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Lycopene-Induced Hydroxyl Radical Causes Oxidative DNA Damage in Escherichia coli
Lycopene-Induced Hydroxyl Radical Causes Oxidative DNA Damage in Escherichia coli
Journal of Microbiology and Biotechnology. 2014. Sep, 24(9): 1232-1237
Copyright © 2014, The Korean Society For Microbiology And Biotechnology
  • Received : June 03, 2014
  • Accepted : July 10, 2014
  • Published : September 28, 2014
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About the Authors
Wonyoung Lee
Dong Gun Lee
dglee222@knu.ac.kr

Abstract
Lycopene, which is a well-known red carotenoid pigment, has been drawing scientific interest because of its potential biological functions. The current study reports that lycopene acts as a bactericidal agent by inducing reactive oxygen species (ROS)-mediated DNA damage in Escherichia coli . Lycopene treatment elevated the level of ROS—in particular, hydroxyl radicals ( OH) —which can damage DNA in E. coli . Lycopene-induced DNA damage in bacteria was confirmed and we also observed cell filamentation caused by cell division arrest, an indirect marker of the DNA damage repair system, in lycopene-treated E. coli . Increased RecA expression was observed, indicating activation of the DNA repair system (SOS response). To summarize, lycopene exerts its antibacterial effects by inducing OH -mediated DNA damage that cannot be ameliorated by the SOS response. Lycopene may be a clinically useful adjuvant for current antimicrobial therapies.
Keywords
Introduction
Infectious diseases have become increasingly difficult to treat because of the prevalence of antibiotic-resistant microorganisms [12] . Therefore, there is a continual need to develop novel methods of treating disease, and to understand the mechanisms underlying antibiotic resistance to address this global public health crisis [1] . Lycopene is a red pigment that belongs to the carotenoid family, found in red fruits and vegetables such as tomatoes, and it could potentially be used to prevent some forms of cancer [2] or to treat microbial infections [4] . Lycopene has also been shown to act synergistically with some antibiotics against various bacteria [13] . Although many research determined the antimicrobial ability of lycopene, we attempted to elucidate the antimicrobial mechanism of lycopene because it remains unclear.
We chose Escherichia coli strains to study the antibacterial properties of lycopene. The gram-negative bacterium E. coli is widely used as a model experimental system in bacterial studies. E. coli lives on the body surface of mammals, and it can occasionally cause infection if the normal flora is disturbed [15] . In the present study, lycopene’s mode of action in E. coli was compared with that of the antibiotic norfloxacin. Norfloxacin executes bacterial cells by causing an accumulation of ·OH, highly devastating molecules that appear to oxidize DNA as well as create lethal doublestrand DNA breaks caused by incomplete repair that can induce cell death in bacteria [3 , 11 , 18] . We therefore investigated lycopene’s antibacterial mode of action. Under the lycopene-treated conditions, E. coli cells died exhibiting ·OH formation (detected with HPF staining) and DNA damage (detected with TUNEL assay). Furthermore, cell division arrest which is inferred from cell filamentation and expression of RecA as a contributor of SOS repair system were investigated as a part of antibacterial mechanism of lycopene.
Materials and Methods
- Preparation of Lycopene and the Bacterial Strains
A stock solution of lycopene (Sigma Chemical Co., St. Louis, MO, USA) was prepared in dimethyl sulfoxide (DMSO). For all the experiments, a final concentration of 2% DMSO was used as the solvent for lycopene. E. coli cells were obtained from ATCC (25922; Manassas, VA, USA). Prior to use, the bacteria were stored in 30% glycerol and were frozen at -70℃. The bacterial cells were grown in Luria-Bertani (LB) broth (Difco Laboratories, Detroit, MI, USA) under aerobic conditions at 37℃. Cell growth was measured by optical density at 620 nm with a microtiter ELISA Reader (Molecular Devices Emax, Sunnyvale, CA, USA).
- Time-Kill Kinetic Analysis
E. coli cells (1 × 10 6 cells/ml of LB) were incubated with lycopene or norfloxacin at their respective minimum inhibitory concentration (MIC). Previous report showed that the MIC of lycopene is 5 μg/ml [26] , and for norfloxacin is 0.125 μg/ml [18] . Aliquots were taken from the cultures after 0, 2, 4, 6, and 8 h of incubation and streaked onto LB agar plates. The numbers of colony-forming units were counted after incubation at 37℃ for 24 h [17] . The percent survival was determined relative to the control treatment. Experiments were performed in triplicate, and the results were expressed as the mean ± standard deviation (SD).
- Assays to Measure Accumulation of Hydroxyl Radicals (•OH)
OH accumulation was measured with the fluorescent dye 3’-( p -hydroxyphenyl) fluorescein (HPF) (Invitrogen, Carlsbad, CA, USA) [29] . E. coli cells (1 × 10 6 cells/ml of LB) were treated with either lycopene or norfloxacin at their respective MIC for 2 h at 37℃. After incubation, the cells were washed with PBS (pH 7.4, 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , and 2 mM KH 2 PO 4 ), stained with 5 μM HPF, and acquired on a FACSCalibur flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA).
- Assays to Detect DNA Damage
DNA strand breaks in the E. coli cells were detected using the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay [22] . Cells (1 × 10 6 cells/ml of LB) treated with either lycopene or norfloxacin for 2 h were washed in PBS, permeabilized (0.1% Triton X-100 and 0.1% sodium citrate) for 2min on ice, and then washed again with PBS. The DNA fragments were labeled for 1 h at 37℃ with an in situ FITC-conjugated dUTP cell death detection kit. The stained cells were observed using a fluorescent microscope (Nikon Eclipse Ti-S; Nikon, Japan), and the fluorescence intensity was measured with a FACSCalibur flow cytometer.
- Cell Division Arrest Assay
To ascertain whether cell cycle arrest occurred, chromatin condensation was analyzed using 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma) staining [20] . E. coli (1 × 10 6 cells/ml of LB) were treated with lycopene or norfloxacin for 2 h. For nuclear staining, the cells were washed twice with PBS, and were then incubated with 1 μg/ml DAPI in the dark for 20 min. The cells were then observed with a fluorescent microscope, and the intensity of fluorescence was measured with a spectrofluorophotometer (Shimadzu RF-5301PC; Shimadzu, Japan).
- Observation of Morphological Changes in E. coli and RecA Expression Patterns
To detect morphological changes caused by cell cycle arrest in E. coli , treated bacterial cells were assayed with a FACSCalibur flow cytometer to compare the forward scatter (FSC) and side scatter (SSC) [28] .
To investigate the activation of SOS response, we performed a RecA expression assay. Purified RecA protein was purchased from Affymetrix as a positive control for assessing the RecA isolated from experimental samples. RecA was isolated and used for western blot analysis, performed as described previously. Briefly, E. coli cells (1 × 10 6 cells/ml of LB) were cultured in LB medium at 37℃, collected by centrifugation at 300 ×g, and washed twice with PBS. The cells were then separately treated with lycopene or norfloxacin for 2 h at 37℃. The treated cells were lysed using sonication, and were then centrifuged at 12,000 × g for 20 min to remove any remaining intact cells and cellular debris. Trichloroacetic acid (TCA) was used to precipitate the cellular proteins from the supernatant. The washed TCA-precipitated proteins were used to assay for RecA by western blotting [23] . The protein content of the precipitate was estimated using a NanoVue Plus Spectrophotometer (GE Healthcare, UK). Ten milligram of protein from each sample was resolved by 7.5% SDS-PAGE. The separated proteins were transferred to a nitrocellulose membrane and analyzed by western blotting. A rabbit polyclonal anti-RecA antibody (Abcam, UK) was used for the primary antibody, and horseradish peroxidase-conjugated goat anti-rabbit IgG (Biovision, Milpitas, CA, USA) was used as the secondary antibody. An enhanced-chemiluminescence substrate was used to detect RecA.
Results and Discussion
- Lycopene Exerts Bactericidal Activity
Lycopene has been widely studied because of the benefits associated with this natural compound, but most of the previous works have been performed in the food science field. Although there have been previous reports on the antimicrobial properties of lycopene, the underlying mechanism remains unknown. We focused on understanding the intracellular mechanism of lycopene action in E. coli .
Most of all, a time-kill assay for E. coli was performed to determine whether lycopene possesses antibacterial activity. In our experiment, lycopene was shown to have potent bactericidal effects. All E. coli cells incubated with the MIC of lycopene died after 4 h, while all the cells treated with the MIC of norfloxacin died after 2 h ( Fig. 1 A). These results are consistent with previous reports [4 , 13] , and we could set up the conditions for further experiments on the basis of this result.
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Bactericidal effect of lycopene and hydroxyl radicals (OH) accumulation induced by lycopene in E. coli. (A) Time-kill kinetics of 5 μg/ml lycopene and 0.125 μg/ml norfloxacin in E. coli. All E. coli cells incubated with lycopene at the MIC died after 4 h, whereas all of the bacterial cells incubated with norfloxacin at the MIC died after 2 h. All the experiments were performed three times, and the results are expressed as the mean ± SD. (B) E. coli cells were treated with 5 μg/ml lycopene and 0.125 μg/ml norfloxacin for 2 h at 37℃, with 5 μM HPF dye. The stained cells were measured with FACSCalibur flow cytometer.
- Lycopene Induces Oxidative Stress, Specifically ·OH Accumulation in E. coli
Lycopene has been proven to possess antioxidative capacity aganinst endogenous and H 2 O 2 -induced ROS [7] . However, it was also shown in a cultured human colon cancer cell line that at higher incubation concentrations (>2 μg/ml), lycopene loses its antioxidative properties and may generate oxidative DNA damage instead [19] . On the basis of these reports, we next investigated whether lycopene induces oxidative stress at the MIC of lycopene in E. coli , because this bacterial strain resides in the colon epithelium of mammals. To this end, lycopene-treated E. coli cells were labeled with HPF, which is a highly specific fluorescent probe that is oxidized by OH [6] . The fluorescent intensity of HPF in E. coli cells increased from 26.41% to 35.53% and 50.48% after treatment with lycopene and norfloxacin, respectively ( Fig. 1 B). Bacterial cells have antioxidant machinery that protect them against ROS that are generated under normal conditions from intracellular metabolites, such as hydrogen peroxide (H 2 O 2 ), superoxide anion (O 2 ), nitric oxide (NO), and OH [6] . However, ROS can accumulate and overload the antioxidant system when the cells are placed under adequately stressful conditions [6] . Among the ROS, we focused on OH because the Fenton and Haber-Weiss reactions convert the excess ROS to OH, and these highly destructive molecules attack cell components. In this experiment, the results indicate that lycopene induced accumulation of OH in E. coli .
- DNA Damage Occurs in E. coli Cells Treated with Lycopene
Foti et al . [11] suggested that OH, triggered by antibiotics, led to the catastrophe in cell components such as DNA, lipids, and proteins. However, cell death is predominantly elicited by specific oxidation of guanine, one of the four nucleotides or building blocks of DNA, followed by the formation of double-strand DNA breaks, DNA fragmentation, and cell death [5 , 8 , 14] . Based on this, double-strand DNA breaks were observed in E. coli cells undergoing lycopeneinduced bacterial cell death by using an FITC-conjugated dUTP reagent in the TUNEL assay. In this assay, a modified nucleotide with a fluorescent label is attached to the 3’-hydroxyl terminus of a DNA molecule, catalyzed by terminal deoxynucleotidyl transferase, and it can be used directly to measure double-strand DNA breaks [11] . We measured the population of fluorescently labeled cells using flow cytometry and fluorescence microscopy. The fluorescent intensity of TUNEL in E. coli cells increased from 27.63% to 57.91% and 64.13% after treatment for 2 h with lycopene and norfloxacin, respectively ( Fig. 2 A). This result led us to hypothesize that OH, a crucial mediator of DNA damage, starts oxidative base modifications and leads to double-strand DNA breaks in E. coli treated with lycopene. We also observed more bright green fluorescent cells and morphological changes, in particular filamentation in cells, after treatment with lycopene or norfloxacin ( Fig. 2 B). Because morphological changes are related with inhibition of cell division and it can support DNA damage, we next investigated cell division arrest in E. coli treated with lycopene.
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Lycopene significantly increased TUNEL fluorescence in E. coli treated with lycopene and norfloxacin. (A) Double-strand DNA breaks assay with TUNEL fluorescence using FACSCalibur flow cytometer. (B) Observation of TUNEL fluorescence under fluorescence microscopy. (a) Untreated control cells, (b) cells treated with 5 μg/ml lycopene, and (c) cells treated with 0.125 μg/ml norfloxacin for 2 h at 37℃.
- Lycopene Induces Cell Division Arrest in E. coli
We observed cell division arrest in lycopene-treated E. coli . To quantify the cell cycle arrest, we used the structure-sensitive dye DAPI. DAPI is a conformation-sensitive dye that binds to DNA in condensed chromosomes, which allows it to be an effective indicator of cell cycle arrest [9] . Compared with untreated E. coli cells that showed only slight fluorescence, the lycopene- and norfloxacin-treated E. coli cells exhibited strong fluorescence, indicating the presence of condensed chromosomes in these cells ( Fig. 3 A). We also detected more bright blue fluorescent cells and filamentation in cells, a marker of cell division arrest in E. coli , occurring in stained cells by fluorescence microscopy ( Fig. 3 B). Oxidized DNA needs to be restored by the bacterial cells’ own repair systems. During the repair process, cell division is arrested by cell-cycle check-point regulation to provide more time for repair before the critical phases of DNA replication [16] . While the cell division is arrested, chromosome remains condensed and the filamentous phenomenon occurs. Through the DAPI staining, we confirmed chromosome condensation, indicating replication arrest [10] , and the results strongly support that lycopene triggers DNA damage in E. coli cells as a result of the intracellular OH accumulations.
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Detection of chromosome condensation, indicating cell cycle arrest, in E. coli cells treated with lycopene and norfloxacin for 2 h at 37℃. (A) Relative DAPI fluorescence intensity observed under spectrofluorophotometry. (B) Observation of DAPI fluorescence under fluorescence microscopy. (a) Untreated cells, (b) cells treated with 5 μg/ml lycopene, and (c) cells treated with 0.125 μg/ml norfloxacin.
- E. coli Cells Induce the SOS Response to Repair the DNA Damaged by Lycopene
The lycopene-induced morphological changes in E. coli were further characterized using flow cytometry- to compare the FSC and SSC profiles of lycopene or norfloxacin-treated cells with untreated cells. In general, FSC is an indicator of cell size and SSC is an indicator of granularity. However, SSC is much more sensitive and can resolve particles at least as small as 0.2 microns, and it can be linked to total protein staining as a size indicator [27 , 28] . Fig. 4 A shows that the cells treated with lycopene and norfloxacin had increased SSC values in the 3D density plots relative to the main population of control cells, indicating cell filamentation. Bacterial morphological changes, in particular filamentation, is generally mediated by the cell division inhibitor SulA under stressful conditions, and is evidence that E. coli initiates the SOS response to repair the damage [21] .
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Lycopene-induced morphological changes of cell and SOS response in E. coli cells treated with lycopene and norfloxacin. (A) Three-dimensional flow cytometric contour-plot analysis of E. coli treated with 5 μg/ml lycopene or 0.125 μg/ml norfloxacin. (B) RecA, which is related with SOS response, expression analysis by western blot assay. E. coli cells were treated with 5 μg/ml lycopene or 0.125 μg/ml norfloxacin. Samples were taken at 2 h after treatment and RecA protein was loaded in last lane as a positive control for assessing the RecA isolated from experimental samples.
The SOS system involves the RecA protein, which is stimulated by single-stranded DNA. Under conditions of stress, the active form of RecA leads to induction of the SOS response by assisting with the cleavage of LexA, which inhibits the expression of genes related to the bacterial repair system [24 , 25] . Western blotting was used to determine whether lycopene caused an increase in the intracellular concentration of RecA. As shown in Fig. 4 B, the RecA bands in cells treated with lycopene or norfloxacin were thicker than the corresponding band in untreated cells. Based on this result, we concluded that lycopene induced oxidization in nucleotides and activated the SOS response to repair cells. However, this repair is not without risk: it has to unravel the DNA double helix, and cut one of the chains, to replace the incorrect base. This is not a problem, unless two such repairs happen close to each other: then the DNA suffers double-strand break, which usually kills the cell [11] . To summarize, the cells try to repair the damage when the oxidized guanine is inserted into DNA. However, incomplete repair of spaced 8-oxo-deoxyguanosine lesions result in double-strand DNA breaks and this speeds up their own death.
In the present study, we characterized many aspects of lycopene-induced bacterial cell death, including OH accumulation, double-strand DNA breaks, cell division arrest, and activation of the SOS response ( Fig. 5 ). Our findings suggest that lycopene has a novel mechanism of antibacterial action, which is the ROS-mediated DNA damage in bacteria, and that it shows potential to be used in antimicrobial therapy.
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The ROS formation by lycopene in E. coli triggers oxidative DNA damage. Thereafter, double-strand DNA breaks, cell division arrest, and activation of the SOS response occur in E. coli, followed by cell death.
Acknowledgements
This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korea Government (MSIP) (No. 2008-0062618).
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