Hydroxyurea induces hydroxyl radical-mediated cell death in Escherichia coli

Abstract

Hydroxyurea (HU) specifically inhibits class I ribonucleotide reductase (RNR), depleting dNTP pools and leading to replication fork arrest. Although HU inhibition of RNR is well recognized, the mechanism by which it leads to cell death remains unknown. To investigate the mechanism of HU-induced cell death, we used a systems-level approach to determine the genomic and physiological responses of E. coli to HU treatment. Our results suggest a model by which HU treatment rapidly induces a set of protective responses to manage genomic instability. Continued HU stress activates iron uptake and toxins MazF and RelE, whose activity causes the synthesis of incompletely translated proteins and stimulation of envelope stress responses. These effects alter the properties of one of the cell's terminal cytochrome oxidases, causing an increase in superoxide production. The increased superoxide production, together with the increased iron uptake, fuels the formation of hydroxyl radicals that contribute to HU-induced cell death.

Figure 1

(A) Survival of exponentially growing MC4100 cultures treated with (●) or without (■) 100 mM HU for the indicated times. (B) MC4100 pL(lexO)-GFP treated with 100mM HU for 15 min (i) and 30 min (ii). MC4100 pL(lexO)-GFP did not show GFP fluorescence in the absence of HU (data not shown). MC4100 pL(furO)-GFP treated with 100mM HU for 15 min (iii) and 30 min (iv). MC4100 pL(furO)-GFP did not show GFP fluorescence in the absence of HU (data not shown). (C) WT AB1157 (■) and AB1157 lexA3 (▲) were spotted on LB agar plates containing increasing concentrations of HU. (D) MC4100 carrying P_lac ftsZ-GFP treated with 2.0 μM IPTG +/− 100 mM HU for 1 h.

Figure 2

(A) MC4100 (■) and a ΔtonB mutant (●) were serially diluted and spotted on LB agar plates containing increasing concentrations of HU. (B) Fur-regulated GFP expressing cells were treated with 100 mM HU for 1 h and than imaged. No GFP fluorescence was observed in the absence of HU (data not shown). (C) Hydroxyl radical formation measured by HPF fluorescence in MC4100 treated +/−100 mM HU. The relative fluorescence values were determined by taking the mean fluorescence value for each sample at each time point and normalizing that value to the maximum mean fluorescence value that the wild type sample achieved over the time course. Each data point represents the average of three independent measurements of these relative fluorescence values, and the error bars represent the standard error of the independent measurements. (D) Survival curve of MC4100 treated with 100 mM HU in the presence (▲) or absence (●) of 100 mM thiourea. The growth of MC4100 in the presence of 100 mM thiourea is shown as a control (■). (E) Hydroxyl radical formation measured by HPF fluorescence in MC4100 following treatment with 100 mM HU +/− 100 mM thiourea. Fluorescence for each condition is shown relative to the maximum fluorescence achieved by MC4100 + 100 mM HU. (F) MC4100 (■) and ΔahpC (●) strains were spotted on LB agar plates containing increasing concentrations of HU.

Figure 3

(A) Survival curve of MC4100 treated with (●) or without (■) 100 mM guanazole. (B-C) MC4100 pL(furO)-GFP treated with 100 mM HU or 100 mM guanazole and sorted by flow cytometry as previously described (Dwyer et al., 2007). (D) Comparison of hydroxyl radical formation measured by HPF fluorescence in MC4100 treated with HU (■) or guanazole (●) normalized to MIC. Fluorescence for each strain is shown relative to the maximum fluorescence achieved by MC4100 + 100 mM HU. (E) Survival curve of the dnaE(ts) mutant (●) and parental strain (■) grown at the non permissive temperature. (F) Hydroxyl radical formation measured by HPF fluorescence in the dnaE(ts) (●) and parental strain (■) grown at the non permissive temperature. Fluorescence for each strain is shown relative to the maximum fluorescence achieved by the wild-type strain. (G) MC4100 (■) and ΔcydB (●) strains were spotted on LB agar plates containing increasing concentrations of HU. (H) Hydroxyl radical formation measured by HPF fluorescence in MC4100 (■) and the ΔcydB mutant (●) during HU treatment. Fluorescence for each strain is shown relative to the maximum fluorescence achieved by MC4100 + 100 mM HU.

Figure 4

HU stress assays: (A) MC4100 (■) and ΔmazEF (●), (B) MC4100 (■) and ΔrelBE (●), (C) MC4100 (■) and ΔrelBE ΔmazEF (◆), (D) ΔrelBE ΔmazEF (■) and ΔrelBE ΔmazEF ΔtonB (●). Strains were spotted on LB agar plates containing increasing concentrations of HU. (E) Hydroxyl radical formation measured by HPF fluorescence in MC4100 (■) and the ΔrelBE ΔmazEF ΔtonB mutant (●) in the presence of 100 mM HU using flow cytometry. Fluorescence for each strain is shown relative to the maximum fluorescence achieved by MC4100 + 100 mM HU

Figure 5

(A) MC4100 and the ΔrelBE ΔmazEF mutant carrying the tmRNA-6XHis allele were treated +/− 100 mM HU for 3 h. Equal amounts of total protein lysate were separated by SDS-PAGE and probed with a monoclonal anti-His antbody. Lane 1, MC4100 (−) HU, lane 2 ΔrelBE ΔmazEF (−) HU, lane 3 MC4100 (+) HU, lane 4 ΔrelBE ΔmazEF (+) HU, lane 5 MC4100 without tmRNA-6XHis (+) HU. (B) MC4100 and the ΔrelBE ΔmazEF carrying reporter rpoHP3-lacZ treated +/− HU for 1 h. The plot shows the ratio of lacZ activity +HU/−HU for each strain. (C) MC4100 (■), ΔcpxR (●) and ΔdegP (▲) strains were spotted on LB agar plates containing increasing concentrations of HU. (D) Hydroxyl radical formation measured by HPF fluorescence in MC4100 (■) and the ΔcpxA mutant (●) in the presence of 100 mM HU using flow cytometry. (E-G) Fur-GFP containing cells were treated with 100 mM HU for 3 h. The culture was then stained with DiBAC. (E) Nomarsky image of cells. (F) Image showing DIBAC staining. (G) Image showing Fur-regulated GFP expression. (H) MC4100 pL(soxR)-GFP (■) and ΔrelBE ΔmazEF pL(soxR)-GFP (●) treated with 100 mM HU and sorted by flow cytometry.

Figure 6

Schematic representation of cellular response to HU treatment. In E. coli, HU rapidly inhibits RNR arresting replication fork progression (A). Subsequent activation of toxins MazF and RelE leads to improperly translated proteins, membrane stress and membrane stress responses (B). These effects disrupt respiratory chain activity causing an increase in superoxide production (C) eventually leading to increased OH^• production (D) eventual cell death. Misregulation of the HU-induced iron uptake response (E) further fuel this processes by increasing the amount of free iron available for Fenton chemistry.