Generation of reactive oxygen species by lethal attacks from competing microbes

Dong, Tao; Dong, Shaojun; Catalano, Christy; Moore, Richard A.; Liang, Xiaoye; Mekalanos, John J.

doi:10.1073/pnas.1425007112

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

2015

DOI: 10.1073/pnas.1425007112

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Generation of reactive oxygen species by lethal attacks from competing microbes

Tao Dong

Shaojun Dong

Christy Catalano

et al.

Abstract: Whether antibiotics induce the production of reactive oxygen species (ROS) that contribute to cell death is an important yet controversial topic. Here, we report that lethal attacks from bacterial and viral species also result in ROS production in target cells. Using soxS as an ROS reporter, we found soxS was highly induced in Escherichia coli exposed to various forms of attacks mediated by the type VI secretion system (T6SS), P1vir phage, and polymyxin B. Using a fluorescence ROS probe, we found enhanced ROS … Show more

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Cited by 140 publications

(137 citation statements)

References 40 publications

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“…Each antibacterial effector coexists with an antagonistic immunity protein that confers protection during T6SS-mediated attacks between sister cells (3,18,24). Interestingly, T6SS-mediated lethal attacks induce the generation of reactive oxygen species in the prey cells (25), similar to cells treated with antibiotics (26,27).…”

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confidence: 99%

Identification of divergent type VI secretion effectors using a conserved chaperone domain

Liang

Moore

Wilton

et al. 2015

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

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140

186

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The type VI secretion system (T6SS) is a lethal weapon used by many bacteria to kill eukaryotic predators or prokaryotic competitors. Killing by the T6SS results from repetitive delivery of toxic effectors. Despite their importance in dictating bacterial fitness, systematic prediction of T6SS effectors remains challenging due to high effector diversity and the absence of a conserved signature sequence. Here, we report a class of T6SS effector chaperone (TEC) proteins that are required for effector delivery through binding to VgrG and effector proteins. The TEC proteins share a highly conserved domain (DUF4123) and are genetically encoded upstream of their cognate effector genes. Using the conserved TEC domain sequence, we identified a large family of TEC genes coupled to putative T6SS effectors in Gram-negative bacteria. We validated this approach by verifying a predicted effector TseC in Aeromonas hydrophila. We show that TseC is a T6SS-secreted antibacterial effector and that the downstream gene tsiC encodes the cognate immunity protein. Further, we demonstrate that TseC secretion requires its cognate TEC protein and an associated VgrG protein.Distinct from previous effector-dependent bioinformatic analyses, our approach using the conserved TEC domain will facilitate the discovery and functional characterization of new T6SS effectors in Gram-negative bacteria.interspecies interaction | colicin | antitoxin | toxin | protein secretion

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mentioning

confidence: 99%

Identification of divergent type VI secretion effectors using a conserved chaperone domain

Liang

Moore

Wilton

et al. 2015

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

Self Cite

140

186

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“…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).…”

mentioning

confidence: 99%

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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“…Reactive oxygen species (ROS) have been proposed to be key factors in antimicrobial lethality (3)(4)(5), and substantial evidence supports this proposition (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19). However, their role in lethality has been challenged (20,21).…”

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confidence: 99%

Dimethyl Sulfoxide Protects Escherichia coli from Rapid Antimicrobial-Mediated Killing

Wang

Xue

et al. 2016

Antimicrob Agents Chemother

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The contribution of reactive oxygen species (ROS) to antimicrobial lethality was examined by treating Escherichia coli with dimethyl sulfoxide (DMSO), an antioxidant solvent frequently used in antimicrobial studies. DMSO inhibited killing by ampicillin, kanamycin, and two quinolones and had little effect on MICs. DMSO-mediated protection correlated with decreased ROS accumulation and provided evidence for ROS-mediated programmed cell death. These data support the contribution of ROS to antimicrobial lethality and suggest caution when using DMSO-dissolved antimicrobials for short-time killing assays. O ne approach to help stem the emergence of new antimicrobial resistance is to kill bacterial pathogens rapidly, thereby quickly reducing bacterial burden and restricting effects of stressinduced mutagenesis (1, 2). Reactive oxygen species (ROS) have been proposed to be key factors in antimicrobial lethality (3-5), and substantial evidence supports this proposition (3-19). However, their role in lethality has been challenged (20, 21). If ROS are indeed integral to antimicrobial-mediated killing, compounds that act as antioxidants and radical scavengers should reduce antimicrobial lethality. We chose to examine this hypothesis using the radical scavenger dimethyl sulfoxide (DMSO) (22, 23), because it is also a popular solvent that is widely used in the pharmaceutical industry and in antimicrobial research due to its (i) low toxicity, (ii) ability to dissolve both organic and inorganic compounds, (iii) ability to remain in a liquid state over a broad temperature range (e.g., from 19°C to 189°C), (iv) ability to enhance cell membrane permeability, and (v) miscibility in water and a wide range of organic solvents. We report here that DMSO interferes with rapid killing of Escherichia coli and Acinetobacter baumannii by members of three antimicrobial classes.E. coli K-12 strains BW25113 and ATCC 25922 and A. baumannii strain ATCC 17978 were grown in Luria-Bertani (LB) broth or on LB agar at 37°C. LB medium, ampicillin, and kanamycin were obtained from Sangon Biotech Co., Ltd. (Shanghai, China). Oxolinic acid, ciprofloxacin, and DMSO were acquired from Sigma-Aldrich Co. (St. Louis, MO). Meropenem (Sumitomo Dainippon Pharma Co. Ltd.) was obtained from Zhongshan Hospital Pharmacy. The fluorescent probe carboxy-H 2 DCFDA [5(6)-carboxy-2=,7=-dichlorodihydrofluorescein diacetate] was purchased from Invitrogen (Grand Island, NY). All chemical stock solutions were dissolved in sterile water (except carboxy-H 2 DCFDA, which was dissolved in DMSO) and stored at Ϫ80°C until use. MICs were assayed by broth dilution according to CLSI protocols (24); exponentially growing cultures were diluted to 10 5 CFU/ml for MIC determinations. To measure rapid bacterial killing, exponentially growing cultures at about 5 ϫ 10 8 CFU/ml were treated with antimicrobials, after which they were serially diluted and plated on drug-free agar. Viable colony counts were determined after an overnight incubation at 37°C; percentage survival rates were calculat...

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Generation of reactive oxygen species by lethal attacks from competing microbes

Cited by 140 publications

References 40 publications

Identification of divergent type VI secretion effectors using a conserved chaperone domain

Identification of divergent type VI secretion effectors using a conserved chaperone domain

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

Dimethyl Sulfoxide Protects Escherichia coli from Rapid Antimicrobial-Mediated Killing

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