DSBs) into the bacterial chromosome (Malik et al. 2006). Cells repair DSBs largely through the DNA-damage-inducible SOS gene network (Radman 1975;Friedberg et al. 2006). The su...

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Persister‐promoting bacterial toxin TisB produces anion‐selective pores in planar lipid bilayers

Cited by 92 publications

References 23 publications

Examination of Enterococcus faecalis Toxin-Antitoxin System Toxin Fst Function Utilizing a Pheromone-Inducible Expression Vector with Tight Repression and Broad Dynamic Range

Examination of Enterococcus faecalis Toxin-Antitoxin System Toxin Fst Function Utilizing a Pheromone-Inducible Expression Vector with Tight Repression and Broad Dynamic Range

Role of Oxidative Stress in Persister Tolerance

Tolerance ofEscherichia colito Fluoroquinolone Antibiotics Depends on Specific Components of the SOS Response Pathway

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