Role of Autofluorescence in Flow Cytometric Analysis of Escherichia coli Treated with Bactericidal Antibiotics

Renggli, Sabine; Keck, Wolfgang; Jenal, Urs; Ritz, Daniel

doi:10.1128/jb.00393-13

Cited by 80 publications

(74 citation statements)

References 47 publications

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“…Fourth, we used two dyes based on different chemistries that have been widely used for ROS detection in previous studies (3, 12, 25): 3′-(p-hydroxyphenyl) fluorescein (HPF) and 5/6-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA), whose fluorescence has been shown to correlate with O 2 − production determined by direct measurement (24). For both dyes, we observed statistically significant increases in fluorescence compared with controls for maltose-induced autofluorescence and morphology changes (26,27) in the absence of a dye (Fig. 2D).…”

Section: Male-lacz Induction Is Not Lethal In Anaerobic Conditions Bumentioning

confidence: 56%

Lethality of MalE-LacZ hybrid protein shares mechanistic attributes with oxidative component of antibiotic lethality

Takahashi

Gruber

Yang

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Downstream metabolic events can contribute to the lethality of drugs or agents that interact with a primary cellular target. In bacteria, the production of reactive oxygen species (ROS) has been associated with the lethal effects of a variety of stresses including bactericidal antibiotics, but the relative contribution of this oxidative component to cell death depends on a variety of factors. Experimental evidence has suggested that unresolvable DNA problems caused by incorporation of oxidized nucleotides into nascent DNA followed by incomplete base excision repair contribute to the ROSdependent component of antibiotic lethality. Expression of the chimeric periplasmic-cytoplasmic MalE-LacZ 72-47 protein is an historically important lethal stress originally identified during seminal genetic experiments that defined the SecY-dependent protein translocation system. Multiple, independent lines of evidence presented here indicate that the predominant mechanism for MalE-LacZ lethality shares attributes with the ROS-dependent component of antibiotic lethality. MalE-LacZ lethality requires molecular oxygen, and its expression induces ROS production. The increased susceptibility of mutants sensitive to oxidative stress to MalE-LacZ lethality indicates that ROS contribute causally to cell death rather than simply being produced by dying cells. Observations that support the proposed mechanism of cell death include MalE-LacZ expression being bacteriostatic rather than bactericidal in cells that overexpress MutT, a nucleotide sanitizer that hydrolyzes 8-oxo-dGTP to the monophosphate, or that lack MutM and MutY, DNA glycosylases that process base pairs involving 8-oxo-dGTP. Our studies suggest stress-induced physiological changes that favor this mode of ROS-dependent death.A gents or conditions that inhibit important biological processes can kill cells. However, physiological processes metabolically downstream of the initial inhibition can also contribute to cell death (1). For example, β-lactam antibiotics not only inhibit penicillin-binding proteins leading to lysis; they also induce a futile cycle of cell wall synthesis and degradation that contributes to their killing (2). In another example, lethal attacks on Escherichia coli mediated by the type VI secretion system P1vir phage and the antimicrobial peptide polymyxin B elicit the production of reactive oxygen species (ROS) that contribute to cell death (3). In bacteria, ROS production has been associated with the lethal effects of diverse stresses (4). In most cases, the detailed mechanisms responsible for ROS production are poorly understood; however, a variety of futile metabolic cycles can elicit H 2 O 2 production, illustrating the breadth of possible metabolic perturbations that could potentially induce oxidative stress (5).Despite widespread evidence that endogenous ROS produced as a consequence of metabolic stress can be lethal to bacteria and eukaryotes, the application of this concept to antibiotic lethality has been complicated. Evidence from multiple investiga...

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Section: Male-lacz Induction Is Not Lethal In Anaerobic Conditions Bumentioning

confidence: 56%

Lethality of MalE-LacZ hybrid protein shares mechanistic attributes with oxidative component of antibiotic lethality

Takahashi

Gruber

Yang

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…It is unlikely that activation of all dyes would result from nonspecific or confounding chemical events and oxidizing species. We found that for the majority of dyes tested, wild-type cells treated with ampicillin (a β-lactam), gentamicin (an aminoglycoside), or norfloxacin (a fluoroquinolone) exhibited statistically significant increases in fluorescence compared with controls for antibiotic treatment-related autofluorescence (55), in which no dye was added. Collectively, the broad activation of these dyes suggests several different reactive species that may damage biomolecules are produced in response to antibiotic treatment.…”

Section: Resultsmentioning

confidence: 95%

Antibiotics induce redox-related physiological alterations as part of their lethality

Dwyer

Belenky

Yang

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

737

683

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Deeper understanding of antibiotic-induced physiological responses is critical to identifying means for enhancing our current antibiotic arsenal. Bactericidal antibiotics with diverse targets have been hypothesized to kill bacteria, in part by inducing production of damaging reactive species. This notion has been supported by many groups but has been challenged recently. Here we robustly test the hypothesis using biochemical, enzymatic, and biophysical assays along with genetic and phenotypic experiments. We first used a novel intracellular H 2 O 2 sensor, together with a chemically diverse panel of fluorescent dyes sensitive to an array of reactive species to demonstrate that antibiotics broadly induce redox stress. Subsequent gene-expression analyses reveal that complex antibiotic-induced oxidative stress responses are distinct from canonical responses generated by supraphysiological levels of H 2 O 2 . We next developed a method to quantify cellular respiration dynamically and found that bactericidal antibiotics elevate oxygen consumption, indicating significant alterations to bacterial redox physiology. We further show that overexpression of catalase or DNA mismatch repair enzyme, MutS, and antioxidant pretreatment limit antibiotic lethality, indicating that reactive oxygen species causatively contribute to antibiotic killing. Critically, the killing efficacy of antibiotics was diminished under strict anaerobic conditions but could be enhanced by exposure to molecular oxygen or by the addition of alternative electron acceptors, indicating that environmental factors play a role in killing cells physiologically primed for death. This work provides direct evidence that, downstream of their target-specific interactions, bactericidal antibiotics induce complex redox alterations that contribute to cellular damage and death, thus supporting an evolving, expanded model of antibiotic lethality.reactive oxygen species | DNA repair | mutagenesis T he increasing incidence of antibiotic-resistant infections coupled with a declining antibiotic pipeline has created a global public health threat (1-6). Therefore there is a pressing need to expand our conceptual understanding of how antibiotics act and to use insights gained from such efforts to enhance our antibiotic arsenal. It has been proposed that different classes of bactericidal antibiotics, regardless of their drug-target interactions, generate varying levels of deleterious reactive oxygen species (ROS) that contribute to cell killing (7,8). This unanticipated notion, built upon important prior work (9-11), has been extended and supported by multiple laboratories investigating wide-ranging drug classes (e.g., β-lactams, aminoglycosides, and fluoroquinolones) and bacterial species (e.g., Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica, Mycobacterium tuberculosis, Bacillus subtilis, Staphylococcus aureus, Acinetobacter baumannii, Burkholderia cepecia, Streptococcus pneumonia, Enterococcus faecalis) using independent lines of evidence (12-39). Importantly,...

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“…Thus, we disregarded the results of HPF assays. Similar criticism about the use of HPF was raised recently concerning the interference between the autofluorescence of cells with the actual fluorescence in the presence of the probe, especially upon antibiotic treatment (32).…”

Section: Resultsmentioning

confidence: 99%

Putrescine Reduces Antibiotic-Induced Oxidative Stress as a Mechanism of Modulation of Antibiotic Resistance in Burkholderia cenocepacia

El-Halfawy

Valvano

2014

Antimicrob Agents Chemother

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cCommunication of antibiotic resistance among bacteria via small molecules is implicated in transient reduction of bacterial susceptibility to antibiotics, which could lead to therapeutic failures aggravating the problem of antibiotic resistance. Released putrescine from the extremely antibiotic-resistant bacterium Burkholderia cenocepacia protects less-resistant cells from different species against the antimicrobial peptide polymyxin B (PmB). Exposure of B. cenocepacia to sublethal concentrations of PmB and other bactericidal antibiotics induces reactive oxygen species (ROS) production and expression of the oxidative stress response regulator OxyR. We evaluated whether putrescine alleviates antibiotic-induced oxidative stress. The accumulation of intracellular ROS, such as superoxide ion and hydrogen peroxide, was assessed fluorometrically with dichlorofluorescein diacetate, while the expression of OxyR and putrescine synthesis enzymes was determined in luciferase assays using chromosomal promoter-lux reporter system fusions. We evaluated wild-type and isogenic deletion mutant strains with defects in putrescine biosynthesis after exposure to sublethal concentrations of PmB and other bactericidal antibiotics. Exogenous putrescine protected against oxidative stress induced by PmB and other antibiotics, whereas reduced putrescine synthesis resulted in increased ROS generation and a parallel increased sensitivity to PmB. Of the 3 B. cenocepacia putrescine-synthesizing enzymes, PmB induced only BCAL2641, an ornithine decarboxylase. This study reveals BCAL2641 as a critical component of the putrescine-mediated communication of antibiotic resistance and as a plausible target for designing inhibitors that would block the communication of such resistance among different bacteria, ultimately reducing the window of therapeutic failure in treating bacterial infections.

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Role of Autofluorescence in Flow Cytometric Analysis of Escherichia coli Treated with Bactericidal Antibiotics

Cited by 80 publications

References 47 publications

Lethality of MalE-LacZ hybrid protein shares mechanistic attributes with oxidative component of antibiotic lethality

Lethality of MalE-LacZ hybrid protein shares mechanistic attributes with oxidative component of antibiotic lethality

Antibiotics induce redox-related physiological alterations as part of their lethality

Putrescine Reduces Antibiotic-Induced Oxidative Stress as a Mechanism of Modulation of Antibiotic Resistance in Burkholderia cenocepacia

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