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Membrane disruption and DNA binding of Staphylococcus aureus cell induced by a novel antimicrobial peptide produced by Lactobacillus paracasei subsp. tolerans FX-6
Food Control 59 (2016) 609e613
Contents lists available at ScienceDirect
Food Control journal homepage: www.elsevier.com/locate/foodcont
Membrane disruption and DNA binding of Staphylococcus aureus cell induced by a novel antimicrobial peptide produced by Lactobacillus paracasei subsp. tolerans FX-6 Jianyin Miao a, b, Jianliang Zhou a, Guo Liu a, Feilong Chen a, Yunjiao Chen a, Xiangyang Gao a, William Dixon b, Mingyue Song b, Hang Xiao b, **, Yong Cao a, * a b
College of Food Science, South China Agricultural University, Guangzhou 510642, PR China Department of Food Science, University of Massachusetts, Amherst, MA 01003, United States
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 March 2015 Received in revised form 18 June 2015 Accepted 20 June 2015 Available online 23 June 2015
Previously, we have isolated a novel bacteriocin, peptide F1 from Tibetan Kefir, and demonstrated its superior antimicrobial activity. However, its antimicrobial mechanism is still undefined. This study was aimed to elucidate the antimicrobial mechanism of peptide F1 against Staphylococcus aureus. The antimicrobial effects of peptide F1 were characterized by the following methods: chemical assay to quantify cytoplasmic b-galactosidase leakage, atomic absorption spectrometry to measure the released potassium ions, transmission electron microscopy to visualize the cellular morphological changes, and electrophoresis analysis and atomic force microscopy together to exam the DNA binding activity. Our results revealed that peptide F1 exerted its bactericidal effects by damaging bacterial cell membranes and by binding to the genomic DNA in the cytoplasm, which both led to rapid cell death. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Antimicrobial peptide Staphylococcus aureus Cell membrane DNA Antimicrobial mechanism
1. Introduction The microbial contamination in the food industry has been the cause of huge economic losses each year. The number of foodborne illness caused by 31 major foodborne pathogens in the United States was estimated at 9.4 million per year (Scallan, et al., 2011), costing the U.S. between 50 and 77 billion each year (Lanzas, Lu, & €hn, 2011). Additionally, there are approximately 1.6 million Gro cases of foodborne illnesses occurring related to 30 specified pathogens each year in Canada (Thomas, et al., 2013). Among the most widespread foodborne pathogenic organisms, Staphylococcus aureus is an important foodborne pathogen causing foodborne illnesses (Mariam et al., 2014; Wang, et al., 2013; Wang, et al., 2014). Conventional food safety intervention includes the use of cold chain, high salt, high sugar, acidic environments, chemical preservatives, and other modern technologies. Recently, bio-control is becoming a popular option for food safety practice because of its
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (H. Xiao), caoyong2181@scau. edu.cn (Y. Cao). http://dx.doi.org/10.1016/j.foodcont.2015.06.044 0956-7135/© 2015 Elsevier Ltd. All rights reserved.
high efficacy and label friendliness. Addition of bacteriocinproducing lactic acid bacteria or the bacteriocins produced by lactic acid bacteria to the food products as bio-preservatives have been proven to be effective in achieving bio-control in food (De Vuyst & Leroy, 2007; Jin, Zhang, & Boyd, 2010). Bacteriocins are a group of small antimicrobial peptides against other bacteria (Anastasiadou, Papagianni, Filiousis, Ambrosiadis, & Koidis, 2008; Kruger, Barbosa, Miranda, Landgraf, Destro, Todorov, et al., 2013). Nisin, the first reported bacteriocin produced by lactic acid bacteria, is an antimicrobial peptide approved for use in over 40 countries and has been used as a food biopreservative for over 50 years. Peptide F1 is a novel bacteriocin discovered in our previous study, and it is produced by Lactobacillus paracasei subsp. tolerans FX-6 isolated from Tibetan kefir (Miao et al., 2014). We demonstrated that peptide F1 has a wide antimicrobial spectrum against fungi, gram-positive and gram-negative bacteria. Moreover, peptide F1 has excellent heat stability (with standing 20 min at 121 C, and 60 min at 100 C), pH stability (pH 3.0e9.0), and resistance against protease treatment. However, the actual antimicrobial mechanisms of peptide F1 are still unknown. The aim of this study was to elucidate antimicrobial mechanisms of peptide F1 against
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S. aureus, which will contribute to expand the theoretical research on the mechanisms of antimicrobial peptides. 2. Materials and methods 2.1. Materials Peptide F1 used in this study is a bacteriocin obtained from Tibetan kefir in our previous research, and its minimal inhibition concentration (MIC) was 125 mg/mL (0.06 mol/mL) against S. aureus ATCC 63589 (Miao et al., 2014). S. aureus ATCC 63589 was stored in the microbial culture laboratory in the College of Food Science at the South China Agricultural University, Guangzhou, China. Fluorescein isothiocyanate (FITC) and o-nitrophenyl-b-D-galactopyranoside (ONPG) were purchased from SigmaeAldrich (Shanghai, China). 2.2. Cell membrane permeability assay Cell membrane permeability assay was performed by measuring the released cytoplasmic b-galactosidase from S. aureus cells into the culture medium using ONPG as the substrate as previously reported with modification (Creaser, 1955; Marri, Dallai, & Marchini, 1996; Tsuji, et al., 2001). After overnight incubation at 37 C in LuriaeBertani broth, S. aureus cultures were centrifuged at 3000 g, and the harvested cells were incubated in M9 lactose medium (1.28 g Na2HPO4, 0.3 g KH2PO4, 0.05 g NaCl, 0.1 g NH4Cl, 0.05 g MgSO4, 0.001 g CaCl2, and 0.5 g lactose were dissolved in 100 mL of double-distilled water) at 37 C for 8 h. After centrifugation at 3000 g for 1 min, bacterial cells were collected, washed twice with sterile saline, resuspended to an optical density (OD) of 0.2 at 600 nm in the assay buffer (0.8 g NaCl, 0.02 g KCl, 0.29 g Na2HPO4, 0.024 g KH2PO4, 0.025 g MgSO4, and 0.39 g b-mercaptoethanol were dissolved in 100 mL of double-distilled water), and ONPG was added to a final concentration of 0.1 mg/L. Finally peptide F1 was added to a final concentration of 1 MIC, and the cell suspension without peptide F1 was used as the control. The cell suspension was incubated at 37 C. The production of o-nitrophenol over time was measured using a microplate reader (Multiskan MK3, Thermo, USA) at 420 nm. 2.3. Measurement of the released potassium ions
The interaction between peptide F1 and genomic DNA of S. aureus was investigated by the DNA gel retardation assay and the atomic force microscopy imaging analysis. After overnight incubation at 37 C in LuriaeBertani broth, S. aureus cultures were centrifuged at 3000 g for 10 min at 4 C, and the pelleted cells were used to extract genomic DNA by a bacterial genomic DNA extraction kit (Sangon Biotech Co., Ltd, Shanghai, China). The purity of the extracted genomic DNA was evaluated by the ratio of optical density at 260 and 280 nm (OD260/OD280 1.90). The extracted DNA was dissolved in TE buffer (10 mM TriseHCl, 1 mM EDTA, and pH 8.0) at a final concentration of 3 mg/mL. In the DNA gel retardation assay, 3 mL of DNA (3 mg/mL) was mixed with increasing amounts of peptide F1 in 3 mL at 30 C for 10 min. After adding 1 mL of 6 loading buffer, the mixture was subjected to electrophoresis on a 0.8% agarose gel. Gel retardation was visualized under UV illumination using a GelDoc XR gel imaging system (Bio-Rad, USA). In the atomic force microscopy experiments, 3 mL DNA (3 mg/mL) was mixed with 3 mL of peptide F1 (50 mg/mL) at 30 C for 10 min, and then the mixture was imaged by CSPM5500 scanning probe microscope (Guangzhou Primitive Nano Instrument, China).
3. Result and discussion 3.1. Peptide F1 caused leakage of cytoplasmic content in Staphylococcus aureus cells A previous study demonstrated that the presence of lactose and galactose in the culture medium could induce S. aureus cells to produce b-galactosidase in the cytoplasm (Creaser, 1955). Generally, cytoplasmic b-galactosidase can not pass through the integral cell membrane of bacteria. However, if the cell membrane is damaged, cytoplasmic b-galactosidase can be detected extracellularly due to its leakage through damaged cell membrane. In the current study, we utilized M9 lactose medium to induce the production of cytoplasmic b-galactosidase in S. aureus. After adding peptide F1 to the culture medium, we measured the enzymatic activity of b-galactosidase. As shown in Fig. 1, the b-galactosidase activity in the culture medium was detected at 40 min after treatment with peptide F1, and its activity kept increasing during the test time period (up to 130 min). No b-galactosidase activity was detected in the culture medium of control bacterial cells that was not treated with peptide F1. These results suggested that peptide F1 increased the cell membrane permeability of S. aureus, which led to the leakage of cytoplasmic content to the culture medium.
0.0141 0.0121 0.0101 OD
S. aureus was grown overnight in LuriaeBertani broth. The bacterial cells in the exponential phase were centrifuged, washed, and resuspended (108 CFU/mL) in 0.9% sterile saline（0.9 g NaCl were dissolved in 100 mL of deionized water）. Peptide F1 at 1 MIC was added to the cell suspension. A cell suspension in 0.9% sterile saline（0.9 g NaCl were dissolved in 100 mL of deionized water） was used as the control. At various time intervals (30, 60, 90, 120, and 150 min), the suspensions were centrifuged. The supernatants were subjected to measurement by atomic absorption spectrometer (S7-AA-7000, Shimadzu, Japan).
2.5. Interaction between peptide F1 and DNA
2.4. Transmission electron microscopy 8
The S. aureus cells in the exponential phase (10 CFU/mL of LuriaeBertani broth) were treated with peptide F1 at the final concentration of 5 MIC at 37 C for three different periods (10, 30, and 60 min). Cells were pelleted by centrifugation at 3000 g for 10 min and then collected. The cell pellets were processed for transmission electron microscopy as described in a previous study (Duan, Jin, Zhang, Li, & Xiang, 2014). The ultrathin sections were examined by transmission electron microscope (Hitachi H-7000, Japan) at magnification of 11,000 .
0.0081 0.0061 0.0041 0.0021 0.0001 10
40
70
100
130
Time min Fig. 1. b-Galactosidase activity (measured from the absorbance at 420 nm) in the culture medium of Staphylococcus aureus cells treated with peptide F1. Data were expressed as mean ± standard deviation (N ¼ 3).
J. Miao et al. / Food Control 59 (2016) 609e613
potassium ions to releasing extracellularly. Other antimicrobial peptides showed similar effects on different bacteria. For example, treatments with peptides LtnA1 and LtnA2 for 30 min caused significant potassium ions to release in Lactococcus lactis subsp. cremoris HP (Morgan, O'Connor, Cotter, Ross, & Hill, 2005). Peptide P7 was reported to cause leakage of potassium ions in Salmonella typhimurium cells by inducing ion channel formation in the cell membrane (Li, Shi, Su, & Le, 2012).
0.12
mg/L
0.1
Potassium ions
611
0.08 0.06 0.04
0.02
3.2. The effects of peptide F1 on cell morphology of Staphylococcus aureus cells
0 0
30
60
90
120
150
Time(min) Fig. 2. The extracellular levels of potassium ions released by Staphylococcus aureus cells treated with peptide F1 (A) or without peptide F1 (-) over time.
To further determine the effects of peptide F1 on the integrity of cell membrane of S. aureus cells, the amount of potassium ions released by S. aureus cells were measured. As shown in Fig. 2, in the absence of peptide F1, the extracellular levels of potassium ions were relatively stable at low levels during the test period (150 min). When S. aureus cells were exposed to peptide F1, the extracellular levels of potassium ions were much higher than the control cells. Moreover, the extracellular levels of potassium ions released by S. aureus cells treated with peptide F1 kept increasing during the test time period (up to 150 min). These results demonstrated that peptide F1 had the ability to disturb the cell membrane and cause
To further characterize the bactericidal effects of peptide F1, transmission electron microscopy was used to visualize the morphological changes of S. aureus cells exposed to peptide F1 at 5 MIC. As shown in Fig. 3A, in the absence of peptide F1, the S. aureus cells showed a well-defined cell membrane and a uniform cytoplasm region. After S. aureus cells were treated with peptide F1 for 0.5 h (Fig. 3B) and 1 h (Fig. 3C), the cells gradually lose the clear boundary of cell membrane. Moreover, peptide F1 treatment significantly disturbed uniformity of the cytoplasm region, which was evident by the unevenly distributed cytoplasm materials. After 2 h of treatment with peptide F1, the S. aureus cells were lysed and lost their cellular morphology (Fig. 3D). It has been reported that antibacterial peptides can interact directly with bacteria cell membranes to increase the membrane permeability and cause rapid cell death (Li, Xiang, Zhang, Huang, & Su, 2012). Our results showed that peptide F1 caused significant damages to the cellular structures including cell membrane and cytoplasm region, which
Fig. 3. Transmission electron microscopic images of Staphylococcus aureus cells treated by peptide F1 at 5 MIC for 0 h (A), 0.5 h (B), 1 h (C) and 2 h (D), respectively. Ten different images were taken for each time point, and representative images are shown.
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eventually led to rapid lyses of bacterial cells. Other antimicrobial peptide showed similar effects on cell morphology. For example, the treatment with peptide PGLa led to the lyses of S. aureus cells (Hartmann et al., 2010). However, melittin, an antimicrobial peptide composed of 26 amino acids, had different effects on cell morphology, which caused the cell membrane forming pores to change the cell morphology of S. aureus, rather than leading to the lyses of cells (Park, et al., 2006). 3.3. PeptideF1 interacted with genomic DNA of Staphylococcus aureus cells Besides damaging the cell membrane, antimicrobial peptides may affect the biological functions of other important cellular components, such as nucleic acids and other intracellular biopolymers (Li, Shi, Cheserek, Su, & Le, 2013). To determine if peptide F1 targeted intracellular biopolymers, we characterized the interaction between peptide F1 and genomic DNA of S. aureus cells by the DNA gel retardation assay and the atomic force microscopy. Previous studies have demonstrated the effectiveness of gel retardation assay in evaluating interactions between bacterial DNA and antibacterial agents (Gottschalk, Ifrah, Lerche, Gottlieb, Cohn, Hiasa, et al., 2013; Li et al., 2013; Li et al., 2015; Subbalakshmi & Sitaram, 1998). For our study, the genomic DNA of S. aureus cells was isolated, and mixed with different amounts of peptide F1 (with the weight ratio of peptide F1/DNA was 0, 0.5/3, 5/3, 10/3, 25/3, 50/ 3, 100/3, or 200/3) were shown in Fig. 4A. With the increasing weight ratio of peptide F1/DNA, the binding effect became
progressively more pronounced. When the ratio reached 100/3 and 200/3, the DNA bands were completely retained in the sample wells and were unable to migrate into the gel. These results demonstrated that peptide F1 was able to bind bacterial genomic DNA directly. Furthermore, the results from atomic force microscopy clearly showed that peptide F1 bind to the bacterial DNA of S. aureus cells (Fig. 4B and C). Overall, our results suggested that DNA was an intracellular target of peptide F1. Previous studies have shown that the potential mechanisms of bacterial cell death caused by antimicrobial peptides mainly included cell membrane dysfunction (Hancock & Chapple, 1999), inhibition of extracellular biopolymer synthesis (Chitnis & Prasad, 1990), and inhibition of intracellular functions (del Castillo, del Castillo, & Moreno, 2001). Based on our results, peptide might attack the bacterial cell membrane first and cause damges to the cell membrane. This increased permeability of cell membrane and allowed peptide F1 to enter the bacterial cells. Peptide F1 in the cytoplasm further interacted with important intracellular targets such as DNA and alter their biological functions, which in turn caused cell lysis and cell death. 4. Conclusions In conclusion, our results demonstrated that there were at least two key mechanisms underlying the antimicrobial actions of peptide F1 against S. aureus, including the permeabilization of bacterial cell membrane and the direct binding to genomic DNA. Both mechanisms can potentially cause severe damages to bacterial cells
Fig. 4. DNA binding analysis of peptide F1 against Staphylococcus aureus DNA. (A) showed the result of the gel retardation analysis. Bands 1e8 were for the weight ratio (peptide F1/ DNA) of 0, 0.5/3, 5/3, 10/3, 25/3, 50/3, 100/3, and 200/3, respectively. (B) was the atomic force microscopy image of genomic DNA in the absence of peptide F1, and (C) was the atomic force microscopy image of genomic DNA in the presence of peptide F1. Three different images were taken for each treatment, and representative images are shown.
J. Miao et al. / Food Control 59 (2016) 609e613
and lead to cell death. Having multiple targets means that peptide F1 could be more effective in killing bacterial cells because the bacteria cells have additional hurdles to overcome to build resistance. Overall, our results indicate that peptide F1 is a promising biological preservative for the agricultural and food industry.
Acknowledgement The authors would like to express their sincere gratitude to the National Natural Science Foundation of China (No. 31171768) for the financial support of this research. References Anastasiadou, S., Papagianni, M., Filiousis, G., Ambrosiadis, I., & Koidis, P. (2008). Pediocin SA-1, an antimicrobial peptide from Pediococcus acidilactici NRRL B5627: production conditions, purification and characterization. Bioresource Technology, 99(13), 5384e5390. del Castillo, F. J., del Castillo, I., & Moreno, F. (2001). Construction and characterization of mutations at codon 751 of the Escherichia coli gyrB gene that confer resistance to the antimicrobial peptide microcin B17 and alter the activity of DNA gyrase. Journal of Bacteriology, 183(6), 2137e2140. Chitnis, S., & Prasad, K. (1990). Seminalplasmin, an antimicrobial protein from bovine seminal plasma, inhibits peptidoglycan synthesis in Escherichia coli. FEMS Microbiology Letters, 72(3), 281e284. Creaser, E. (1955). The induced (adaptive) biosynthesis of b-galactosidase in Staphylococcus aureus. Journal of General Microbiology, 12(2), 288e297. De Vuyst, L., & Leroy, F. (2007). Bacteriocins from lactic acid bacteria: production, purification, and food applications. Journal of Molecular Microbiology and Biotechnology, 13(4), 194e199. Duan, H., Jin, S., Zhang, Y., Li, F., & Xiang, J. (2014). Granulocytes of the red claw crayfish Cherax quadricarinatus can endocytose beads, E. coli and WSSV, but in different ways. Developmental & Comparative Immunology, 46(2), 186e193. Gottschalk, S., Ifrah, D., Lerche, S., Gottlieb, C. T., Cohn, M. T., Hiasa, H., et al. (2013). The antimicrobial lysine-peptoid hybrid LP5 inhibits DNA replication and induces the SOS response in Staphylococcus aureus. BMC Microbiology, 13(1), 1e8. Hancock, R. E., & Chapple, D. S. (1999). Peptide antibiotics. Antimicrobial Agents and Chemotherapy, 43(6), 1317e1323. Hartmann, M., Berditsch, M., Hawecker, J., Ardakani, M. F., Gerthsen, D., & Ulrich, A. S. (2010). Damage of the bacterial cell envelope by antimicrobial peptides gramicidin S and PGLa as revealed by transmission and scanning electron microscopy. Antimicrobial Agents and Chemotherapy, 54(8), 3132e3142. Jin, T., Zhang, H., & Boyd, G. (2010). Incorporation of preservatives in polylactic acid films for inactivating Escherichia coli O157: H7 and extending microbiological shelf life of strawberry puree. Journal of Food Protection, 73(5), 812e818. Kruger, M. F., Barbosa, M. d. S., Miranda, A., Landgraf, M., Destro, M. T., Todorov, S. D., et al. (2013). Isolation of bacteriocinogenic strain of Lactococcus lactis subsp. lactis from rocket salad (Eruca sativa Mill.) and evidences of production of a variant of nisin with modification in the leader-peptide. Food control, 33(2), 467e476.
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€hn, Y. T. (2011). Mathematical modeling of the transmission Lanzas, C., Lu, Z., & Gro and control of foodborne pathogens and antimicrobial resistance at preharvest. Foodborne Pathogens and Disease, 8(1), 1e10. Li, L., Shi, Y., Cheserek, M. J., Su, G., & Le, G. (2013). Antibacterial activity and dual mechanisms of peptide analog derived from cell-penetrating peptide against Salmonella typhimurium and Streptococcus pyogenes. Applied Microbiology and Biotechnology, 97(4), 1711e1723. Li, L., Shi, Y., Cheserek, M. J., Xia, S., Cheng, X., & Le, G. (2015). A cell-penetrating peptide analogue, P7, exerts antimicrobial activity against Escherichia coli ATCC25922 via penetrating cell membrane and targeting intracellular DNA. Food Chemistry, 166, 231e239. Li, L., Shi, Y., Su, G., & Le, G. (2012a). Selectivity for and destruction of Salmonella typhimurium via a membrane damage mechanism of a cell-penetrating peptide ppTG20 analogue. International Journal of Antimicrobial Agents, 40(4), 337e343. Li, Y., Xiang, Q., Zhang, Q., Huang, Y., & Su, Z. (2012b). Overview on the recent study of antimicrobial peptides: origins, functions, relative mechanisms and application. Peptides, 37(2), 207e215. Mariam, S. H., Zegeye, N., Tariku, T., Andarge, E., Endalafer, N., & Aseffa, A. (2014). Potential of cell-free supernatants from cultures of selected lactic acid bacteria and yeast obtained from local fermented foods as inhibitors of Listeria monocytogenes, Salmonella spp. and Staphylococcus aureus. BMC Research Notes, 7(1), 606. Marri, L., Dallai, R., & Marchini, D. (1996). The novel antibacterial peptide ceratotoxin A alters permeability of the inner and outer membrane of Escherichia coli K-12. Current Microbiology, 33(1), 40e43. Miao, J., Guo, H., Ou, Y., Liu, G., Fang, X., Liao, Z., et al. (2014). Purification and characterization of bacteriocin F1, a novel bacteriocin produced by Lactobacillus paracasei subsp. tolerans FX-6 from Tibetan kefir, a traditional fermented milk from Tibet, China. Food Control, 42, 48e53. Morgan, S. M., O'Connor, P. M., Cotter, P. D., Ross, R. P., & Hill, C. (2005). Sequential actions of the two component peptides of the lantibiotic lacticin 3147 explain its antimicrobial activity at nanomolar concentrations. Antimicrobial Agents and Chemotherapy, 49(7), 2606e2611. Park, S.-C., Kim, J.-Y., Shin, S.-O., Jeong, C.-Y., Kim, M.-H., Shin, S. Y., et al. (2006). Investigation of toroidal pore and oligomerization by melittin using transmission electron microscopy. Biochemical and Biophysical Research Communications, 343(1), 222e228. Scallan, E., Hoekstra, R. M., Angulo, F. J., Tauxe, R. V., Widdowson, M.-A., Roy, S. L., et al. (2011). Foodborne illness acquired in the United Statesdmajor pathogens. Emerging Infectious Diseases, 17(1). Subbalakshmi, C., & Sitaram, N. (1998). Mechanism of antimicrobial action of indolicidin. FEMS Microbiology Letters, 160(1), 91e96. Thomas, M. K., Murray, R., Flockhart, L., Pintar, K., Pollari, F., Fazil, A., et al. (2013). Estimates of the burden of foodborne illness in Canada for 30 specified pathogens and unspecified agents, circa 2006. Foodborne Pathogens and Disease, 10(7), 639e648. Tsuji, Y., Aoyama, T., Takeuchi, K., Homma, K.-i., Takahashi, H., Nakajima, Y., et al. (2001). Identification and characterization of an antibacterial peptide of the 26kDa protease of Sarcophaga peregrina with antibacterial activity. Journal of Biochemistry, 130(2), 313e318. Wang, Y., Chen, Q., Cui, S., Xu, X., Zhu, J., Luo, H., et al. (2014). Enumeration and characterization of Salmonella isolates from retail chicken carcasses in Beijing, China. Foodborne Pathogens and Disease, 11(2), 126e132. Wang, X., Tao, X., Xia, X., Yang, B., Xi, M., Meng, J., et al. (2013). Staphylococcus aureus and methicillin-resistant Staphylococcus aureus in retail raw chicken in China. Food Control, 29(1), 103e106.
Contents lists available at ScienceDirect
Food Control journal homepage: www.elsevier.com/locate/foodcont
Membrane disruption and DNA binding of Staphylococcus aureus cell induced by a novel antimicrobial peptide produced by Lactobacillus paracasei subsp. tolerans FX-6 Jianyin Miao a, b, Jianliang Zhou a, Guo Liu a, Feilong Chen a, Yunjiao Chen a, Xiangyang Gao a, William Dixon b, Mingyue Song b, Hang Xiao b, **, Yong Cao a, * a b
College of Food Science, South China Agricultural University, Guangzhou 510642, PR China Department of Food Science, University of Massachusetts, Amherst, MA 01003, United States
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 March 2015 Received in revised form 18 June 2015 Accepted 20 June 2015 Available online 23 June 2015
Previously, we have isolated a novel bacteriocin, peptide F1 from Tibetan Kefir, and demonstrated its superior antimicrobial activity. However, its antimicrobial mechanism is still undefined. This study was aimed to elucidate the antimicrobial mechanism of peptide F1 against Staphylococcus aureus. The antimicrobial effects of peptide F1 were characterized by the following methods: chemical assay to quantify cytoplasmic b-galactosidase leakage, atomic absorption spectrometry to measure the released potassium ions, transmission electron microscopy to visualize the cellular morphological changes, and electrophoresis analysis and atomic force microscopy together to exam the DNA binding activity. Our results revealed that peptide F1 exerted its bactericidal effects by damaging bacterial cell membranes and by binding to the genomic DNA in the cytoplasm, which both led to rapid cell death. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Antimicrobial peptide Staphylococcus aureus Cell membrane DNA Antimicrobial mechanism
1. Introduction The microbial contamination in the food industry has been the cause of huge economic losses each year. The number of foodborne illness caused by 31 major foodborne pathogens in the United States was estimated at 9.4 million per year (Scallan, et al., 2011), costing the U.S. between 50 and 77 billion each year (Lanzas, Lu, & €hn, 2011). Additionally, there are approximately 1.6 million Gro cases of foodborne illnesses occurring related to 30 specified pathogens each year in Canada (Thomas, et al., 2013). Among the most widespread foodborne pathogenic organisms, Staphylococcus aureus is an important foodborne pathogen causing foodborne illnesses (Mariam et al., 2014; Wang, et al., 2013; Wang, et al., 2014). Conventional food safety intervention includes the use of cold chain, high salt, high sugar, acidic environments, chemical preservatives, and other modern technologies. Recently, bio-control is becoming a popular option for food safety practice because of its
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (H. Xiao), caoyong2181@scau. edu.cn (Y. Cao). http://dx.doi.org/10.1016/j.foodcont.2015.06.044 0956-7135/© 2015 Elsevier Ltd. All rights reserved.
high efficacy and label friendliness. Addition of bacteriocinproducing lactic acid bacteria or the bacteriocins produced by lactic acid bacteria to the food products as bio-preservatives have been proven to be effective in achieving bio-control in food (De Vuyst & Leroy, 2007; Jin, Zhang, & Boyd, 2010). Bacteriocins are a group of small antimicrobial peptides against other bacteria (Anastasiadou, Papagianni, Filiousis, Ambrosiadis, & Koidis, 2008; Kruger, Barbosa, Miranda, Landgraf, Destro, Todorov, et al., 2013). Nisin, the first reported bacteriocin produced by lactic acid bacteria, is an antimicrobial peptide approved for use in over 40 countries and has been used as a food biopreservative for over 50 years. Peptide F1 is a novel bacteriocin discovered in our previous study, and it is produced by Lactobacillus paracasei subsp. tolerans FX-6 isolated from Tibetan kefir (Miao et al., 2014). We demonstrated that peptide F1 has a wide antimicrobial spectrum against fungi, gram-positive and gram-negative bacteria. Moreover, peptide F1 has excellent heat stability (with standing 20 min at 121 C, and 60 min at 100 C), pH stability (pH 3.0e9.0), and resistance against protease treatment. However, the actual antimicrobial mechanisms of peptide F1 are still unknown. The aim of this study was to elucidate antimicrobial mechanisms of peptide F1 against
610
J. Miao et al. / Food Control 59 (2016) 609e613
S. aureus, which will contribute to expand the theoretical research on the mechanisms of antimicrobial peptides. 2. Materials and methods 2.1. Materials Peptide F1 used in this study is a bacteriocin obtained from Tibetan kefir in our previous research, and its minimal inhibition concentration (MIC) was 125 mg/mL (0.06 mol/mL) against S. aureus ATCC 63589 (Miao et al., 2014). S. aureus ATCC 63589 was stored in the microbial culture laboratory in the College of Food Science at the South China Agricultural University, Guangzhou, China. Fluorescein isothiocyanate (FITC) and o-nitrophenyl-b-D-galactopyranoside (ONPG) were purchased from SigmaeAldrich (Shanghai, China). 2.2. Cell membrane permeability assay Cell membrane permeability assay was performed by measuring the released cytoplasmic b-galactosidase from S. aureus cells into the culture medium using ONPG as the substrate as previously reported with modification (Creaser, 1955; Marri, Dallai, & Marchini, 1996; Tsuji, et al., 2001). After overnight incubation at 37 C in LuriaeBertani broth, S. aureus cultures were centrifuged at 3000 g, and the harvested cells were incubated in M9 lactose medium (1.28 g Na2HPO4, 0.3 g KH2PO4, 0.05 g NaCl, 0.1 g NH4Cl, 0.05 g MgSO4, 0.001 g CaCl2, and 0.5 g lactose were dissolved in 100 mL of double-distilled water) at 37 C for 8 h. After centrifugation at 3000 g for 1 min, bacterial cells were collected, washed twice with sterile saline, resuspended to an optical density (OD) of 0.2 at 600 nm in the assay buffer (0.8 g NaCl, 0.02 g KCl, 0.29 g Na2HPO4, 0.024 g KH2PO4, 0.025 g MgSO4, and 0.39 g b-mercaptoethanol were dissolved in 100 mL of double-distilled water), and ONPG was added to a final concentration of 0.1 mg/L. Finally peptide F1 was added to a final concentration of 1 MIC, and the cell suspension without peptide F1 was used as the control. The cell suspension was incubated at 37 C. The production of o-nitrophenol over time was measured using a microplate reader (Multiskan MK3, Thermo, USA) at 420 nm. 2.3. Measurement of the released potassium ions
The interaction between peptide F1 and genomic DNA of S. aureus was investigated by the DNA gel retardation assay and the atomic force microscopy imaging analysis. After overnight incubation at 37 C in LuriaeBertani broth, S. aureus cultures were centrifuged at 3000 g for 10 min at 4 C, and the pelleted cells were used to extract genomic DNA by a bacterial genomic DNA extraction kit (Sangon Biotech Co., Ltd, Shanghai, China). The purity of the extracted genomic DNA was evaluated by the ratio of optical density at 260 and 280 nm (OD260/OD280 1.90). The extracted DNA was dissolved in TE buffer (10 mM TriseHCl, 1 mM EDTA, and pH 8.0) at a final concentration of 3 mg/mL. In the DNA gel retardation assay, 3 mL of DNA (3 mg/mL) was mixed with increasing amounts of peptide F1 in 3 mL at 30 C for 10 min. After adding 1 mL of 6 loading buffer, the mixture was subjected to electrophoresis on a 0.8% agarose gel. Gel retardation was visualized under UV illumination using a GelDoc XR gel imaging system (Bio-Rad, USA). In the atomic force microscopy experiments, 3 mL DNA (3 mg/mL) was mixed with 3 mL of peptide F1 (50 mg/mL) at 30 C for 10 min, and then the mixture was imaged by CSPM5500 scanning probe microscope (Guangzhou Primitive Nano Instrument, China).
3. Result and discussion 3.1. Peptide F1 caused leakage of cytoplasmic content in Staphylococcus aureus cells A previous study demonstrated that the presence of lactose and galactose in the culture medium could induce S. aureus cells to produce b-galactosidase in the cytoplasm (Creaser, 1955). Generally, cytoplasmic b-galactosidase can not pass through the integral cell membrane of bacteria. However, if the cell membrane is damaged, cytoplasmic b-galactosidase can be detected extracellularly due to its leakage through damaged cell membrane. In the current study, we utilized M9 lactose medium to induce the production of cytoplasmic b-galactosidase in S. aureus. After adding peptide F1 to the culture medium, we measured the enzymatic activity of b-galactosidase. As shown in Fig. 1, the b-galactosidase activity in the culture medium was detected at 40 min after treatment with peptide F1, and its activity kept increasing during the test time period (up to 130 min). No b-galactosidase activity was detected in the culture medium of control bacterial cells that was not treated with peptide F1. These results suggested that peptide F1 increased the cell membrane permeability of S. aureus, which led to the leakage of cytoplasmic content to the culture medium.
0.0141 0.0121 0.0101 OD
S. aureus was grown overnight in LuriaeBertani broth. The bacterial cells in the exponential phase were centrifuged, washed, and resuspended (108 CFU/mL) in 0.9% sterile saline（0.9 g NaCl were dissolved in 100 mL of deionized water）. Peptide F1 at 1 MIC was added to the cell suspension. A cell suspension in 0.9% sterile saline（0.9 g NaCl were dissolved in 100 mL of deionized water） was used as the control. At various time intervals (30, 60, 90, 120, and 150 min), the suspensions were centrifuged. The supernatants were subjected to measurement by atomic absorption spectrometer (S7-AA-7000, Shimadzu, Japan).
2.5. Interaction between peptide F1 and DNA
2.4. Transmission electron microscopy 8
The S. aureus cells in the exponential phase (10 CFU/mL of LuriaeBertani broth) were treated with peptide F1 at the final concentration of 5 MIC at 37 C for three different periods (10, 30, and 60 min). Cells were pelleted by centrifugation at 3000 g for 10 min and then collected. The cell pellets were processed for transmission electron microscopy as described in a previous study (Duan, Jin, Zhang, Li, & Xiang, 2014). The ultrathin sections were examined by transmission electron microscope (Hitachi H-7000, Japan) at magnification of 11,000 .
0.0081 0.0061 0.0041 0.0021 0.0001 10
40
70
100
130
Time min Fig. 1. b-Galactosidase activity (measured from the absorbance at 420 nm) in the culture medium of Staphylococcus aureus cells treated with peptide F1. Data were expressed as mean ± standard deviation (N ¼ 3).
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potassium ions to releasing extracellularly. Other antimicrobial peptides showed similar effects on different bacteria. For example, treatments with peptides LtnA1 and LtnA2 for 30 min caused significant potassium ions to release in Lactococcus lactis subsp. cremoris HP (Morgan, O'Connor, Cotter, Ross, & Hill, 2005). Peptide P7 was reported to cause leakage of potassium ions in Salmonella typhimurium cells by inducing ion channel formation in the cell membrane (Li, Shi, Su, & Le, 2012).
0.12
mg/L
0.1
Potassium ions
611
0.08 0.06 0.04
0.02
3.2. The effects of peptide F1 on cell morphology of Staphylococcus aureus cells
0 0
30
60
90
120
150
Time(min) Fig. 2. The extracellular levels of potassium ions released by Staphylococcus aureus cells treated with peptide F1 (A) or without peptide F1 (-) over time.
To further determine the effects of peptide F1 on the integrity of cell membrane of S. aureus cells, the amount of potassium ions released by S. aureus cells were measured. As shown in Fig. 2, in the absence of peptide F1, the extracellular levels of potassium ions were relatively stable at low levels during the test period (150 min). When S. aureus cells were exposed to peptide F1, the extracellular levels of potassium ions were much higher than the control cells. Moreover, the extracellular levels of potassium ions released by S. aureus cells treated with peptide F1 kept increasing during the test time period (up to 150 min). These results demonstrated that peptide F1 had the ability to disturb the cell membrane and cause
To further characterize the bactericidal effects of peptide F1, transmission electron microscopy was used to visualize the morphological changes of S. aureus cells exposed to peptide F1 at 5 MIC. As shown in Fig. 3A, in the absence of peptide F1, the S. aureus cells showed a well-defined cell membrane and a uniform cytoplasm region. After S. aureus cells were treated with peptide F1 for 0.5 h (Fig. 3B) and 1 h (Fig. 3C), the cells gradually lose the clear boundary of cell membrane. Moreover, peptide F1 treatment significantly disturbed uniformity of the cytoplasm region, which was evident by the unevenly distributed cytoplasm materials. After 2 h of treatment with peptide F1, the S. aureus cells were lysed and lost their cellular morphology (Fig. 3D). It has been reported that antibacterial peptides can interact directly with bacteria cell membranes to increase the membrane permeability and cause rapid cell death (Li, Xiang, Zhang, Huang, & Su, 2012). Our results showed that peptide F1 caused significant damages to the cellular structures including cell membrane and cytoplasm region, which
Fig. 3. Transmission electron microscopic images of Staphylococcus aureus cells treated by peptide F1 at 5 MIC for 0 h (A), 0.5 h (B), 1 h (C) and 2 h (D), respectively. Ten different images were taken for each time point, and representative images are shown.
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eventually led to rapid lyses of bacterial cells. Other antimicrobial peptide showed similar effects on cell morphology. For example, the treatment with peptide PGLa led to the lyses of S. aureus cells (Hartmann et al., 2010). However, melittin, an antimicrobial peptide composed of 26 amino acids, had different effects on cell morphology, which caused the cell membrane forming pores to change the cell morphology of S. aureus, rather than leading to the lyses of cells (Park, et al., 2006). 3.3. PeptideF1 interacted with genomic DNA of Staphylococcus aureus cells Besides damaging the cell membrane, antimicrobial peptides may affect the biological functions of other important cellular components, such as nucleic acids and other intracellular biopolymers (Li, Shi, Cheserek, Su, & Le, 2013). To determine if peptide F1 targeted intracellular biopolymers, we characterized the interaction between peptide F1 and genomic DNA of S. aureus cells by the DNA gel retardation assay and the atomic force microscopy. Previous studies have demonstrated the effectiveness of gel retardation assay in evaluating interactions between bacterial DNA and antibacterial agents (Gottschalk, Ifrah, Lerche, Gottlieb, Cohn, Hiasa, et al., 2013; Li et al., 2013; Li et al., 2015; Subbalakshmi & Sitaram, 1998). For our study, the genomic DNA of S. aureus cells was isolated, and mixed with different amounts of peptide F1 (with the weight ratio of peptide F1/DNA was 0, 0.5/3, 5/3, 10/3, 25/3, 50/ 3, 100/3, or 200/3) were shown in Fig. 4A. With the increasing weight ratio of peptide F1/DNA, the binding effect became
progressively more pronounced. When the ratio reached 100/3 and 200/3, the DNA bands were completely retained in the sample wells and were unable to migrate into the gel. These results demonstrated that peptide F1 was able to bind bacterial genomic DNA directly. Furthermore, the results from atomic force microscopy clearly showed that peptide F1 bind to the bacterial DNA of S. aureus cells (Fig. 4B and C). Overall, our results suggested that DNA was an intracellular target of peptide F1. Previous studies have shown that the potential mechanisms of bacterial cell death caused by antimicrobial peptides mainly included cell membrane dysfunction (Hancock & Chapple, 1999), inhibition of extracellular biopolymer synthesis (Chitnis & Prasad, 1990), and inhibition of intracellular functions (del Castillo, del Castillo, & Moreno, 2001). Based on our results, peptide might attack the bacterial cell membrane first and cause damges to the cell membrane. This increased permeability of cell membrane and allowed peptide F1 to enter the bacterial cells. Peptide F1 in the cytoplasm further interacted with important intracellular targets such as DNA and alter their biological functions, which in turn caused cell lysis and cell death. 4. Conclusions In conclusion, our results demonstrated that there were at least two key mechanisms underlying the antimicrobial actions of peptide F1 against S. aureus, including the permeabilization of bacterial cell membrane and the direct binding to genomic DNA. Both mechanisms can potentially cause severe damages to bacterial cells
Fig. 4. DNA binding analysis of peptide F1 against Staphylococcus aureus DNA. (A) showed the result of the gel retardation analysis. Bands 1e8 were for the weight ratio (peptide F1/ DNA) of 0, 0.5/3, 5/3, 10/3, 25/3, 50/3, 100/3, and 200/3, respectively. (B) was the atomic force microscopy image of genomic DNA in the absence of peptide F1, and (C) was the atomic force microscopy image of genomic DNA in the presence of peptide F1. Three different images were taken for each treatment, and representative images are shown.
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and lead to cell death. Having multiple targets means that peptide F1 could be more effective in killing bacterial cells because the bacteria cells have additional hurdles to overcome to build resistance. Overall, our results indicate that peptide F1 is a promising biological preservative for the agricultural and food industry.
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