Gyrase inhibitors induce an oxidative damage cellular death pathway in Escherichia coli

Daniel J Dwyer^1,2,a, Michael A Kohanski^2,3,4,a, Boris Hayete^2,5 & James J Collins^1,2,3,5

^aThese authors contributed equally to this work

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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.

Figure 2

Transcriptome response to gyrase inhibition. Highlighted is a portion of the functionally enriched gene expression response of norfloxacin-treated or CcdB-expressing wild-type cells. Relative weighted z-scores (a measure of standard deviation) were calculated for each gene, based on comparison to mean expression values derived from a large (500) database of microarray data; these values were then normalized by subtracting the corresponding uninduced sample z-scores. Using biochemical pathway and transcription factor regulatory classifications, we identified significantly upregulated functional units that responded in a coordinated manner. For each functional unit, genes that exhibited a weighted z-score 2 standard deviations are shown; scale is shown on left. This analysis is described in greater detail in Materials and methods and Supplementary information. Additionally, all gyrase inhibitor microarray results can be found in Supplementary information.

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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.

Figure 8

Oxidative damage cell death pathway model. A model for iron misregulation and reactive oxygen species generation following gyrase inhibition and DNA damage formation. (A) Gyrase inhibitors (red triangles), such as norfloxacin and CcdB, target DNA-bound gyrase (yellow circles). The resultant complex induces double-stranded breakage and loss of chromosomal supercoiling by preventing strand rejoining by the gyrase enzyme. (B) Gyrase poisoning promotes the generation of superoxide (O₂^–), which (C) oxidatively attacks iron–sulfur clusters (three-dimensional cube depicts [4Fe–4S] cluster; iron and sulfur are shown as orange and blue circles, respectively); sustained superoxide attack of iron–sulfur-containing proteins (light blue) leads to functional inactivation (dark blue), destabilization and iron leaching. (D) Repetitious oxidation and repair of clusters, or redox cycling, promotes iron misregulation and may serve to generate a cytoplasmic pool of 'free' ferrous (Fe²⁺) iron. (E) Ferrous iron, via the Fenton reaction, rapidly catalyzes the formation of deleterious hydroxyl radicals (OH), which readily damage DNA, lipids and proteins; the Fenton reaction can thus take place at destabilized iron–sulfur clusters or where 'free' ferrous iron has accumulated. We propose that reactive oxygen species are generated via an oxygen-dependent death pathway that amplifies the primary effect of gyrase inhibition and contributes to cell death following gyrase poisoning.

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