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MODEL
Research in Microbiology xx (2014) 1e10 www.elsevier.com/locate/resmic
Aspidin BB, a phloroglucinol derivative, exerts its antibacterial activity against Staphylococcus aureus by inducing the generation of reactive oxygen species Na Li a,1, Chang Gao b,1, Xiao Peng a, Wei Wang a, Meng Luo a, Yu-jie Fu a,*, Yuan-gang Zu a,** a
Engineering Research Center of Forest Bio-Preparation, Ministry of Education, Northeast Forestry University, Harbin 150040, PR China b College of Basic Medical Science, Peking University Health Science Center, Beijing 100083, PR China Received 1 January 2014; accepted 10 March 2014
Abstract Aspidin BB, a phloroglucinol derivative extracted from Dryopteris fragrans (L.) Schott, has been previously reported to exert high biological activities. In the present study, antibacterial activities and mechanisms of aspidin BB against Staphylococcus aureus were investigated. Aspidin BB exhibited strong antibacterial activity against standard and three clinical S. aureus, with minimal inhibition concentration (MIC) values ranging from 15.63 mg/mL to 62.5 mg/mL. After treatment with aspidin BB for 72 h using the MTT assay, the IC50 value was 48.14 mM (22.11 mg/mL). The timeekill assay indicated that aspidin BB could kill S. aureus completely at 2 MIC (MBC) within 4 h. Aspidin BB was capable to induce an increase in NADPH oxidase activity from 4.03 U/mg to 7.48 U/mg when the concentrations were increased from 1/2 MIC to 4 MIC. Simultaneously, aspidin BB attenuated antioxidant defense by decreasing superoxide dismutase (SOD) activity and glutathione (GSH) levels. The level of reactive oxygen species (ROS) was apparently elevated when measuring OD575. This phenomenon was blocked by pretreatment of S. aureus with the antioxidant compound catalase (200 U/mL) and the survival rate of S. aureus increased from 3.95% to 73.04%. These results showed that ROS was indeed an important mediator in the antibacterial action of aspidin BB. In addition, aspidin-BB-induced peroxidation of membranes, DNA damage and protein degradation of S. aureus were all verified in a dose-dependent manner. In conclusion, aspidin BB induced generation of ROS by activating NADPH oxidase activity and inhibiting SOD activity and GSH levels, damaging the membrane, DNA and proteins and finally led to cell death. Ó 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
Keywords: Dryopteris fragrans; Aspidin BB; Reactive oxygen species; Membrane; DNA; Protein
1. Introduction
* Corresponding author. Engineering Research Center of Forest BioPreparation, Ministry of Education, Northeast Forestry University, Box 332, Hexing Road 26, Harbin 150040, PR China. Tel./fax: þ86 451 82190535. ** Corresponding author. Engineering Research Center of Forest BioPreparation, Ministry of Education, Northeast Forestry University, Box 332, Hexing Road 26, Harbin 150040, PR China. Tel.: þ86 451 82191517; fax: þ86 451 82102082. E-mail addresses: [email protected] (Y.-j. Fu), [email protected] (Y.-g. Zu). 1 These authors contributed equally to this work.
Staphylococcus is a group of bacteria that can cause a diverse array of diseases as a result of various tissue infections. Among these, Staphylococcus aureus, which causes a number of infections, with prevalence rates ranging from 4.6% to 54.4%, is a major human pathogen in hospitals and communities [1]. It causes massive infections ranging from superficial skin infections to life-threatening diseases such as sepsis, dermatitis, folliculitis decalvans, pneumonia, endocarditis, osteomyelitis and arthritis [2]. The employment of antibiotics can decrease the danger of these diseases and save lives, yet they may also result in drug resistance which has become a
http://dx.doi.org/10.1016/j.resmic.2014.03.002 0923-2508/Ó 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. Please cite this article in press as: Li N, et al., Aspidin BB, a phloroglucinol derivative, exerts its antibacterial activity against Staphylococcus aureus by inducing the generation of reactive oxygen species, Research in Microbiology (2014), http://dx.doi.org/10.1016/j.resmic.2014.03.002
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N. Li et al. / Research in Microbiology xx (2014) 1e10
worldwide clinical threat [3]. Therefore, there is a continuing need to develop safe and effective antibacterials with low drug resistance. ROS, including O2 ; H2 O2 and OH , are by-products of normal metabolism and xenobiotic exposure of living organisms [4]. Imbalance between the generation of ROS and the antioxidant system can cause oxidative stress. Enzymic and non-enzymic antioxidant defense may be activated to inhibit the imbalance. SOD and GSH are representative enzymic and non-enzymic antioxidants, respectively. In addition, overproduced ROS can damage the membrane, DNA and proteins, leading to cell death [5]. Natural products are a source of great interest for curing bacterial infection, with low toxicity and high efficiency. Dryopteris fragrans (L.) Schott, known as Xianglinmaojue in China, has been historically used in folk medicine for treating dermatosis, rheumatoid arthritis and cancer [6]. Aspidin BB is a typically active phloroglucinol derivative extracted from D. fragrans and has been reported to exert anti-cancer, anthelmintic and antivirus activities [7]. As we have seen, most phloroglucinol derivatives possess strong antibacterial activity, especially against S. aureus [8]. Our previous data showed that dryofragin, a phloroglucinol derivative, can induce apoptosis in human breast cancer MCF-7 cells through the ROSmediated mitochondrial pathway [9]. Based on this, we sought to determine know whether aspidin BB, a phloroglucinol derivative, could exert antibacterial activity against S. aureus by inducing the generation of ROS.
2.3. Measurement of MICs and MBCs of aspidin BB Minimum inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) were measured by serial twofold dilutions method based on guidelines in the literature [10]. Aspidin BB was dissolved in DMSO and then added to S. aureus suspension to obtain the final concentration of 0.5% (v/ v) DMSO. S. aureus (105 CFU/mL) was incubated with aspidin BB at concentrations of twofold serial dilutions (0.98 mg/mLe500 mg/mL) in a 96-well microplate (100 mL per well) under aerobic conditions at 37 C for 24 h. The negative control received only 0.5% (v/v) DMSO. MIC was defined as the lowest concentration of aspidin BB that prevented the growth of S. aureus. Then, all MIC tubes (10 mL of culture from each tube) were used for spreading on nutrient plates for colony counting. The concentration at which inoculated bacterial were completely killed was considered to be the MBC. Erythromycin was used as a positive control. 2.4. Cell culture and cytotoxicity assays
2. Materials and methods
African green monkey kidney cells (Vero) were grown in Dulbecco’s modified Eagle’s medium (Sigma) supplemented with 10% fetal bovine serum and 100 U/mL penicillin and 100 mg/mL streptomycin. The cells were incubated in a humidified incubator at 37 C with 5% CO2. The cytotoxicity profile of aspidin BB was tested against Vero using MTT cell viability assay. In cytotoxicity assays, each sample was tested at least three times, with each concentration tested in triplicate per experiment.
2.1. Materials
2.5. Timeekill dynamics assay
Aspidin BB (purity > 95%) was isolated from D. fragrans (L.) Schott using a macroporous resin column chromatography method described by our group [8]. Erythromycin (purity > 99%) was used as positive control and was purchased from Sigma Chemical Co. (St. Louis, MO, USA). 3(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), NADPH, nitro blue tetrazolium (NBT) and catalase were obtained from Sigma Chemicals (Shanghai, China). All kits were purchased from the Beyotime Institute of Biotechnology. The other chemicals were procured locally and were of analytical reagent grade.
Timeekill dynamics of S. aureus treated by aspidin BB (corresponding to control, 1/2 MIC, 1 MIC and 2 MIC) was studied based on the number of viable cells determined through a colony counting method in plates [11]. Each assay included a growth control without a test sample. Timeekill curves were constructed by plotting log10 CFU/mL against time (h).
2.2. Culture of S. aureus S. aureus (ATCC 6538) was purchased from the Institute of Applied Microbiology, Heilongjiang Academy of Science (China). Three clinical bacteria (S. aureus MRSA 1a, S. aureus MRSA 2a and S. aureus MRSA 3a) were isolated from Harbin Medical University (HRBMU). They were maintained on agar slants at 4 C and cultured on nutrient agar at 37 C for 24 h. Prior to each experiment, S. aureus was refreshed from nutrient agar in nutrient broth at 37 C and allowed to grow up to early log phase at an OD600 of 0.5 (1 108 colony-forming units (cfu)/mL) for further study.
2.6. Measurement of enzymic and non-enzymic substances of S. aureus after aspidin BB treatment 2.6.1. NADPH oxidase activity assay NADPH oxidase activity was assayed by the method previously described by Streker et al. [12], with slight modification. Bacterial cultures grown in nutrient broth with different concentrations of aspidin BB (1/2 MIC, 1 MIC, 2 MIC and 4 MIC) were harvested by centrifugation at 4500 g for 5 min, washed and resuspended in phosphate buffer saline (PBS 50 mM; pH 7.0). Control samples were prepared similarly without treatment. Then, bacteria were lysed in 0.5 mg/mL lysozyme at 4 C for 30 min. Aliquots (0.1 mL) were incubated at room temperature in the same buffer (2.8 mL) containing NADPH (0.15 mM). The OD340 of the samples monitoring oxidation of NADPH was determined for
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N. Li et al. / Research in Microbiology xx (2014) 1e10
NADPH oxidase activity. Simultaneously, protein concentrations were quantified using a BCA protein assay kit. One unit of NADPH oxidase was defined as the amount of enzyme (mg protein) that catalyzed the oxidation of 1.0 mmol NADPH min1. 2.6.2. Superoxide dismutase activity assay This activity was assayed by xanthine oxidase and conversion of NBT to NBT formazan. S. aureus cells were cultured in nutrient broth with aeration for 24 h at 37 C. After aspidin BB (1/2 MIC, 1 MIC, 2 MIC, 4 MIC) treatment, the cells were harvested by centrifugation at 4500 g for 5 min, washed and resuspended in PBS. Cells without aspidin BB treatment were considered as the blank control. Then, lysozyme at 0.5 mg/mL was used to lyse cells at 4 C for 30 min. After centrifugation at 13,400 g for 5 min, supernatants were collected for total SOD activity measurement. SOD activity was determined by adding the master mix and xanthine supplied in the kit, followed by incubation at 37 C for 20 min and measured by a microplate reader at 560 nm. Then, the percent inhibition was calculated based on instructions and 50% inhibition corresponded to one unit of enzyme activity. Protein concentration was quantified using a BCA protein assay kit. SOD activity was expressed as unit/mg protein. 2.6.3. Glutathione quantification Bacterial cells treated by different concentrations (1/2 MIC, 1 MIC, 2 MIC and 4 MIC) of aspidin BB were harvested by centrifugation at 4500 g for 10 min and washed with PBS. Cells without aspidin BB treatment were regarded as the blank control. Then, the pellets were suspended and deproteinated in protein removal agent S by brief sonication. After rapidly freezeethawing twice between 37 C and liquid nitrogen, the suspensions were centrifuged at 10,000 g for 10 min. The supernatants were collected for total glutathione measurement. GSH was determined by adding the reaction mix and 0.16 mg/ mL NADPH supplied in the kit, followed by incubation at 25 C for 25 min and measured by a microplate reader at 412 nm. The total amount of GSH was determined by means of a calibration curve. Protein concentrations were quantified using a BCA protein assay kit. The quantification of GSH was expressed as mmol/mg protein. 2.7. Measurement of reactive oxygen species (ROS) Intracellular ROS in the bacterial cells were measured by the protocol described by Pramanik et al. [13]. Bacterial cultures were pelleted by centrifugation at 4500 g for 10 min and resuspended in Hanks’ balanced salt solution (HBSS). Then, different concentrations of aspidin BB (1/2 MIC, 1 MIC, 2 MIC and 4 MIC) were added and incubated at 37 C for 4 h, and later, 1 mg/mL NBT for 30 min at 37 C. Control samples were prepared similarly without aspidin BB treatment. Afterwards, 0.1 mL of 0.1 M HCl was added to stop the reaction and the tubes were centrifuged at 1500 g for 10 min. The pellets were then treated with 600 mmol DMSO to extract the reduced NBT and 500 mmol HBSS were added. Ultimately,
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formazan blues obtained from cells were measured at 575 nm. Simultaneously, different concentrations of antioxidant catalase (100, 200 U/mL) as a scavenger of ROS were also used to measure the changes in ROS. 2.8. Measurement of growth of S. aureus in the presence of antioxidant catalase The effects of antioxidant catalase on the antibacterial activity of aspidin BB against S. aureus were determined by a colony formation assay as previously described by Helmerhorst et al. [14], with slight modification. Different concentrations of antioxidant catalase were added to logarithmically growing S. aureus cells to obtain concentrations of 100 and 200 U/mL. After incubation at 37 C for 15 min, different volumes of aspidin BB were added from stock solution to obtain a concentration of 1/2 MIC, 1 MIC, 2 MIC and 4 MIC. Then, 10 mL aliquots were plated on nutrient agar plates after incubation for 4 h at 37 C. CFUs were counted after 24 h of incubation at 37 C. Percent survival was calculated as the ratio of the number of CFUs after treatment with aspidin BB compared to the initial inoculum. 2.9. Membrane damage 2.9.1. Atomic force microscope assay The morphological changes in S. aureus induced by aspidin BB were examined by atomic force microscopy (AFM) [15]. S. aureus treated with different concentrations of aspidin BB (1/2 MIC, 1 MIC, 2 MIC and 4 MIC) was incubated for 4 h at 37 C. Then, cells were harvested by centrifugation at 4500 g for 10 min and washed with PBS. The untreated cells were considered to be the control. For AFM analysis, 10 mL of the samples were applied to a freshly cleaved mica surface, dried for 15 min, softly washed with deionized water three times and ultimately dried in the air for examination. Individual S. aureus of each sample solution were randomly selected and imaged by AFM. Furthermore, the morphological parameters including length, width and height were precisely measured by using the off-line analysis commands provided by Nano-Scope Software. To quantitatively describe the topography of the bacterial surface, average surface roughness was calculated using the root-mean-square (RMS) average of the height deviations. The equation was qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi PN 2 Rrms ¼ i¼1 ðZi Zm Þ =ðN 1Þ, where N is the total number of data points, Zi is the height of the ith point and Zm is the mean height. A fixed size of 500 500 nm2 of S. aureus surface was measured for determination of roughness values. At least 15 cells were processed to calculate mean values for each parameter. 2.9.2. Integrity of the cell membrane Cell membrane integrity of S. aureus was examined by measuring the release of UV-absorbing materials at 260 nm [16]. Bacterial cultures grown in nutrient broth were harvested by centrifugation at 11,000 g for 10 min, washed and
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resuspended in PBS. Different concentrations of aspidin BB (MIC and MBC) were added to cell suspensions. Control samples were prepared similarly without aspidin BB treatment. The released materials absorbing at 260 nm over time were monitored with a UVeVIS spectrophotometer. 2.9.3. Measurement of protein leakage of S. aureus Cell membrane damage to S. aureus was examined by concentration changes in proteins leakage [17]. Logarithmically growing S. aureus were treated with different concentrations of aspidin BB (1 MIC, 2 MIC and 4 MIC) for 4 h at 37 C. Controls were run without aspidin BB treatment. Supernatants were collected by centrifugation at 13,400 g for 10 min. The protein fraction contained in the supernatants was condensed using TCA/acetone and boiled at 20 mL of sample buffer (0.06 M TriseHCl (pH 6.8), 5% mercaptoethanol, 10% glycerol and 2% SDS, 0.001% bromophenol blue) for 10 min. In addition, the BCA protein assay kit was used to quantify protein concentrations. SDS-PAGE was performed with a 5% stacking gel and a 12% separating gel followed by silver staining [18]. 2.9.4. Measurement of lipid peroxidation This assay was based on the chromogenic reaction of malonyldialdehyde (MDA) and thiobarbituric acid (TBA). S. aureus cells were cultured in nutrient broth with aeration for 24 h at 37 C. After aspidin BB (1/2 MIC, 1 MIC, 2 MIC, 4 MIC) treatment, cells were harvested by centrifugation at 4500 g for 5 min, washed and resuspended in PBS. Cells without aspidin BB were considered to be the blank control. Then, lysozyme at 0.5 mg/mL was used to lyse cells at 4 C for 30 min. After centrifugation at 1600 g for 10 min, the supernatants were collected for lipid peroxidation measurement. Lipid peroxidation was determined by adding the master mix supplied in the kit and samples collected from the supernatants, followed by boiling for 15 min and cooling to room temperature. The suspensions were centrifuged at 1000 g for 10 min and the OD532 of the supernatants was determined by a microplate reader to quantify the MDA-TBA adduct. Then, the total amount of MDA was determined by means of a calibration curve. Protein concentrations were quantified using a BCA protein assay kit. Production of MDA was expressed as nmol/mg protein. 2.10. DNA fragmentation assay DNA fragmentation was measured using the DNA ladder assay [19]. DNA from the aspidin BB-treated S. aureus was isolated using the Genomic DNA Mini Preparation kit with a spin column. Cleavage products in TAE buffer (4.84 g Tris base, pH 8.0, 0.5 M EDTA/1 L) were analyzed by 0.8% agarose gel electrophoresis at 50 V for 40 min. The gels were stained with ethidium bromide to visualize the DNA. 2.11. SDS-PAGE of whole-cell protein lysates The degradation of S. aureus proteins after aspidin BB treatment was determined by SDS-PAGE. The method for
extracting proteins was the same as described previously. However, precipitations of whole-cell proteins were left and washed twice with PBS. In addition, concentrations of wholecell protein lysates were estimated using a BCA protein assay kit and were adjusted to the same concentration. SDS-PAGE was performed with a 5% stacking gel and a 12% separating gel followed by silver staining [18]. 2.12. Statistical analysis All values were expressed as means standard deviation (SD) of three experiments. Data were analyzed using the oneway ANOVA test. The photographs of AFM, DNA and SDSPAGE shown in the figure were only representative. In all cases, a value of p < 0.05 was considered statistically significant. 3. Results 3.1. Antibacterial activity against S. aureus The antibacterial activity of aspidin BB was investigated against S. aureus. As shown in Table 1, MIC values of aspidin BB ranged from 15.63 mg/mL to 62.5 mg/mL for a standard strain and three clinical strains. When compared with erythromycin as positive control, aspidin BB showed comparative antibacterial activity. Due to its great sensitivity, the standard strain (S. aureus ATCC 6538) was chosen to study the antibacterial mechanism of aspidin BB. The cytotoxicity of aspidin BB and positive control erythromycin against Vero was determined by the MTT assay. The IC50 values were, respectively, 48.14 mM (22.11 mg/mL) and 97.81 mM (71.78 mg/mL) for aspidin BB and erythromycin after 72 h treatment (Fig. 2-A). The growth patterns of S. aureus treated by aspidin BB at 1/ 2 MIC, 1 MIC and 2 MIC (MBC) within 24 h were assayed (Fig. 2-B). Results indicated that aspidin BB time- and dosedependently inhibited growth of S. aureus. Moreover, the bacteria were completely killed at MBCs within 4 h. 3.2. Investigating the ROS level Changes in enzymatic (NADPH oxidase and SOD) and non-enzymatic (GSH) substances regulating the level of ROS for S. aureus were determined by microplate reader using kits.
Table 1 MICs and MBCs of aspidin BB against S. aureus. Bacteria
S. S. S. S. a
aureus aureus aureus aureus
(ATCC 6538) (MRSA 1a) (MRSA 2a) (MRSA 3a)
Aspidin BB (mg/mL)
Positive control erythromycin (mg/mL)
MIC
MBC
MIC
MBC
15.63 62.5 31.25 31.25
31.25 125 62.5 62.5
1.95 15.63 15.63 7.82
7.82 31.25 62.5 31.25
Clinical isolates from Harbin Medical University (HRBMU).
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This indicated that catalase prevented aspidin-BB-induced ROS generation. Furthermore, the survival rate of S. aureus treated with 4 MIC aspidin BB was only 3.95%, but it increased to 63.25% and 73.04% when pretreated with catalase at 100 and 200 U/mL, respectively (Fig. 4-B). These data showed that the key event in aspidin BB-induced killing of S. aureus was the generation of ROS. Fig. 1. Chemical structure of aspidin BB.
A concentration-dependent, statistically significant (p < 0.05) increase in NADPH oxidase activity of aspidin BB-treated S. aureus was observed (Fig. 3). It was 3.34 U/mg in the absence of aspidin BB and finally increased to 7.48 U/mg after aspidin BB treatment (Fig. 3A). Then SOD activity underwent a considerable decrease from 36.71 U/mg to 10.31 U/mg when the concentrations of aspidin BB were changed from 1 MIC to 4 MIC. However, GSH levels continued to slightly decrease. To determine whether ROS induction was directly involved in antibacterial activity, the effect of antioxidant catalase on aspidin BB-induced ROS generation and its antibacterial activity against S. aureus was assessed. As shown in Fig. 4-A, intracellular ROS was increased significantly in a dosedependent manner when S. aureus was treated with aspidin BB for 4 h. In the presence of catalase, ROS only increased slightly when the concentration of aspidin BB was increased.
Fig. 2. (A) Effect of aspidin BB upon Vero was determined by the MTT assay. Cells were treated with specified concentrations of aspidin BB (0, 1.5625, 3.125, 6.25, 12.5, 25, 50, and 100 mM) for the indicated time (72 h). Data are expressed as mean values SD from three independent experiments. (B) Timeekill kinetics of aspidin BB (1/2 MIC, 1 MIC and 2 MIC) against S. aureus.
3.3. ROS-induced membrane damage and DNA and protein degradation after aspidin BB treatment The membrane damage to S. aureus induced by aspidin BB was studied through a combination of different methods, including AFM, integrity of the cell membrane, protein leakage and lipid peroxidation assays. AFM was first used to visually observe the morphological changes in S. aureus after aspidin BB treatment. The topography and phase images are shown in Fig. 5. Untreated cells (Fig. 5-C) had smooth surfaces without notable ruptures or large pores. However, irregularity on the surfaces and even membrane integrity loss caused by aspidin BB were observed at 2 MIC and 4 MIC treatment. In addition, morphological parameters were also precisely measured by Nano-Scope software (Molecular Imaging Inc.). With increased concentrations of aspidin BB, the average RMS roughness was increased from 1.26 0.34 for control cells to 11.42 0.41 for 4 MIC-treated cells. (Table 2) Leakage of proteins, integrity of the cell membrane and lipid peroxidation assays further verified the membrane damage. Fig. 6-A shows the increased protein leakage of S. aureus treated by aspidin BB and that it was dose-dependent. The protein leakage of control cells was 13.2 mg/mL and increased to 85.31 mg/mL after 4 MIC treatment (Fig. 6-B). Furthermore, the release rates of intracellular components increased sharply within 2 h and then increased at a lower rate before 8 h, as shown in Fig. 6-C. Then again, generations of MDA were increased from 21.44 to 141.84 nmol/mg proteins when the concentrations of aspidin BB were increased to 4 MIC. All these results confirmed that aspidin BB caused membrane damage in a concentration-dependent manner. To determine whether aspidin BB could break down DNA and proteins of S. aureus, agarose gel electrophoresis and SDS-PAGE were applied. DNA and proteins were isolated from S. aureus treated with different concentrations of aspidin BB (1 MIC, 2 MIC and 4 MIC). As shown in Fig. 7-A, specific DNA degraded smearing typical of necrotic degeneration was prominent in S. aureus cells, especially for cells treated with 4 MIC aspidin BB. Fig. 7-B showed that proteins of untreated controls and aspidin-BB-treated S. aureus were different and exhibited apparent degradation after aspidin BB treatment. This indicated that aspidin BB could break down the DNA and proteins of S. aureus. 4. Discussion D. fragrans (L.) contains significant amounts of phloroglucinol derivatives, mainly dryofragin, aspidin BB, aspidin
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Fig. 3. Cell responses to aspidin BB (1/2 MIC, 1 MIC, 2 MIC and 4 MIC) in S. aureus. (A) NADPH oxidase activity; (B) SOD activity; (C) GSH level. The absorbance values were measured by a microplate reader at 340 nm, 560 nm and 412 nm, respectively. Data were expressed as mean values SD from three independent experiments. *p < 0.05, **p < 0.01, compared to control.
PB and aspidinol. Among these, aspidin BB exhibited high antibacterial activity against S. aureus. However, the antibacterial mechanism of aspidin BB (Fig. 1) has not been reported in depth. Therefore, we investigated the antibacterial activity and molecular mechanism of aspidin BB against S. aureus for the first time. Preliminary screening for the antibacterial activity of aspidin BB was performed by the microtiter broth dilution method [20]. As shown in Table 1, the standard strain (S. aureus ATCC 6538) possessed more sensitivity to aspidin BB than three clinical strains, which might be due to their antibiotic susceptibility pattern [10]. Additionally, when compared with positive control erythromycin, aspidin BB possessed comparative antibacterial activity against all strains. Previous research has shown that erythromycin can inhibit the growth of S. aureus by preventing formation of the 50S ribosomal subunit and inhibiting ribosome translation. By destroying the structure or modifying the binding site, S. aureus might be resistant to erythromycin [21]. Some studies have revealed that the ROS-based antibacterial mode is non-specific in terms of its active site and therefore decreases the possibility of resistance generation [22]. Hence, aspidin BB could be more likely to overcome resistance to erythromycin or other antibiotics. In order to exclude nonspecific cytotoxicity of aspidin BB, Vero was used in the MTT assay. In comparison, aspidin BB showed no toxicity
when S. aureus was killed. The same MTT assay was carried out for erythromycin as a positive control. For explaining the speed of bactericidal activity of aspidin BB against S. aureus, timeekill curves were drafted based on MIC and MBC assay. As shown in Fig. 2-B, aspidin BB caused a lethal effect on bacteria at 2 MIC (MBC). Meanwhile, in order to determine the bacteriostatic or bactericidal properties of an agent, tests on its potential antibacterial action in vitro are necessary. For this reason, the most sensitive standard strain was chosen for research on the antibacterial mechanism of aspidin BB. Studies over the past few years have demonstrated that ROS can actively participate in numerous types of biological processes. However, it can also cause a number of diseases such as rheumatoid arthritis, Alzheimer’s, atherosclerosis and Parkinson’s diseases when the degree of ROS exceeds the normal level [23]. Interestingly, this study indicated that aspidin BB induced bactericidal activity against S. aureus with a ROS increase and a SOD and GSH decrease. Aspidin BB at concentrations from 1/ 2 MIC to 4 MIC disrupted intracellular redox homeostasis. Activation of NADPH oxidase is a general means of ROS generation and this has been proven [12]. As shown in Fig. 3-A, NADPH oxidase activity exhibited significant does-dependency when S. aureus was exposed to different concentrations of aspidin BB for 4 h (Fig. 3-A). This might be the principal reason for generation of ROS. However, the excessive rise in ROS may
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Fig. 4. The effect of antioxidant catalase (100, 200 U/mL) on the ROS level (A) and the survival rate (B) of S. aureus after aspidin BB treatment. Data were expressed as mean values SD from three independent experiments. *p < 0.05, **p < 0.01, compared to control.
be accompanied by an immediate compensatory increase in free radical scavenging enzymatic (e.g., superoxide dismutase, catalase, glutathione reductase and glutathione peroxidases) and non-enzymatic (e.g., glutathione, arginine, citrulline and
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zinc) defense systems to protect the cell itself [24]. Among these, SOD and GSH are the representative enzymatic and non-enzymatic antioxidants. As we have seen, SOD can catalyze dismutation of O2 to H2O2 and can be considered to constitute the first line of bacterial defense against ROS [25]. Our studies revealed that SOD activity in aspidin BB-treated S. aureus was reduced in a dose-dependent fashion (Fig. 3-B). Additionally, GSH, which is the most abundant intracellular thiol, can also provide the primary defense against ROS. GSH is still known to be involved in many biological processes, such as protein and DNA synthesis, cell transport and enzyme activity modulation [26]. In addition, H2O2 can be converted to H2O þ O2 by GSH and catalase or to OH via the Fenton and HabereWeiss reactions [27]. Our results indicated that aspidin BB decreased GSH levels in a concentration-dependent manner in S. aureus (Fig. 3-C). Taken together, we proposed that the level of ROS in S. aureus might be elevated after aspidin BB treatment. As shown in Fig. 4-A, aspidin BB promoted the generation of ROS in S. aureus, especially at 2 MIC and 4 MIC. Furthermore, the addition of antioxidant catalase not only prevented ROS generation (Fig. 4-A), but also the antibacterial effect of aspidin BB (Fig. 4-B), indicating that ROS was indeed an important mediator in the antibacterial action of aspidin BB. Indeed, the increased formation of ROS has numerous deleterious effects on bacterial cells, ranging from peroxidation of the membrane to DNA damage, protein degradation and eventually cell death [28]. A series of methods were used to test these effects. A positive correlation between membrane permeability and time and concentration was exhibited by the results of protein leakage and integrity of the cell membrane after aspidin BB treatment (Fig. 6AeC). In addition, this was further substantiated by our AFM study where the rupture of the cell membrane was visually observed (Fig. 5). After this, increased generation of MDA produced during lipid peroxidation of unsaturated fatty acid was observed. As we know, the effect of lipid peroxidation then acts as an amplifier and more radicals form. Some of them, like aldehydes, are highly active and can further damage molecules such as DNA and proteins [29], as
Fig. 5. Representative AFM images of S. aureus treated with different concentrations of aspidin BB (1/2 MIC, 1 MIC, 2 MIC and 4 MIC) for 4 h. (A) topographic image; (B) phase image. Please cite this article in press as: Li N, et al., Aspidin BB, a phloroglucinol derivative, exerts its antibacterial activity against Staphylococcus aureus by inducing the generation of reactive oxygen species, Research in Microbiology (2014), http://dx.doi.org/10.1016/j.resmic.2014.03.002
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Table 2 Measurement of S. aureus dimensions after exposure to aspidin BB at different concentrations (These data were generated by AFM.). Concentration
S. aureus
Length (nm) Width (nm) Height (nm) RMS roughness (nm)
826.25 816.27 94.32 1.26
p Value
Control
1/2 MIC
13.44 8.41 16.47 0.34
931.68 861.94 85.14 3.68
1 MIC 24.35 15.30 5.38 0.17
demonstrated in Fig. 7. By comparing whole-cell protein lysates with National Center for Biotechnology information, some possible target proteins of aspidin BB against S. aureus have been suggested, such as SarA (14.718 kDa), Fib (18.765 kDa), ferritin (19.589 kDa), the ATP-dependent clp proteolytic subunit (21.514 kDa), SOD (Mn/Fe) (23.041 kDa), staphyloxanthin biosynthesis protein (33.376 kDa), the S. aureus sex pheromone (45.381 kDa) and iron-regulated surface determinant protein B (72.192 kDa) and H (100.847 kDa). They are mainly responsible for regulating virulence factors, iron metabolism
854.28 836.96 63.27 4.27
2 MIC
13.46 15.44 9.05 0.52
673.46 592.33 27.14 9.73
4 MIC
13.29 8.04 7.18 0.47
574.16 513.63 18.41 11.42
12.63 14.51 8.26 0.41
MODEL
Research in Microbiology xx (2014) 1e10 www.elsevier.com/locate/resmic
Aspidin BB, a phloroglucinol derivative, exerts its antibacterial activity against Staphylococcus aureus by inducing the generation of reactive oxygen species Na Li a,1, Chang Gao b,1, Xiao Peng a, Wei Wang a, Meng Luo a, Yu-jie Fu a,*, Yuan-gang Zu a,** a
Engineering Research Center of Forest Bio-Preparation, Ministry of Education, Northeast Forestry University, Harbin 150040, PR China b College of Basic Medical Science, Peking University Health Science Center, Beijing 100083, PR China Received 1 January 2014; accepted 10 March 2014
Abstract Aspidin BB, a phloroglucinol derivative extracted from Dryopteris fragrans (L.) Schott, has been previously reported to exert high biological activities. In the present study, antibacterial activities and mechanisms of aspidin BB against Staphylococcus aureus were investigated. Aspidin BB exhibited strong antibacterial activity against standard and three clinical S. aureus, with minimal inhibition concentration (MIC) values ranging from 15.63 mg/mL to 62.5 mg/mL. After treatment with aspidin BB for 72 h using the MTT assay, the IC50 value was 48.14 mM (22.11 mg/mL). The timeekill assay indicated that aspidin BB could kill S. aureus completely at 2 MIC (MBC) within 4 h. Aspidin BB was capable to induce an increase in NADPH oxidase activity from 4.03 U/mg to 7.48 U/mg when the concentrations were increased from 1/2 MIC to 4 MIC. Simultaneously, aspidin BB attenuated antioxidant defense by decreasing superoxide dismutase (SOD) activity and glutathione (GSH) levels. The level of reactive oxygen species (ROS) was apparently elevated when measuring OD575. This phenomenon was blocked by pretreatment of S. aureus with the antioxidant compound catalase (200 U/mL) and the survival rate of S. aureus increased from 3.95% to 73.04%. These results showed that ROS was indeed an important mediator in the antibacterial action of aspidin BB. In addition, aspidin-BB-induced peroxidation of membranes, DNA damage and protein degradation of S. aureus were all verified in a dose-dependent manner. In conclusion, aspidin BB induced generation of ROS by activating NADPH oxidase activity and inhibiting SOD activity and GSH levels, damaging the membrane, DNA and proteins and finally led to cell death. Ó 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
Keywords: Dryopteris fragrans; Aspidin BB; Reactive oxygen species; Membrane; DNA; Protein
1. Introduction
* Corresponding author. Engineering Research Center of Forest BioPreparation, Ministry of Education, Northeast Forestry University, Box 332, Hexing Road 26, Harbin 150040, PR China. Tel./fax: þ86 451 82190535. ** Corresponding author. Engineering Research Center of Forest BioPreparation, Ministry of Education, Northeast Forestry University, Box 332, Hexing Road 26, Harbin 150040, PR China. Tel.: þ86 451 82191517; fax: þ86 451 82102082. E-mail addresses: [email protected] (Y.-j. Fu), [email protected] (Y.-g. Zu). 1 These authors contributed equally to this work.
Staphylococcus is a group of bacteria that can cause a diverse array of diseases as a result of various tissue infections. Among these, Staphylococcus aureus, which causes a number of infections, with prevalence rates ranging from 4.6% to 54.4%, is a major human pathogen in hospitals and communities [1]. It causes massive infections ranging from superficial skin infections to life-threatening diseases such as sepsis, dermatitis, folliculitis decalvans, pneumonia, endocarditis, osteomyelitis and arthritis [2]. The employment of antibiotics can decrease the danger of these diseases and save lives, yet they may also result in drug resistance which has become a
http://dx.doi.org/10.1016/j.resmic.2014.03.002 0923-2508/Ó 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. Please cite this article in press as: Li N, et al., Aspidin BB, a phloroglucinol derivative, exerts its antibacterial activity against Staphylococcus aureus by inducing the generation of reactive oxygen species, Research in Microbiology (2014), http://dx.doi.org/10.1016/j.resmic.2014.03.002
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N. Li et al. / Research in Microbiology xx (2014) 1e10
worldwide clinical threat [3]. Therefore, there is a continuing need to develop safe and effective antibacterials with low drug resistance. ROS, including O2 ; H2 O2 and OH , are by-products of normal metabolism and xenobiotic exposure of living organisms [4]. Imbalance between the generation of ROS and the antioxidant system can cause oxidative stress. Enzymic and non-enzymic antioxidant defense may be activated to inhibit the imbalance. SOD and GSH are representative enzymic and non-enzymic antioxidants, respectively. In addition, overproduced ROS can damage the membrane, DNA and proteins, leading to cell death [5]. Natural products are a source of great interest for curing bacterial infection, with low toxicity and high efficiency. Dryopteris fragrans (L.) Schott, known as Xianglinmaojue in China, has been historically used in folk medicine for treating dermatosis, rheumatoid arthritis and cancer [6]. Aspidin BB is a typically active phloroglucinol derivative extracted from D. fragrans and has been reported to exert anti-cancer, anthelmintic and antivirus activities [7]. As we have seen, most phloroglucinol derivatives possess strong antibacterial activity, especially against S. aureus [8]. Our previous data showed that dryofragin, a phloroglucinol derivative, can induce apoptosis in human breast cancer MCF-7 cells through the ROSmediated mitochondrial pathway [9]. Based on this, we sought to determine know whether aspidin BB, a phloroglucinol derivative, could exert antibacterial activity against S. aureus by inducing the generation of ROS.
2.3. Measurement of MICs and MBCs of aspidin BB Minimum inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) were measured by serial twofold dilutions method based on guidelines in the literature [10]. Aspidin BB was dissolved in DMSO and then added to S. aureus suspension to obtain the final concentration of 0.5% (v/ v) DMSO. S. aureus (105 CFU/mL) was incubated with aspidin BB at concentrations of twofold serial dilutions (0.98 mg/mLe500 mg/mL) in a 96-well microplate (100 mL per well) under aerobic conditions at 37 C for 24 h. The negative control received only 0.5% (v/v) DMSO. MIC was defined as the lowest concentration of aspidin BB that prevented the growth of S. aureus. Then, all MIC tubes (10 mL of culture from each tube) were used for spreading on nutrient plates for colony counting. The concentration at which inoculated bacterial were completely killed was considered to be the MBC. Erythromycin was used as a positive control. 2.4. Cell culture and cytotoxicity assays
2. Materials and methods
African green monkey kidney cells (Vero) were grown in Dulbecco’s modified Eagle’s medium (Sigma) supplemented with 10% fetal bovine serum and 100 U/mL penicillin and 100 mg/mL streptomycin. The cells were incubated in a humidified incubator at 37 C with 5% CO2. The cytotoxicity profile of aspidin BB was tested against Vero using MTT cell viability assay. In cytotoxicity assays, each sample was tested at least three times, with each concentration tested in triplicate per experiment.
2.1. Materials
2.5. Timeekill dynamics assay
Aspidin BB (purity > 95%) was isolated from D. fragrans (L.) Schott using a macroporous resin column chromatography method described by our group [8]. Erythromycin (purity > 99%) was used as positive control and was purchased from Sigma Chemical Co. (St. Louis, MO, USA). 3(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), NADPH, nitro blue tetrazolium (NBT) and catalase were obtained from Sigma Chemicals (Shanghai, China). All kits were purchased from the Beyotime Institute of Biotechnology. The other chemicals were procured locally and were of analytical reagent grade.
Timeekill dynamics of S. aureus treated by aspidin BB (corresponding to control, 1/2 MIC, 1 MIC and 2 MIC) was studied based on the number of viable cells determined through a colony counting method in plates [11]. Each assay included a growth control without a test sample. Timeekill curves were constructed by plotting log10 CFU/mL against time (h).
2.2. Culture of S. aureus S. aureus (ATCC 6538) was purchased from the Institute of Applied Microbiology, Heilongjiang Academy of Science (China). Three clinical bacteria (S. aureus MRSA 1a, S. aureus MRSA 2a and S. aureus MRSA 3a) were isolated from Harbin Medical University (HRBMU). They were maintained on agar slants at 4 C and cultured on nutrient agar at 37 C for 24 h. Prior to each experiment, S. aureus was refreshed from nutrient agar in nutrient broth at 37 C and allowed to grow up to early log phase at an OD600 of 0.5 (1 108 colony-forming units (cfu)/mL) for further study.
2.6. Measurement of enzymic and non-enzymic substances of S. aureus after aspidin BB treatment 2.6.1. NADPH oxidase activity assay NADPH oxidase activity was assayed by the method previously described by Streker et al. [12], with slight modification. Bacterial cultures grown in nutrient broth with different concentrations of aspidin BB (1/2 MIC, 1 MIC, 2 MIC and 4 MIC) were harvested by centrifugation at 4500 g for 5 min, washed and resuspended in phosphate buffer saline (PBS 50 mM; pH 7.0). Control samples were prepared similarly without treatment. Then, bacteria were lysed in 0.5 mg/mL lysozyme at 4 C for 30 min. Aliquots (0.1 mL) were incubated at room temperature in the same buffer (2.8 mL) containing NADPH (0.15 mM). The OD340 of the samples monitoring oxidation of NADPH was determined for
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N. Li et al. / Research in Microbiology xx (2014) 1e10
NADPH oxidase activity. Simultaneously, protein concentrations were quantified using a BCA protein assay kit. One unit of NADPH oxidase was defined as the amount of enzyme (mg protein) that catalyzed the oxidation of 1.0 mmol NADPH min1. 2.6.2. Superoxide dismutase activity assay This activity was assayed by xanthine oxidase and conversion of NBT to NBT formazan. S. aureus cells were cultured in nutrient broth with aeration for 24 h at 37 C. After aspidin BB (1/2 MIC, 1 MIC, 2 MIC, 4 MIC) treatment, the cells were harvested by centrifugation at 4500 g for 5 min, washed and resuspended in PBS. Cells without aspidin BB treatment were considered as the blank control. Then, lysozyme at 0.5 mg/mL was used to lyse cells at 4 C for 30 min. After centrifugation at 13,400 g for 5 min, supernatants were collected for total SOD activity measurement. SOD activity was determined by adding the master mix and xanthine supplied in the kit, followed by incubation at 37 C for 20 min and measured by a microplate reader at 560 nm. Then, the percent inhibition was calculated based on instructions and 50% inhibition corresponded to one unit of enzyme activity. Protein concentration was quantified using a BCA protein assay kit. SOD activity was expressed as unit/mg protein. 2.6.3. Glutathione quantification Bacterial cells treated by different concentrations (1/2 MIC, 1 MIC, 2 MIC and 4 MIC) of aspidin BB were harvested by centrifugation at 4500 g for 10 min and washed with PBS. Cells without aspidin BB treatment were regarded as the blank control. Then, the pellets were suspended and deproteinated in protein removal agent S by brief sonication. After rapidly freezeethawing twice between 37 C and liquid nitrogen, the suspensions were centrifuged at 10,000 g for 10 min. The supernatants were collected for total glutathione measurement. GSH was determined by adding the reaction mix and 0.16 mg/ mL NADPH supplied in the kit, followed by incubation at 25 C for 25 min and measured by a microplate reader at 412 nm. The total amount of GSH was determined by means of a calibration curve. Protein concentrations were quantified using a BCA protein assay kit. The quantification of GSH was expressed as mmol/mg protein. 2.7. Measurement of reactive oxygen species (ROS) Intracellular ROS in the bacterial cells were measured by the protocol described by Pramanik et al. [13]. Bacterial cultures were pelleted by centrifugation at 4500 g for 10 min and resuspended in Hanks’ balanced salt solution (HBSS). Then, different concentrations of aspidin BB (1/2 MIC, 1 MIC, 2 MIC and 4 MIC) were added and incubated at 37 C for 4 h, and later, 1 mg/mL NBT for 30 min at 37 C. Control samples were prepared similarly without aspidin BB treatment. Afterwards, 0.1 mL of 0.1 M HCl was added to stop the reaction and the tubes were centrifuged at 1500 g for 10 min. The pellets were then treated with 600 mmol DMSO to extract the reduced NBT and 500 mmol HBSS were added. Ultimately,
3
formazan blues obtained from cells were measured at 575 nm. Simultaneously, different concentrations of antioxidant catalase (100, 200 U/mL) as a scavenger of ROS were also used to measure the changes in ROS. 2.8. Measurement of growth of S. aureus in the presence of antioxidant catalase The effects of antioxidant catalase on the antibacterial activity of aspidin BB against S. aureus were determined by a colony formation assay as previously described by Helmerhorst et al. [14], with slight modification. Different concentrations of antioxidant catalase were added to logarithmically growing S. aureus cells to obtain concentrations of 100 and 200 U/mL. After incubation at 37 C for 15 min, different volumes of aspidin BB were added from stock solution to obtain a concentration of 1/2 MIC, 1 MIC, 2 MIC and 4 MIC. Then, 10 mL aliquots were plated on nutrient agar plates after incubation for 4 h at 37 C. CFUs were counted after 24 h of incubation at 37 C. Percent survival was calculated as the ratio of the number of CFUs after treatment with aspidin BB compared to the initial inoculum. 2.9. Membrane damage 2.9.1. Atomic force microscope assay The morphological changes in S. aureus induced by aspidin BB were examined by atomic force microscopy (AFM) [15]. S. aureus treated with different concentrations of aspidin BB (1/2 MIC, 1 MIC, 2 MIC and 4 MIC) was incubated for 4 h at 37 C. Then, cells were harvested by centrifugation at 4500 g for 10 min and washed with PBS. The untreated cells were considered to be the control. For AFM analysis, 10 mL of the samples were applied to a freshly cleaved mica surface, dried for 15 min, softly washed with deionized water three times and ultimately dried in the air for examination. Individual S. aureus of each sample solution were randomly selected and imaged by AFM. Furthermore, the morphological parameters including length, width and height were precisely measured by using the off-line analysis commands provided by Nano-Scope Software. To quantitatively describe the topography of the bacterial surface, average surface roughness was calculated using the root-mean-square (RMS) average of the height deviations. The equation was qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi PN 2 Rrms ¼ i¼1 ðZi Zm Þ =ðN 1Þ, where N is the total number of data points, Zi is the height of the ith point and Zm is the mean height. A fixed size of 500 500 nm2 of S. aureus surface was measured for determination of roughness values. At least 15 cells were processed to calculate mean values for each parameter. 2.9.2. Integrity of the cell membrane Cell membrane integrity of S. aureus was examined by measuring the release of UV-absorbing materials at 260 nm [16]. Bacterial cultures grown in nutrient broth were harvested by centrifugation at 11,000 g for 10 min, washed and
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N. Li et al. / Research in Microbiology xx (2014) 1e10
resuspended in PBS. Different concentrations of aspidin BB (MIC and MBC) were added to cell suspensions. Control samples were prepared similarly without aspidin BB treatment. The released materials absorbing at 260 nm over time were monitored with a UVeVIS spectrophotometer. 2.9.3. Measurement of protein leakage of S. aureus Cell membrane damage to S. aureus was examined by concentration changes in proteins leakage [17]. Logarithmically growing S. aureus were treated with different concentrations of aspidin BB (1 MIC, 2 MIC and 4 MIC) for 4 h at 37 C. Controls were run without aspidin BB treatment. Supernatants were collected by centrifugation at 13,400 g for 10 min. The protein fraction contained in the supernatants was condensed using TCA/acetone and boiled at 20 mL of sample buffer (0.06 M TriseHCl (pH 6.8), 5% mercaptoethanol, 10% glycerol and 2% SDS, 0.001% bromophenol blue) for 10 min. In addition, the BCA protein assay kit was used to quantify protein concentrations. SDS-PAGE was performed with a 5% stacking gel and a 12% separating gel followed by silver staining [18]. 2.9.4. Measurement of lipid peroxidation This assay was based on the chromogenic reaction of malonyldialdehyde (MDA) and thiobarbituric acid (TBA). S. aureus cells were cultured in nutrient broth with aeration for 24 h at 37 C. After aspidin BB (1/2 MIC, 1 MIC, 2 MIC, 4 MIC) treatment, cells were harvested by centrifugation at 4500 g for 5 min, washed and resuspended in PBS. Cells without aspidin BB were considered to be the blank control. Then, lysozyme at 0.5 mg/mL was used to lyse cells at 4 C for 30 min. After centrifugation at 1600 g for 10 min, the supernatants were collected for lipid peroxidation measurement. Lipid peroxidation was determined by adding the master mix supplied in the kit and samples collected from the supernatants, followed by boiling for 15 min and cooling to room temperature. The suspensions were centrifuged at 1000 g for 10 min and the OD532 of the supernatants was determined by a microplate reader to quantify the MDA-TBA adduct. Then, the total amount of MDA was determined by means of a calibration curve. Protein concentrations were quantified using a BCA protein assay kit. Production of MDA was expressed as nmol/mg protein. 2.10. DNA fragmentation assay DNA fragmentation was measured using the DNA ladder assay [19]. DNA from the aspidin BB-treated S. aureus was isolated using the Genomic DNA Mini Preparation kit with a spin column. Cleavage products in TAE buffer (4.84 g Tris base, pH 8.0, 0.5 M EDTA/1 L) were analyzed by 0.8% agarose gel electrophoresis at 50 V for 40 min. The gels were stained with ethidium bromide to visualize the DNA. 2.11. SDS-PAGE of whole-cell protein lysates The degradation of S. aureus proteins after aspidin BB treatment was determined by SDS-PAGE. The method for
extracting proteins was the same as described previously. However, precipitations of whole-cell proteins were left and washed twice with PBS. In addition, concentrations of wholecell protein lysates were estimated using a BCA protein assay kit and were adjusted to the same concentration. SDS-PAGE was performed with a 5% stacking gel and a 12% separating gel followed by silver staining [18]. 2.12. Statistical analysis All values were expressed as means standard deviation (SD) of three experiments. Data were analyzed using the oneway ANOVA test. The photographs of AFM, DNA and SDSPAGE shown in the figure were only representative. In all cases, a value of p < 0.05 was considered statistically significant. 3. Results 3.1. Antibacterial activity against S. aureus The antibacterial activity of aspidin BB was investigated against S. aureus. As shown in Table 1, MIC values of aspidin BB ranged from 15.63 mg/mL to 62.5 mg/mL for a standard strain and three clinical strains. When compared with erythromycin as positive control, aspidin BB showed comparative antibacterial activity. Due to its great sensitivity, the standard strain (S. aureus ATCC 6538) was chosen to study the antibacterial mechanism of aspidin BB. The cytotoxicity of aspidin BB and positive control erythromycin against Vero was determined by the MTT assay. The IC50 values were, respectively, 48.14 mM (22.11 mg/mL) and 97.81 mM (71.78 mg/mL) for aspidin BB and erythromycin after 72 h treatment (Fig. 2-A). The growth patterns of S. aureus treated by aspidin BB at 1/ 2 MIC, 1 MIC and 2 MIC (MBC) within 24 h were assayed (Fig. 2-B). Results indicated that aspidin BB time- and dosedependently inhibited growth of S. aureus. Moreover, the bacteria were completely killed at MBCs within 4 h. 3.2. Investigating the ROS level Changes in enzymatic (NADPH oxidase and SOD) and non-enzymatic (GSH) substances regulating the level of ROS for S. aureus were determined by microplate reader using kits.
Table 1 MICs and MBCs of aspidin BB against S. aureus. Bacteria
S. S. S. S. a
aureus aureus aureus aureus
(ATCC 6538) (MRSA 1a) (MRSA 2a) (MRSA 3a)
Aspidin BB (mg/mL)
Positive control erythromycin (mg/mL)
MIC
MBC
MIC
MBC
15.63 62.5 31.25 31.25
31.25 125 62.5 62.5
1.95 15.63 15.63 7.82
7.82 31.25 62.5 31.25
Clinical isolates from Harbin Medical University (HRBMU).
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This indicated that catalase prevented aspidin-BB-induced ROS generation. Furthermore, the survival rate of S. aureus treated with 4 MIC aspidin BB was only 3.95%, but it increased to 63.25% and 73.04% when pretreated with catalase at 100 and 200 U/mL, respectively (Fig. 4-B). These data showed that the key event in aspidin BB-induced killing of S. aureus was the generation of ROS. Fig. 1. Chemical structure of aspidin BB.
A concentration-dependent, statistically significant (p < 0.05) increase in NADPH oxidase activity of aspidin BB-treated S. aureus was observed (Fig. 3). It was 3.34 U/mg in the absence of aspidin BB and finally increased to 7.48 U/mg after aspidin BB treatment (Fig. 3A). Then SOD activity underwent a considerable decrease from 36.71 U/mg to 10.31 U/mg when the concentrations of aspidin BB were changed from 1 MIC to 4 MIC. However, GSH levels continued to slightly decrease. To determine whether ROS induction was directly involved in antibacterial activity, the effect of antioxidant catalase on aspidin BB-induced ROS generation and its antibacterial activity against S. aureus was assessed. As shown in Fig. 4-A, intracellular ROS was increased significantly in a dosedependent manner when S. aureus was treated with aspidin BB for 4 h. In the presence of catalase, ROS only increased slightly when the concentration of aspidin BB was increased.
Fig. 2. (A) Effect of aspidin BB upon Vero was determined by the MTT assay. Cells were treated with specified concentrations of aspidin BB (0, 1.5625, 3.125, 6.25, 12.5, 25, 50, and 100 mM) for the indicated time (72 h). Data are expressed as mean values SD from three independent experiments. (B) Timeekill kinetics of aspidin BB (1/2 MIC, 1 MIC and 2 MIC) against S. aureus.
3.3. ROS-induced membrane damage and DNA and protein degradation after aspidin BB treatment The membrane damage to S. aureus induced by aspidin BB was studied through a combination of different methods, including AFM, integrity of the cell membrane, protein leakage and lipid peroxidation assays. AFM was first used to visually observe the morphological changes in S. aureus after aspidin BB treatment. The topography and phase images are shown in Fig. 5. Untreated cells (Fig. 5-C) had smooth surfaces without notable ruptures or large pores. However, irregularity on the surfaces and even membrane integrity loss caused by aspidin BB were observed at 2 MIC and 4 MIC treatment. In addition, morphological parameters were also precisely measured by Nano-Scope software (Molecular Imaging Inc.). With increased concentrations of aspidin BB, the average RMS roughness was increased from 1.26 0.34 for control cells to 11.42 0.41 for 4 MIC-treated cells. (Table 2) Leakage of proteins, integrity of the cell membrane and lipid peroxidation assays further verified the membrane damage. Fig. 6-A shows the increased protein leakage of S. aureus treated by aspidin BB and that it was dose-dependent. The protein leakage of control cells was 13.2 mg/mL and increased to 85.31 mg/mL after 4 MIC treatment (Fig. 6-B). Furthermore, the release rates of intracellular components increased sharply within 2 h and then increased at a lower rate before 8 h, as shown in Fig. 6-C. Then again, generations of MDA were increased from 21.44 to 141.84 nmol/mg proteins when the concentrations of aspidin BB were increased to 4 MIC. All these results confirmed that aspidin BB caused membrane damage in a concentration-dependent manner. To determine whether aspidin BB could break down DNA and proteins of S. aureus, agarose gel electrophoresis and SDS-PAGE were applied. DNA and proteins were isolated from S. aureus treated with different concentrations of aspidin BB (1 MIC, 2 MIC and 4 MIC). As shown in Fig. 7-A, specific DNA degraded smearing typical of necrotic degeneration was prominent in S. aureus cells, especially for cells treated with 4 MIC aspidin BB. Fig. 7-B showed that proteins of untreated controls and aspidin-BB-treated S. aureus were different and exhibited apparent degradation after aspidin BB treatment. This indicated that aspidin BB could break down the DNA and proteins of S. aureus. 4. Discussion D. fragrans (L.) contains significant amounts of phloroglucinol derivatives, mainly dryofragin, aspidin BB, aspidin
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Fig. 3. Cell responses to aspidin BB (1/2 MIC, 1 MIC, 2 MIC and 4 MIC) in S. aureus. (A) NADPH oxidase activity; (B) SOD activity; (C) GSH level. The absorbance values were measured by a microplate reader at 340 nm, 560 nm and 412 nm, respectively. Data were expressed as mean values SD from three independent experiments. *p < 0.05, **p < 0.01, compared to control.
PB and aspidinol. Among these, aspidin BB exhibited high antibacterial activity against S. aureus. However, the antibacterial mechanism of aspidin BB (Fig. 1) has not been reported in depth. Therefore, we investigated the antibacterial activity and molecular mechanism of aspidin BB against S. aureus for the first time. Preliminary screening for the antibacterial activity of aspidin BB was performed by the microtiter broth dilution method [20]. As shown in Table 1, the standard strain (S. aureus ATCC 6538) possessed more sensitivity to aspidin BB than three clinical strains, which might be due to their antibiotic susceptibility pattern [10]. Additionally, when compared with positive control erythromycin, aspidin BB possessed comparative antibacterial activity against all strains. Previous research has shown that erythromycin can inhibit the growth of S. aureus by preventing formation of the 50S ribosomal subunit and inhibiting ribosome translation. By destroying the structure or modifying the binding site, S. aureus might be resistant to erythromycin [21]. Some studies have revealed that the ROS-based antibacterial mode is non-specific in terms of its active site and therefore decreases the possibility of resistance generation [22]. Hence, aspidin BB could be more likely to overcome resistance to erythromycin or other antibiotics. In order to exclude nonspecific cytotoxicity of aspidin BB, Vero was used in the MTT assay. In comparison, aspidin BB showed no toxicity
when S. aureus was killed. The same MTT assay was carried out for erythromycin as a positive control. For explaining the speed of bactericidal activity of aspidin BB against S. aureus, timeekill curves were drafted based on MIC and MBC assay. As shown in Fig. 2-B, aspidin BB caused a lethal effect on bacteria at 2 MIC (MBC). Meanwhile, in order to determine the bacteriostatic or bactericidal properties of an agent, tests on its potential antibacterial action in vitro are necessary. For this reason, the most sensitive standard strain was chosen for research on the antibacterial mechanism of aspidin BB. Studies over the past few years have demonstrated that ROS can actively participate in numerous types of biological processes. However, it can also cause a number of diseases such as rheumatoid arthritis, Alzheimer’s, atherosclerosis and Parkinson’s diseases when the degree of ROS exceeds the normal level [23]. Interestingly, this study indicated that aspidin BB induced bactericidal activity against S. aureus with a ROS increase and a SOD and GSH decrease. Aspidin BB at concentrations from 1/ 2 MIC to 4 MIC disrupted intracellular redox homeostasis. Activation of NADPH oxidase is a general means of ROS generation and this has been proven [12]. As shown in Fig. 3-A, NADPH oxidase activity exhibited significant does-dependency when S. aureus was exposed to different concentrations of aspidin BB for 4 h (Fig. 3-A). This might be the principal reason for generation of ROS. However, the excessive rise in ROS may
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Fig. 4. The effect of antioxidant catalase (100, 200 U/mL) on the ROS level (A) and the survival rate (B) of S. aureus after aspidin BB treatment. Data were expressed as mean values SD from three independent experiments. *p < 0.05, **p < 0.01, compared to control.
be accompanied by an immediate compensatory increase in free radical scavenging enzymatic (e.g., superoxide dismutase, catalase, glutathione reductase and glutathione peroxidases) and non-enzymatic (e.g., glutathione, arginine, citrulline and
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zinc) defense systems to protect the cell itself [24]. Among these, SOD and GSH are the representative enzymatic and non-enzymatic antioxidants. As we have seen, SOD can catalyze dismutation of O2 to H2O2 and can be considered to constitute the first line of bacterial defense against ROS [25]. Our studies revealed that SOD activity in aspidin BB-treated S. aureus was reduced in a dose-dependent fashion (Fig. 3-B). Additionally, GSH, which is the most abundant intracellular thiol, can also provide the primary defense against ROS. GSH is still known to be involved in many biological processes, such as protein and DNA synthesis, cell transport and enzyme activity modulation [26]. In addition, H2O2 can be converted to H2O þ O2 by GSH and catalase or to OH via the Fenton and HabereWeiss reactions [27]. Our results indicated that aspidin BB decreased GSH levels in a concentration-dependent manner in S. aureus (Fig. 3-C). Taken together, we proposed that the level of ROS in S. aureus might be elevated after aspidin BB treatment. As shown in Fig. 4-A, aspidin BB promoted the generation of ROS in S. aureus, especially at 2 MIC and 4 MIC. Furthermore, the addition of antioxidant catalase not only prevented ROS generation (Fig. 4-A), but also the antibacterial effect of aspidin BB (Fig. 4-B), indicating that ROS was indeed an important mediator in the antibacterial action of aspidin BB. Indeed, the increased formation of ROS has numerous deleterious effects on bacterial cells, ranging from peroxidation of the membrane to DNA damage, protein degradation and eventually cell death [28]. A series of methods were used to test these effects. A positive correlation between membrane permeability and time and concentration was exhibited by the results of protein leakage and integrity of the cell membrane after aspidin BB treatment (Fig. 6AeC). In addition, this was further substantiated by our AFM study where the rupture of the cell membrane was visually observed (Fig. 5). After this, increased generation of MDA produced during lipid peroxidation of unsaturated fatty acid was observed. As we know, the effect of lipid peroxidation then acts as an amplifier and more radicals form. Some of them, like aldehydes, are highly active and can further damage molecules such as DNA and proteins [29], as
Fig. 5. Representative AFM images of S. aureus treated with different concentrations of aspidin BB (1/2 MIC, 1 MIC, 2 MIC and 4 MIC) for 4 h. (A) topographic image; (B) phase image. Please cite this article in press as: Li N, et al., Aspidin BB, a phloroglucinol derivative, exerts its antibacterial activity against Staphylococcus aureus by inducing the generation of reactive oxygen species, Research in Microbiology (2014), http://dx.doi.org/10.1016/j.resmic.2014.03.002
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N. Li et al. / Research in Microbiology xx (2014) 1e10
Table 2 Measurement of S. aureus dimensions after exposure to aspidin BB at different concentrations (These data were generated by AFM.). Concentration
S. aureus
Length (nm) Width (nm) Height (nm) RMS roughness (nm)
826.25 816.27 94.32 1.26
p Value
Control
1/2 MIC
13.44 8.41 16.47 0.34
931.68 861.94 85.14 3.68
1 MIC 24.35 15.30 5.38 0.17
demonstrated in Fig. 7. By comparing whole-cell protein lysates with National Center for Biotechnology information, some possible target proteins of aspidin BB against S. aureus have been suggested, such as SarA (14.718 kDa), Fib (18.765 kDa), ferritin (19.589 kDa), the ATP-dependent clp proteolytic subunit (21.514 kDa), SOD (Mn/Fe) (23.041 kDa), staphyloxanthin biosynthesis protein (33.376 kDa), the S. aureus sex pheromone (45.381 kDa) and iron-regulated surface determinant protein B (72.192 kDa) and H (100.847 kDa). They are mainly responsible for regulating virulence factors, iron metabolism
854.28 836.96 63.27 4.27
2 MIC
13.46 15.44 9.05 0.52
673.46 592.33 27.14 9.73
4 MIC
13.29 8.04 7.18 0.47
574.16 513.63 18.41 11.42
12.63 14.51 8.26 0.41
