Role of Reactive Oxygen Species in Escherichia coli Inactivation by

Sep 22, 2012 - Inactivation of biofilms on RO membranes by copper ion in combination with norspermidine. Hye-Jin Lee , Jiwon Seo , Min Sik Kim , Chang...
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Role of Reactive Oxygen Species in Escherichia coli Inactivation by Cupric Ion Hee-Jin Park,†,§ Thuy T. M. Nguyen,‡,§ Jeyong Yoon,*,† and Changha Lee*,‡ †

World Class University (WCU) program of Chemical Convergence for Energy & Environment (C2E2), School of Chemical and Biological Engineering, College of Engineering, Institute of Chemical Process, Seoul National University (SNU), Daehak-dong, Gwanak-gu, Seoul 151-744, Republic of Korea ‡ School of Urban and Environmental Engineering, Ulsan National Institute of Science and Technology (UNIST), 100 Banyeon-ri, Eonyang-eup, Ulju-gun, Ulsan 698-805, Republic of Korea S Supporting Information *

ABSTRACT: This study demonstrated Escherichia coli inactivation by cupric ion (Cu[II]), focusing on intracellular generation and consumption of reactive oxygen species (ROS) including superoxide and hydroxyl radials. In the presence of Cu(II), intracellular superoxide levels of E. coli decreased in a concentration-dependent manner, indicating that superoxide radical was used to reduce Cu(II) to Cu(I) in cells. The variation in the hydroxyl radical level by adding Cu(II) was negligible. Molecular oxygen and hydroxyl radical scavengers did not affect the inactivation efficacy of E. coli by Cu(II), excluding the possibility that hydroxyl radicals induced by the copper-mediated reduction of oxygen contributed to the microbiocidal action of Cu(II). However, the inactivation of E. coli by Cu(II) was considerably inhibited and accelerated by a Cu(I)-chelating agent and a Cu(II)-reducing agent, respectively. Our results suggest that the microbiocidal action of Cu(II) is attributable to the cytotoxicity of cellularly generated Cu(I), which does not appear to be associated with oxidative damage by Cu(I)-driven ROS.

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INTRODUCTION Copper plays many significant roles in microorganisms and is consequently known as an essential micronutrient.1,2 In particular, Escherichia coli uses copper as an integral part of several important enzymes including amine oxidase, copper−zinc superoxide dismutase, cytochrome oxidase, and P-type ATPase.3 Nevertheless, microorganisms can be inactivated by elevated concentrations of copper; hence, copper ions and its complexes have been widely used as antimicrobial agents for decades due to their high antimicrobial activity.4,5 Copper has been introduced to control microorganisms in agriculture,6 hospitals,7 paints,8 and ship hulls.9 The direct interactions between copper species and cell components have been proposed to induce copper cytotoxicity. Copper is known to denature proteins10,11 and DNA12,13 by binding to the reactive groups of these biomolecules. The antimicrobial action of copper has also been frequently explained by reactive oxygen species (ROS) generated via redox cycling of the cuprous (Cu[I]) and cupric (Cu[II]) couple.5,14,15 In bacterial cells, Cu(II) is reduced by sulfhydryls,16 superoxide radical (O2−•), or reducing agents such as ascorbic acid or glutathione (GSH)15 to generate Cu(I), which catalyzes the conversion of intracellular hydrogen peroxide (H2O2) into hydroxyl radical (•OH) via a Fenton-like reaction (reactions 1 and 2).17,18 © 2012 American Chemical Society

X red + Cu(II) → Xox + Cu(I)

(1) −

•

Cu(I) + H 2O2 → Cu(II) + OH + OH

(2)

• OH is a powerful and nonselective oxidizing species and causes oxidative damage to biological molecules including DNA, proteins, and lipids.5,20 In addition, Cu(II) induces the generation of O2−•,10,21 which selectively damages cell components by oxidation. However, despite these reports, direct experimental data for the generation of ROS in the presence of Cu(II) are scarce. In particular, little is known about the roles of intra and extracellular ROS in the microbiocidal action of Cu(II). In this study, to improve understanding of bactericidal action of Cu(II) we (i) evaluated intra/extracellular ROS generation by Cu(II); (ii) determined the contribution of ROS on antimicrobial activity of Cu(II); and (iii) confirmed Cu(II) reduction to Cu(I) in bacterial cells and its effect on bacterial inactivation. E. coli was selected as a surrogate bacterium, and the cell-permeable fluorescent probes hydroethidine (HE, for O2−•) 19

Received: Revised: Accepted: Published: 11299

June 14, 2012 August 24, 2012 September 22, 2012 September 22, 2012 dx.doi.org/10.1021/es302379q | Environ. Sci. Technol. 2012, 46, 11299−11304

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and 3′-(p-hydroxyphenyl) fluorescein (HPF, for •OH) were used to detect intra/extracellular ROS. A series of experiments was carried out using oxidant scavengers, copper-chelating agents, and a Cu(II)-reducing agent to evaluate the effects of cellularly generated ROS and Cu(I) on bactericidal activity of Cu(II).

ions, which was established by sparging with N2 gas for 10 min before and throughout the Cu(II) treatment. After the exposure, 1 mL of E. coli suspension exposed to Cu(II) was withdrawn into 25 μL of 0.082 mM EDTA to quench the Cu(II) reaction during the incubation period. The number of viable cells was quantified by the plate counting method (CFU/mL). The Cu(I) concentration was quantified by the neocuproine method24 with slight modification; the reductant (hydroxylamine-hydrochloride) was not used and an excess DMP concentration (2 mM) was employed to prevent rapid oxidation of Cu(I) to Cu(II) in the presence of oxygen. The absorbance of the solution was measured at 454 nm using an Agilent 8453 UV/vis spectrophotometer (Agilent Technologies, Stuttgart, Germany). Transmission Electron Microscopy (TEM) Analysis. E. coli TEM specimens were prepared using chemicals from Electron Microscopy Sciences Co. (Hatfield, PA), as described elsewhere.29 E. coli cells exposed to Cu(II) were harvested by centrifugation at 2500g for 2 min. After removing the supernatant, the cells were fixed in 2% glutaraldehyde and 0.05 M sodium cacodylate buffer (pH 7.2) for 2 h and washed three times with 0.05 M sodium cacodylate buffer. Then, the samples were treated with 1% osmium tetroxide in 0.05 M sodium cacodylate buffer for 2 h and stained with 2% uranyl acetate for 18 h. After staining, the samples were gradually dehydrated with ethanol, infiltrated with propylene oxide, embedded in Spurr’s resin, and polymerized at 70°C for 24 h. The polymerized samples were sectioned using an ultramicrotome (MT-X, RMC, New York, NY) and stained with 2% uranyl acetate and Reynold’s lead citrate. The prepared specimens were examined by TEM (JEM-3010, JEOL, Tokyo, Japan).

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EXPERIMENTAL SECTION Reagents. Copper sulfate (CuSO4), 1,4-piperazinediethanesulfonic acid (PIPES), methanol (MeOH), tert-butyl alcohol(t-BuOH), ethylenediaminetetraacetic acid (EDTA), 2,9-dimethyl-1,10-phenanthroline (DMP), and hydroxylamine were purchased from Sigma-Aldrich Co. (St. Louis, MO). CuSO4 was dissolved in deionized water (Millipore, Molsheim France) and used as a source of Cu(II). PIPES buffer (pH 7.0) was prepared as described previously22 and then used for all experiments, as this buffer has negligible metal-chelating properties.23 DMP dissolved in deionized water was used for scavenging and measuring Cu(I).24 Other reagents were dissolved in deionized water and used as stock solutions. Culture and Analysis of Bacteria. E. coli (ATCC 8739) was selected as the indicator microorganism, as it is widely used for water research due to its similar characteristics to major waterborne bacteria (e.g., Salmonella and Shigella).25,26 A single colony of E. coli ATCC 8739 was inoculated in 40 mL of nutrient broth medium (Difco Co., Detroit, MI) and incubated at 37°C for 18 h to prepare the bacterial stock suspension. The bacterial cells were collected by centrifugation at 1000g for 10 min and washed three times with 40 mL of 150 mM phosphatebuffered saline (pH 7.2). Then, the suspension was diluted to an initial population of 107 CFU/mL in 1 mM PIPES buffer (pH 7.0). The number of bacterial cells was determined by the plate counting method (colony forming units, CFU/mL). ROS Detection. Levels of O2−• and •OH were measured using the cell-permeable fluorescence probes HE and HPF (Invitrogen, Carlsbad, CA), respectively.27 We detected ROS generation in three systems: cell-free condition (without E. coli), the intracellular region, and the intra/extracellular region of E. coli cells. To detect ROS generation under a cell-free condition, 10 μM of the probe compound was employed in a solution without E. coli cells. For intracellular or intra/extracellular ROS detection, we prepared the fluorescence probe compound taken up by E. coli cells following a method reported previously.28 HE or HPF (10 μM) was mixed with an E. coli suspension (2 × 107 CFU/mL) for 1 h at 100 rpm in the dark, and the bacterial cells were collected by centrifugation at 1000g for 10 min. For measuring intracellular ROS, the probes in the extracellular region were removed and washed with 1 mM PIPES buffer (pH 7.0), and intra/extracellular ROS were detected without the removal step. The prepared fluorescent probes with or without E. coli cells were exposed to various concentrations of Cu(II) for 1 h. Then, fluorescence intensity was measured using a microplate reader (Tecan, AT/Genios Pro., Zurich, Switzerland) with 535 or 485 nm excitation and 590 nm or a 535 nm emission filter for HE and HPF, respectively. Inactivation Experiment. Bactericidal experiments were conducted with 50 mL of E. coli suspension in 1 mM PIPES buffer (pH 7.0) at room temperature (25 ± 0.5°C) in the dark under vigorous stirring, and initiated by adding an aliquot of Cu(II) stock solution. Inactivation of E. coli was examined under both aerobic and anaerobic conditions to determine the effect of molecular oxygen on bactericidal activity of copper

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RESULTS ROS Generation in the Presence of Cu(II). Prior to examining Cu(II)-induced ROS generation, control values were measured in the absence of Cu(II) under the three conditions (i.e., cell-free, intracellular, and intra/extracellular conditions) to identify background signals (Figure 1). Figure 1a shows the HE fluorescence intensities for measuring O2−• under the three conditions. The fluorescence intensity for the intracellular condition was higher than that for the cell-free condition, even though the HE background signal should be higher under the cell-free condition than that under the intracellular condition, because the concentration of HE in the cells is usually much lower than that in bulk solution. The fluorescence intensity under the intra/extracellular conditions equaled the sum of the values for the cell free and intracellular conditions. In contrast, the HPF fluorescence intensity used to measure • OH was not affected by the presence of E. coli (Figure 1b). Fluorescence intensity under the intracellular condition was considerably lower than that of the other conditions, which may have been attributable to the relatively low HE concentration in the cells compared to that in the bulk solution. Fluorescence intensity under the intra/extracellular condition was almost the same as that for the cell free condition. ROS generation in the presence of Cu(II) was examined under the three conditions (Figure 2a−c). As the control values differed depending on the condition (Figure 1a and b), the fluorescence intensity ratio normalized with each control value was used to evaluate the ROS level. Under the cell-free condition (Figure 2a), the O2−• level increased slightly after adding Cu(II), probably due to the partial oxidation of HE by the 11300

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Figure 1. Detection of (a) O2−• (hydroethidine, HE) and (b) •OH (3′-(p-hydroxyphenyl) fluorescein, HPF) under the cell-free condition, intra/extracellular space, and intracellular space (pH0 7.0 (1 mM PIPES buffer), treatment time = 60 min, [E. coli]0 = 2 × 107 CFU/mL).

direct interaction with Cu(II). In the presence of E. coli cells (intracellular and intra/extracellular conditions) (Figure 2b and c), the O2−• level decreased significantly with increasing Cu(II) concentration. It appeared that the intra/extracellular condition resulted in a slower decrease in O2−• level than that of the intracellular condition due to the background fluorescence intensity of the extracellular phase. In contrast, the •OH level measured by HPF was not significantly different after adding Cu(II) under all conditions (Figure 2a−c). The •OH fluorescence intensity did not increase even at a more elevated concentration of Cu(II) (10 mM, data not shown). Inactivation of E. coli in the Presence of Cu(II). E. coli was inactivated by approximately 4 log in 30 min in the presence of 50 μM Cu(II) (Figure 3a), and the inactivation efficacy increased with increasing the concentration of Cu(II) (Figure S1 in the Supporting Information). Deaeration using N2 sparging and introducing excess oxidant scavengers (MeOH and t-BuOH) did not significantly influence the inactivation efficacy of E. coli, indicating that molecular oxygen and reactive oxidants such as •OH did not directly affect the bactericidal action of Cu(II). However, adding copper-chelating agents (i.e., EDTA and DMP, Cu(II)- and Cu(I)-chelating agents, respectively) almost completely blocked the inactivation of E. coli (Figure 3b). When hydroxylamine was used to test the toxicity of Cu(I) produced by the reduction of Cu(II), the E. coli inactivation rate was greatly enhanced (Figure 3c). Similar to the case without hydroxylamine (Figure 3a), MeOH and t-BuOH exhibited negligible effects (Figure 3c).

Figure 2. Effect of Cu(II) on reactive oxygen species (ROS) (O2−• and •OH) level. (a) Cell-free condition. (b) Intracellular space. (c) Intra/extracellular space (pH0 7.0 (1 mM PIPES buffer), treatment time = 60 min, [E. coli]0 = 2 × 107 CFU/mL).

Morphological Changes in E. coli. The morphological changes in the E. coli cells following Cu(II) treatment were examined by TEM. Figure 4a−c show the morphology of the E. coli cells before and after exposure to low (5 μM) and high concentrations (100 μM) of Cu(II) for 30 min. At a low concentration (5 μM), an electron-light region was partially generated in the E. coli cytoplasm, and this region was enlarged by increasing the Cu(II) concentration to 100 μM. However, no significant physical disruption of the cells was observed, and the cell walls appeared to be mostly undamaged.

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DISCUSSION Role of O2−•. Fluorescent probes without any ROS generating factors (e.g., cell-free condition in Figure 1) exhibited background fluorescence intensity, probably due to partial auto-oxidation of the probe compounds. Usually this background signal was lower under the intracellular condition 11301

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consumed by the reaction with Cu(II). This reaction proceeds via a single-electron transfer from O2−• to Cu(II) to form Cu(I) (reaction 4). Subsequently, another equivalent of O2−• is consumed by the reaction with Cu(I) (reaction 5). Cu(II) + O2−• → Cu(I) + O2

(4)

Cu(I) + O2−• + 2H+ → Cu(II) + H 2O2

(5)

Both processes (reactions 4 and 5) are diffusion-controlled reactions (109−1010 M−1s−1).31 However, the decrease in the O2−• level by Cu(II) contrasts with the previous assertion that the toxicity of Cu(II) results from O2−•-mediated oxidative stress.10,21,30 According to previous studies, the O2−• sensor protein, SoxR, is considerably activated in the presence of Cu(II)30,32 One possible explanation for the discrepancy is that SoxR clusters may have been oxidized directly by Cu(II) rather than O2−•; the in vivo estimate of the SoxR redox potential is −225 mV,33 and the redox potential of the Cu(II)/Cu(I) couple is +159 mV.34 The insignificant involvement of O2−•-mediated oxidative stress in the bactericidal action of Cu(II) is also supported by a previous observation that superoxide dismutase (SOD) does not mitigate the toxicity of Cu(II) to E. coli.35 In contrast, O2−• can be generated outside the cells in the presence of Cu(II). O2−• can be produced via the reduction of oxygen by Cu(I) (reaction 6, k = 3 × 104 M−1s−1);36 Cu(I) formed in the vicinity of the cells may be driven into bulk solution. Cu(I) + O2 → Cu(II) + O2−•

(6)

Indeed, approximately 3 μM of Cu(I) was detected in the bulk phase when 50 μM Cu(II) was added in the presence of E. coli (measured in situ with DMP in the reaction solution, data not shown). However, although extracellular O2−• was formed, it may not lethally damage the cells, which is supported by the negligible effect of deaeration (N2 sparging) on E. coli inactivation (Figure 3a). Role of •OH. In the presence of Cu(II), •OH is generated by catalytic conversion of intracellular H2O2 (reactions 1 and 2). Additionally, the Cu(I) produced increases the intracellular H2O2 level by reducing O2−• (reaction 5) or oxygen (reaction 6 followed by reaction 5). However, the level of intracellular •OH measured by HPF was not affected by adding Cu(II) (Figure 2a−c), and the fluorescence intensity did not change even at a higher concentration of Cu(II) (10 mM, data not shown). Although a low •OH concentration below the detection limit may arise by Cu(II), such a level of •OH does not appear to generate enough oxidative stress to influence cell viability. When 1 mM H2O2 was added with 0.1 mM Cu(II), the HPF-based fluorescence intensity ratio increased up to 28. In addition, adding 0.1 mM Fe(II) increased the fluorescence intensity ratio up to approximately 3, leading to less than 0.5 log inactivation of E. coli in 1 h (data not shown). In contrast, 50 μM Cu(II), which negligibly influences fluorescence intensity, achieved more than 4 log inactivation of E. coli in 30 min (Figure 3a). A small amount of extracellular •OH, which is undetectable by the HPF assay, may be produced by reducing extracellular O2−• (reaction 5) and the subsequent Fenton-like reaction (reaction 2). However, the possibility that such a level of •OH may affect the inactivation of E. coli was ruled out by the negligible effects of the oxidant scavengers (MeOH and tBuOH) (Figure 3a). In addition, no significant disruption of cell integrity in the presence of Cu(II) (Figure 4b and c) is

Figure 3. Effect of (a) molecular oxygen, •OH scavengers (MeOH and t-BuOH), (b) Cu chelating agents (EDTA and DMP, Cu(II)and Cu(I)-chelating agents, respectively), and (c) hydroxylamine (HA) on E. coli inactivation by Cu(II) (pH0 7.0 (1 mM PIPES buffer), [E. coli]0 = 107 CFU/mL, [MeOH]0 = [t-BuOH]0 = 200 mM, [EDTA]0 = [DMP]0 = 2 mM, [HA] = 10 μM, [Cu(II)]0 = 50 μM (a, b); 5 μM (c)).

than that under the other conditions (cell free and intra/ extracellular conditions), as shown in Figure 1b, because the amount of the probe compound present inside the cells is relatively small. For this reason, the higher fluorescence intensity under the intracellular condition compared to that under the cell-free condition (Figure 1a) indicates that the E. coli cells generated O2−•. Intracellular O2−• is generated via reduction of oxygen during respiration by E. coli.30 The intracellularly generated O2−• did not influence extracellular fluorescence intensity (i.e., the fluorescence intensity under the intra/extracellular conditions was equivalent to the sum of the values for the cell-free and intracellular conditions in Figure 1a), indicating that the O2−• produced was unable to escape from the cell membranes. Intracellular O2−• level decreased significantly in the presence of Cu(II) (Figure 2b), suggesting that O2−• may be directly 11302

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Figure 4. Transmission electron micrograph (TEM) images of (a) E. coli cells (control) and Cu(II) treated cells at (b) 5 μM and (c) 100 μM (pH0 7.0 (1 mM PIPES buffer), [E. coli]0 = 108 CFU/mL, treatment time = 30 min).

susceptible to the combined system of Cu(II) with H2O2 which significantly generates ROS.18

indirect evidence that the attack by extracellular oxidants was not critical. Bacteria treated with strong oxidants such as ozone and oxidizing radicals usually exhibit strongly disrupted cell membranes.37,38 Bactericidal Activity of Cu(I). The minor influences of copper-driven ROS lead to the conclusion that the bactericidal action of Cu(II) mainly results from the direct interactions between copper species (i.e., Cu(II) or Cu(I)) and cell components. Based on the assumption that the influences of ROS are minor, Cu(I) appears to be the major species responsible for inactivating E. coli. The complete quenching of E. coli inactivation by DMP, which selectively complexes with Cu(I), is a strong evidence (Figure 3b). Enhanced inactivation of E. coli after adding hydroxylamine (Figure 3c) indicates that Cu(I) was much more toxic than Cu(II), which is in agreement with a previous result obtained under anoxic conditions.39 Even in the presence of hydroxylamine, the oxidant scavenger (MeOH) failed to inhibit E. coli inactivation (Figure 3c), suggesting that the enhanced production of extracellular •OH (due to the enhanced production of Cu(I) in the bulk phase) had minor effects on the inactivation of E. coli. The morphology of E. coli cells treated by Cu(II) (Figure 4b and c) was similar to that of Ag(I)-treated cells41 in that both metal ions induced damage to the cytoplasm (the electron-light region in the TEM images). No significant disruption of cell integrity was observed. It appears that Cu(II) penetrates the cell membranes through porins40 and is reduced into Cu(I) by intracellular O2−• or biomolecules.15,16 Similar to Cu(I), Ag(I) interacts with thiol groups.42,44 However, the bactericidal action of Ag(I) is also related to the oxidative stress by intracellular ROS.43 Due to ROS generation, Ag(I) may induce more damage in the cytoplasm than that of Cu(II); only 6 μM Ag(I) resulted in a similar degree of disruption to the cytoplasm41 compared to 100 μM Cu(II) (Figure 4c). The major findings in this work suggest that the strategies for improving the efficiency of copper-based disinfection systems should be based on the reduction of Cu(II) to Cu(I) rather than the generation of ROS, which may be extrapolated to entire bacterial species. However, a different approach may be needed for other types of microorganisms such as viruses. Viruses have lack of intracellular reducing agents capable of reducing Cu(II) into Cu(I) (e.g., ascorbic acid, GSH, or O2−•), and moreover have fewer Cu(I) binding sites such as a thiol group. Indeed, it has been reported that MS2 coliphage is resistant to the treatment by Cu(II) alone, but is much more

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ASSOCIATED CONTENT

S Supporting Information *

Inactivation of E. coli at various concentrations of Cu(II) (Figure S1) is available as Supporting Information. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +82-2-880-8927 ( J.Y.); +82-52-217-2812 (C.L.). Fax: +82-2-876-8911 ( J.Y.); +82-52-217-2809 (C.L.). E-mail: [email protected] ( J.Y.); [email protected] (C.L.). Author Contributions §

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by WCU (World Class University) program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (R31-10013). This work was also supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2012006581) and by Korea Ministry of Environment as “The GAIA Project (No. 2012000550021)”. These contributions are greatly appreciated.

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dx.doi.org/10.1021/es302379q | Environ. Sci. Technol. 2012, 46, 11299−11304