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 ₂O 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 ₂O 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.
Mast cells are evolutionarily ancient sentinel cells. Like basophils, mast cells express the high-affinity receptor for immunoglobulin E (IgE) and have been linked to host defense and diverse ...immune-system-mediated diseases. To better characterize the function of these cells, we assessed the transcriptional profiles of mast cells isolated from peripheral connective tissues and basophils isolated from spleen and blood. We found that mast cells were transcriptionally distinct, clustering independently from all other profiled cells, and that mast cells demonstrated considerably greater heterogeneity across tissues than previously appreciated. We observed minimal homology between mast cells and basophils, which shared more overlap with other circulating granulocytes than with mast cells. The derivation of mast-cell and basophil transcriptional signatures underscores their differential capacities to detect environmental signals and influence the inflammatory milieu.
The alarming spread of bacterial strains exhibiting resistance to current antibiotic therapies necessitates that we elucidate the specific genetic and biochemical responses underlying drug-mediated ...cell killing, so as to increase the efficacy of available treatments and develop new antibacterials. Recent research aimed at identifying such cellular contributions has revealed that antibiotics induce changes in metabolism that promote the formation of reactive oxygen species, which play a role in cell death. Here we discuss the relationship between drug-induced oxidative stress, the SOS response and their potential combined contribution to resistance development. Additionally, we describe ways in which these responses are being taken advantage to combat bacterial infections and arrest the rise of resistant strains.
Aminoglycoside antibiotics, such as gentamicin and kanamycin, directly target the ribosome, yet the mechanisms by which these bactericidal drugs induce cell death are not fully understood. Recently, ...oxidative stress has been implicated as one of the mechanisms whereby bactericidal antibiotics kill bacteria. Here, we use systems-level approaches and phenotypic analyses to provide insight into the pathway whereby aminoglycosides ultimately trigger hydroxyl radical formation. We show, by disabling systems that facilitate membrane protein traffic, that mistranslation and misfolding of membrane proteins are central to aminoglycoside-induced oxidative stress and cell death. Signaling through the envelope stress-response two-component system is found to be a key player in this process, and the redox-responsive two-component system is shown to have an associated role. Additionally, we show that these two-component systems play a general role in bactericidal antibiotic-mediated oxidative stress and cell death, expanding our understanding of the common mechanism of killing induced by bactericidal antibiotics.
Murine mast cells (MCs) contain two lineages: inducible bone marrow-derived mucosal MCs (MMCs) and constitutive embryonic-derived connective tissue MCs (CTMCs). Here, we use RNA sequencing, flow ...cytometry, and genetic deletion in two allergic lung inflammation models to define these two lineages. We found that inducible MCs, marked by β7 integrin expression, are highly distinct from airway CTMCs at rest and during inflammation and unaffected by targeted CTMC deletion. β7High MCs expand and mature during lung inflammation as part of a TGF-β-inducible transcriptional program that includes the MMC-associated proteases Mcpt1 and Mcpt2, the basophil-associated protease Mcpt8, granule components, and the epithelial-binding αE integrin. In vitro studies using bone marrow-derived MCs (BMMCs) identified a requirement for SCF in this this TGF-β-mediated development and found that epithelial cells directly elicit TGF-β-dependent BMMC up-regulation of mMCP-1 and αE integrin. Thus, our findings characterize the expansion of a distinct inducible MC subset in C57BL/6 mice and highlight the potential for epithelium to direct MMC development.
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Aspirin-exacerbated respiratory disease (AERD) is a severe disease involving dysregulated type 2 inflammation. However, the role other inflammatory pathways play in AERD is poorly ...understood.
We sought to broadly define the inflammatory milieu of the upper respiratory tract in AERD and to determine the effects of IL-4Rα inhibition on mediators of nasal inflammation.
Twenty-two AERD patients treated with dupilumab for 3 months were followed over 3 visits and compared to 10 healthy controls. Nasal fluid was assessed for 45 cytokines and chemokines using Olink Target 48. Blood neutrophils and cultured human mast cells, monocytes/macrophages, and nasal fibroblasts were assessed for response to IL-4/13 stimulation in vitro.
Of the nasal fluid cytokines measured, nearly one third were higher in AERD patients compared to healthy controls, including IL-6 and the IL-6 family–related cytokine oncostatin M (OSM), both of which correlated with nasal albumin levels, a marker of epithelial barrier dysregulation. Dupilumab significantly decreased many nasal mediators, including OSM and IL-6. IL-4 stimulation induced OSM production from mast cells and macrophages but not from neutrophils, and OSM and IL-13 stimulation induced IL-6 production from nasal fibroblasts.
In addition to type 2 inflammation, innate and IL-6–related cytokines are also elevated in the respiratory tract in AERD. Both OSM and IL-6 are locally produced in nasal polyps and likely promote pathology by negatively affecting epithelial barrier function. IL-4Rα blockade, although seemingly directed at type 2 inflammation, also decreases mediators of innate inflammation and epithelial dysregulation, which may contribute to dupilumab’s therapeutic efficacy in AERD.
The cause of severe nasal polyposis in aspirin-exacerbated respiratory disease (AERD) is unknown. Elevated antibody levels have been associated with disease severity in nasal polyps, but upstream ...drivers of local antibody production in nasal polyps are undetermined.
We sought to identify upstream drivers and phenotypic properties of local antibody-expressing cells in nasal polyps from subjects with AERD.
Sinus tissue was obtained from subjects with AERD, chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP), CRS without nasal polyps, and controls without CRS. Tissue antibody levels were quantified via ELISA and immunohistochemistry and were correlated with disease severity. Antibody-expressing cells were profiled with single-cell RNA sequencing, flow cytometry, and immunofluorescence, with IL-5Rα function determined through IL-5 stimulation and subsequent RNA sequencing and quantitative PCR.
Tissue IgE and IgG4 levels were elevated in AERD compared with in controls (P < .01 for IgE and P < .001 for IgG4 vs CRSwNP). Subjects with AERD whose nasal polyps recurred rapidly had higher IgE levels than did subjects with AERD, with slower regrowth (P = .005). Single-cell RNA sequencing revealed increased IL5RA, IGHG4, and IGHE in antibody-expressing cells from patients with AERD compared with antibody-expressing cells from patients with CRSwNP. There were more IL-5Rα+ plasma cells in the polyp tissue from those with AERD than in polyp tissue from those with CRSwNP (P = .026). IL-5 stimulation of plasma cells in vitro induced changes in a distinct set of transcripts.
Our study identifies an increase in antibody-expressing cells in AERD defined by transcript enrichment of IL5RA and IGHG4 or IGHE, with confirmed surface expression of IL-5Rα and functional IL-5 signaling. Tissue IgE and IgG4 levels are elevated in AERD, and higher IgE levels are associated with faster nasal polyp regrowth. Our findings suggest a role for IL-5Rα+ antibody-expressing cells in facilitating local antibody production and severe nasal polyps in AERD.
Modulation of bacterial chromosomal supercoiling is a function of DNA gyrase‐catalyzed strand breakage and rejoining. This reaction is exploited by both antibiotic and proteic gyrase inhibitors, ...which trap the gyrase molecule at the DNA cleavage stage. Owing to this interaction, double‐stranded DNA breaks are introduced and replication machinery is arrested at blocked replication forks. This immediately results in bacteriostasis and ultimately induces cell death. Here we demonstrate, through a series of phenotypic and gene expression analyses, that superoxide and hydroxyl radical oxidative species are generated following gyrase poisoning and play an important role in cell killing by gyrase inhibitors. We show that superoxide‐mediated oxidation of iron–sulfur clusters promotes a breakdown of iron regulatory dynamics; in turn, iron misregulation drives the generation of highly destructive hydroxyl radicals via the Fenton reaction. Importantly, our data reveal that blockage of hydroxyl radical formation increases the survival of gyrase‐poisoned cells. Together, this series of biochemical reactions appears to compose a maladaptive response, that serves to amplify the primary effect of gyrase inhibition by oxidatively damaging DNA, proteins and lipids.
Synopsis
The topological state of the Escherichia coli chromosome is actively maintained by DNA topoisomerases, which enzymatically modulate the degree of DNA supercoiling by catalyzing strand breakage and rejoining reactions (Champoux, 2001). This activity is critical to the processes of DNA replication (by promoting progression of the replication fork) and RNA transcription (by promoting local melting during initiation); additionally, topoisomerases play important roles in the decatenation of linked DNA and straightening of knotted DNA (Wang, 1996). Perhaps the most well‐characterized member of the DNA topoisomerase family is topoisomerase II or DNA gyrase (Gellert et al, 1976; Cozzarelli, 1980). Gyrase is responsible for the introduction of biologically essential negative DNA supercoils and accomplishes this task, in part, by inducing double‐stranded DNA breaks in an ATP‐dependent reaction (Reece and Maxwell, 1991).
DNA gyrase, and more specifically the reaction it catalyzes, is the target of both synthetic quinolone antibiotic‐ and natural proteic‐based inhibition (Bernard and Couturier, 1992; Chen et al, 1996). The interaction between gyrase inhibitors and DNA‐bound gyrase results in the formation of a stable open cleavage complex which sterically prohibits the passage of DNA and RNA polymerases, while generating widespread DNA damage (Critchlow et al, 1997; Drlica and Zhao, 1997). The combinatorial effect of replication fork stalling and double‐stranded DNA break accumulation is rapid entry into bacteriostasis, and ultimately is considered sufficient to induce cell death.
In this work, we performed a series of phenotypic and gene expression studies on E. coli treated with quinolone or norfloxacin or expressing the peptide toxin, CcdB, in order to identify genetic and biochemical events that contribute to gyrase inhibitor‐induced cell death. Taking a systems biology approach, we enriched our microarray‐derived transcriptome data using biological pathway classifications and transcription factor regulatory connections (Ashburner et al, 2000; Boyle et al, 2004; Camon et al, 2004; Salgado et al, 2006; Faith et al, 2007) to distinguish significantly changing functional units from background gene expression patterns (highlighted in Figure 2). As anticipated, this analysis showed that expression of the DNA damage response and repair program was highly upregulated soon after gyrase inhibition. Unexpectedly, however, our systems‐level approach revealed that genes associated with iron uptake, iron–sulfur cluster synthesis and oxidative stress also exhibited significantly increased expression upon norfloxacin treatment and CcdB expression. The results of our gene expression analysis were then used to guide follow‐up experiments that explored these varied responses to gyrase poisoning.
Using the fluorescent reporter dye, HPF (Setsukinai et al, 2003), we demonstrate that gyrase inhibition by either norfloxacin or CcdB induces the formation of highly deleterious hydroxyl radicals, which oxidatively damage DNA, proteins and membrane lipids. Importantly, using the iron chelator, o‐phenanthroline, we show that hydroxyl radical generation occurs through the Fenton reaction (in which hydrogen peroxide is reduced by ferrous iron; Imlay, 2003), and that blockage of the Fenton reaction leads to an increase in the survival of gyrase‐inhibited E. coli.
To determine the Fenton‐reactive iron source contributing to gyrase inhibitor‐mediated cell death, we employed a variety of promoter‐reporter gene fusions (including constructs that sense changes in the cellular superoxide response, iron regulation and iron–sulfur cluster synthesis) and performed growth studies of several single‐gene knockout strains. The results of these experiments highlighted critical steps in the formation of hydroxyl radicals—first, superoxide is indeed generated following inhibition of DNA gyrase; second, a breakdown of iron regulatory dynamics, likely related to iron–sulfur cluster oxidation by superoxide, occurs upon gyrase poisoning; third, and perhaps most importantly, the number and redox status of iron–sulfur clusters present intracellularly is critical to hydroxyl radical generation and thus, gyrase inhibitor‐induced cell death.
Together, these data have provided for the construction of an oxidative damage cell death pathway model (Figure 8). This series of events appears to constitute a maladaptive response to gyrase inhibition by E. coli, growing in an oxygen‐rich environment, which amplifies the primary effect of gyrase poisons like norfloxacin and CcdB. These results may provide new targets for novel antibacterial therapies with enhanced bactericidal activity.
We performed phenotypic, genetic and microarray analyses on Escherichia coli challenged with the synthetic fluoroquinolone antibiotic, norfloxacin, and the proteic toxin, CcdB, to identify additional biochemical contributions to cell death resulting from DNA gyrase inhibition.
We show that gyrase poisoning by either norfloxacin or CcdB results in the formation of highly deleterious hydroxyl radicals, via the Fenton reaction, which play a key role in inhibitor‐induced cell death.
Taking a systems biology approach, we employed functional pathway classifications and transcription factor regulatory connections to enrich our gene expression data set, and identified coordinated biochemical responses and secondary networks activated by gyrase inhibition which lead to formation of hydroxyl radicals.
Through a series of experiments involving single‐gene knockout strains and promoter‐reporter gene constructs, we show that the redox status and number of iron‐sulfur clusters is critical to gyrase inhibitor‐induced hydroxyl radical formation, and ultimately, cell death.
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