The rapid emergence of antibiotic resistance and microbial contamination in recent years has become a crucial problem worldwide in food safety as well as in food industries [1
]. Thus, the development of new classes of antimicrobial agents has become a primary focus of many researchers [4
]. Antimicrobial peptides (AMPs), also called host defense peptides, are primordial constituents of the innate immune system found in eukaryotic organisms and indicate broad-spectrum antimicrobial activity against microorganisms like viruses, bacteria, parasites, and fungi [5
]. Moreover, antimicrobial peptides have been isolated from a wide range of sources including animals, plants, and bacteria [8
]. Most of the AMPs show common characteristics because of having a positive charge. For example, AMPs generally consist of 12–50 amino acids, with 2–9 cationic residues and up to 50% hydrophobic amino acids [9
]. Additionally, AMPs have low molecular weights, ranging from approximately 1 to 5 kDa [11
]; these features (net charge and size) are important properties of peptides that allow AMPs to bind to or insert themselves into the membranes of bacterial [12
], causing damage to bacterial membranes that lead to cell death [2
]. Therefore, AMPs are presently receiving significant attention as potential alternatives to conventional antibiotics [13
]. However, some AMPs are also cytotoxic to mammalian cells, which may limit the direct use of these peptides as therapeutics [15
In recent years, AMPs, including various protein-derived peptides, have been identified from a diverse range of food sources such as milk products, cereals, soybeans, and fish, owing to their low hemolysis and toxicity [16
]. AMPs from bovine milk proteins have been studied widely, and many AMPs have been shown to be generated by hydrolysis of milk proteins, particularly, bovine casein [18
]. The earliest milk-derived antibacterial peptides were obtained by enzymatic hydrolysis of αs1-casein by chymotrypsin to obtain antibacterial properties. Protein-derived peptides could have potential applications as food biopreservatives [19
]. For example, the protein-derived peptide Cp1 (LRLKKYKVPQL) was obtained from the hydrolysis of bovine αS1
-casein, which intercepted the 11-amino acid of αS1-casein and showed higher antibacterial activity, as reported by McCann et al. [20
] and Tang et al. [21
]. Cp1 may have applications as a potential antimicrobial food biopreservative owing to low molecular mass and high antimicrobial activity compared with that of other protein-derived peptides. Although the isolation and characterization of Cp1 have been previously reported [21
], the hemolytic activity, low cytotoxicity against human cells, and the exact mechanism by which the Cp1 exerts its antimicrobial effects have not been described. Moreover, the general antimicrobial mechanisms of protein-derived peptides have not been elucidated.
Thus, in order to resolve the problem of antibiotic resistance, which is produced by multicellular organisms as a defense mechanism against antibiotics, AMPs appear to be excellent candidates. Therefore, the objective of this study was to evaluate and examine the antimicrobial activity, hemolytic activity, low cytotoxicity against human cells, sensitivity, and exact mechanisms of Cp1 in order to improve our understanding of the antimicrobial mechanisms of protein-derived peptides and provide evidence for protein-derived peptides as a food biopreservative. Moreover, this study focused on obtaining more evidence to support the application of αS1-casein antimicrobial peptides as a biopreservative food byproduct as well as in the food industry.
Adoption of secondary structure in the membrane-mimicking milieu is thought to account for the antimicrobial activity and cytotoxicity of AMPs. In this study, we found that the secondary structure of Cp1 in different environments showed an unordered conformation, indicating that the mechanism through which Cp1 disrupted cell membranes was random coil formation. Moreover, although melittin in sodium phosphate buffer exhibited an unordered conformation, it showed an α-helix structure in the membrane-like environment of TFE and SDS solutions, confirming that the structure of melittin disrupted cell membranes through formation of α-helix structures as the buffer changed from sodium phosphate buffer to TFE and SDS, similar to the results described by Pandey et al. [24
] and Asthana et al. [25
]. Ma et al. [26
] reported that α-helical AMPs have higher antimicrobial activity, which may explain why the antimicrobial activity of Cp1 was lower than that of melittin. However, the helicity of AMPs is associated with hemolytic activity [27
], which may be why the hemolytic activity of Cp1 was lower than that of melittin. Oren et al. [29
] also found that melittin diastereomers lost their α-helical structure, abrogating their hemolytic activity toward human RBCs.
As reported in previous studies, most naturally occurring AMPs are cationic [30
]. Cationic AMPs have a net positive charge due to the absence of acidic residues (glutamate or aspartate) and excess of cationic (arginine, lysine, or histidine) residues and the presence of approximately 30–50% hydrophobic residues [31
]. The bacterial membrane contains zwitterionic phospholipids and carries a slight negative charge, which would attract cationic AMPs, allowing them to insert into membranes at the membrane interface, leading to increased membrane permeability and subsequent cell death [32
]. Cationic residues can increase electrostatic interactions between negatively charged surface of the bacterial membrane and peptides [34
]; the increasing hydrophobicity of AMPs facilitates their insertion into the membrane lipid bilayer within a certain range [36
]. In this study, Cp1 was characterized as a cationic AMP having four cationic (arginine and lysine) residues and five hydrophobic (leucine, valine, and proline) residues. As observed in antimicrobial assays, Cp1 had broad-spectrum antimicrobial activity against gram-positive and gram-negative bacteria in the range from 64 to 640 μM. McCann et al. [20
] reported that Cp1 exhibits MICs ranging from 90 to greater than 720 μM against various gram-negative and gram-positive bacteria; these MICs were slightly lower than those in our current study, possibly because of differences in peptide purity and bacterial strains. Melittin exhibited higher antimicrobial activity against all bacterial strains, which is similar to reports by Asthana et al. [25
] and Zhu et al. [38
]. Although the antimicrobial activity of Cp1 was lower than that of melittin, Cp1 had higher antimicrobial activity than other protein-derived peptides, such as isracidin and VYQHQKAMKPWIQPKTKVIPYVRYL, originating from αs1
-casein and αs2
-casein, with MICs of 100–1000 and 332–4664 μg/mL, respectively [20
Hemolysis and cytotoxicity are major parameters used to assess peptide toxicity in mammalian cells [37
]. In this study, we found that the hemolytic activities and cytotoxicity of Cp1 were low, such that Cp1 had high cell selectivity. These results indicated that this peptide could be developed as a promising food biopreservative. Notably, the effects of cations such as Na+
can hamper the electrostatic interactions between peptides and membranes of bacteria and may therefore affect AMP activity [40
]. Many studies have reported that some cations decrease the antimicrobial activity of peptides. For example, Bellamy et al. [43
] found that the antibacterial activity of lactoferricin B was decreased in the presence of Na+
, or Ca2+
ions. Human cathelicidin LL-37 and pleurocidin isolated from Pleuronectes americanus
are also salt sensitive [44
]. In this study, we found that Ca2+
exhibited a higher antagonistic effect on Cp1 against E. coli
ATCC 25922 and L. monocytogenes
CMCC 54004 at the physiological concentration. Mg2+
also inhibited the activity of Cp1 against E. coli
ATCC 25922 and L. monocytogenes
CMCC 54004, respectively. However, the results showed that salts did not affect the antimicrobial activity of melittin. A previous study demonstrated that the inhibition of antibacterial activity by Mg2+
was lower than that by Ca2+
], consistent with our current findings. However, we found that two monovalent cations (Na+
) did not affect the antimicrobial activity of Cp1 when used at their physiological concentrations. This may be due to the net positive charge, which could slightly decrease the effects of Na+
on electrostatic interactions [46
]. Moreover, divalent cations interfere with opposition for membrane binding between peptides and cations and can increase membrane rigidity [47
]. Taken together, our findings suggested that Cp1 may have lower sensitivity to salts, making it particularly promising for use as a food biopreservative.
Previous studies have shown that the predominant antimicrobial mechanism of cationic AMPs lies in membrane destabilization or transmembrane pore formation [48
]. Phosphatidylglycerol (PG), cardiolipin (CL), and phosphatidylserine (PS) are predominant components in the bacterial membrane, maintaining the negative net charge and promoting the binding of cationic AMPs [49
]. Then, AMPs perturb the lipid bilayer through membrane permeabilization, possibly by surface insertion, followed by membrane thinning [51
], and disrupt the bacterial cell membrane, causing outflow of cytoplasmic contents and ultimate cell death [37
]. Membrane permeabilization is the most essential characteristic of peptide-membrane interactions and plays an important role in measuring the activity of cationic AMPs [38
]. In this study, we also found that Cp1 and melittin had concentration-dependent effects on the permeabilization of the outer membrane of E. coli
UB1005. Both Cp1 and melittin had the ability to permeabilize the inner membrane to ONPG at 1× MIC and 1/2 MIC, indicative of strong membrane-permeabilizing ability, with concentration-dependent effects. From flow cytometric analysis, we found that Cp1 and melittin killed bacteria by damaging cytoplasmic membrane integrity. Our SEM and TEM results further confirmed that Cp1 and melittin had potent interactions with the membrane structure, disrupted the cell membrane, and allowed the intracellular contents of the bacterial cells to leak out through the membrane. Taken together, these results demonstrated that Cp1 and melittin caused damage to the cytoplasmic membrane.
4. Materials and Methods
The peptide Cp1 and melittin were purchased and synthesized by GL Biochem (Shanghai, China) and identified via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS, Linear Scientific Inc., Duquesne, PA, USA), using a-cyano-4-hydroxycinnamic acid (HCCA) as the matrix. The purity of peptide was confirmed to be greater than 95% by analytical reversed-phase high-performance liquid chromatography (RP-HPLC) (LC 3000, Beijing, China). By using electrospray ionization mass spectrometry (ESI-MS) analysis, tentative molecular masses of the peptides were assessed.
Essential sequences, mean hydrophobicity, hydrophobic moment values, and helical wheel projections were evaluated using the methods of Zhu et al. [38
]. Before assessments, peptide was liquefied in deionized water at a concentration of 2.56 mM.
E.coli ATCC 25922, E. coli UB1005, S. pullorum C7913, S. Typhimurium CMCC 50071, Staphylococcus aureus ATCC 29213, and Listeria monocytogenes CMCC 54004 were obtained from Harbin Veterinary Research Institute, CAAS (Harbin, China). From healthy blood donors red blood cells were extracted. The human embryonic kidney epithelial 293 cells used in this study were also obtained from Harbin Veterinary Research Institute, CAAS.
Mueller–Hilton broth (MHB) and Mueller–Hilton Agar (MHA) powder were bought from AoBoX (Beijing, China). Sodium dodecyl sulfate (SDS), trifluoroethyl alcohol (TFE), Triton X-100, o-nitrophenyl-b-d-galactopyranoside (ONPG), 3,30-dipropylthiadicarbocyanine (diSC3-5), HEPES, N-phenyl-1-napthylamine (NPN), propidium iodide (PI) were obtained from Sigma-Aldrich (Shanghai, China). NaCl, KCl, NH4Cl, MgCl2 and FeCl3 were all of analytical grade and obtained from Kermel (Tianjin, China).
4.1. Antimicrobial Assay
As described previously, by the CLSI broth microdilution method the MICs of the peptides were measured [53
]. Concisely, the bacteria were diluted to a final concentration of 105
CFU/mL in MHB. Two-fold serial dilutions of peptides in 12 serial 96-well plates were diluted in bovine serum albumin (BSA; 0.2% with 0.01% acetic acid). Fifty microliters of diluted bacteria were added to 50 μL BSA containing two-fold serial dilutions of peptides in each well. For a period of 18 h the plates were incubated at 37 °C. The minimum peptide concentration (MIC) was defined as the point at which no bacterial growth was observed.
4.2. Hemolytic Activity
For the measurement of hemolytic activity through the method of Stark et al., each peptide was measured [55
]. Briefly, fresh human RBCs were obtained and stored at 4 °C. One milliliter of human RBCs was washed three times and resuspended in 10 PBS, and 50 μL of the human red blood cell solution were added to 50 μL PBS containing two-fold serial dilutions of peptides in each well. For 1 h the plates were kept in an incubator at 37 °C. By centrifugation and removal of supernatants, the discharge of hemoglobin was determined by calculating the absorbance using a microplate reader. As negative and positive controls, erythrocyte suspensions in PBS and 0.1% Triton X-100, were used separately.
The cytotoxicity of each peptide was determined in 293 cells using MTT assays, as reported previously [56
]. The preparation and storage methods for MTT and cell suspensions were previously described by Schmidtchen et al. [57
]. Briefly, 293 cells were incubated overnight in 96-well plates with 10% fetal calf serum and DMEM at 37 °C in 5% CO2
. By using DMEM two-fold serial dilutions of peptides were prepared. AMPs added on the plates and subjected to incubator for 20–24 h at 37 °C containing 5% CO2
. Next, 40 μL MTT solution was added to every well, and plates were subjected to incubator for 4 h at 37 °C. Finally, 150 μL DMSO added to the wells, for 10 min the plates were shaken, and absorbance was measured by using a microplate reader at 492 nm.
4.4. Salt Sensitivity Assays
For checking salt sensitivity standard methods was adopted. Salts can affect the MIC values of peptides. Therefore, the salt sensitivities of the peptide was dignified using the way reported by Zhu et al. [38
], with modifications. To inspect the effect of each salt on the antibacterial activities of the peptides different concentrations of physiological salts (150 mM NaCl, 4.5 mM KCl, 6 μM NH4
Cl, 1 mM MgCl2
, 2 mM CaCl2
, and 4 μM FeCl3
) were added to the incubation buffer. The same step was repeated for each peptide and findings were noted.
4.5. Circular Dichroism (CD) Measurements
To inspect the secondary structures of the peptides CD spectra were noted using a Jasco J-810 Spectropolarimeter with a 1-mm quartz cuvette. The peptides were dissolved in 10 mM PBS (pH 7.4), 50% TFE, and 30 mM SDS micelles. Spectra were monitored at standard temperature and speed, i.e., at 25 °C and noted in the range of 190 to 250 nm [11
4.6. Outer Membrane Permeability Assay
The standard procedure was adopted for the assay, as given in the company booklet. The major steps were: By calculating integration of the fluorescent dye NPN into the outer membrane of E. coli
UB1005, the ability of Cp1 and melittin to improve the outer membrane permeability of gram-negative bacteria was determined as described by Xu et al. [49
] and Subbalakshmi et al. [58
]. Briefly, the basic steps for assay were: cultures of E. coli
UB1005 were diluted in MHB medium at 37 °C and grown to an OD600
of 0.2 in HEPES buffer overnight. The cell suspension was then mixed with 1 mM NPN, and the background fluorescence was noted at standard values of wavelengths until there was no additional increase in fluorescence.
4.7. Inner Membrane Permeability Assay
The standard procedure was adopted for the detection of permeability of inner membrane. The major steps were: By measuring β-galactosidase activity utilizing ONPG as a substrate, the permeability of the inner membrane of E. coli
UB1005 by the peptides was determined [27
]. E. coli
UB1005 was cultured in MHB (containing 2% lactose) until reaching the logarithmic phase and obtained by centrifugation (5000× g
, 5 min). The bacteria were diluted to an OD600
of 0.05 with 10 mM PBS (containing 1.5 mM ONPG). Different amounts of peptide (1× MIC and 1/2 MIC) were added and data were recorded every 2 min from 0 to 30 min at OD420
4.8. Cytoplasmic Membrane Depolarization Assay
The steps for depolarization assay were performed according to standard procedure as given in company booklet. By using the dye diSC3-5, by modifying of the method of Friedrich et al., the depolarization activity of the cytoplasmic membrane of E. coli
UB1005 by the peptides was noted [60
], as described by Dong et al. [46
]. E. coli
UB1005 cells in the logarithmic phase were diluted with 5 mM HEPES (containing 20 mM glucose), and 0.4 mM diSC3
-5 was added. KCl was then added to a concentration of 100 mM. Different amounts of peptide were added, and the fluorescence intensity was monitored from 0 to 600 s.
4.9. Flow Cytometry
For analysis of membrane integrity after peptide treatment, E. coli
ATCC 25922 cells in the mid-log phase in MHB medium under continuous trembling at 200 rpm were resuspended in PBS (pH 7.2) to an OD600
of 0.2. The next step was mixing with peptides at 1 × MIC and incubated for 30 min at 37 ℃ with constant shaking (140 rpm). The control sample lacked the peptide. The bacterial cells were gained by centrifugation and then washed with PBS, subjected to an incubator with the bacterial suspension at a fixed PI concentration of 10 mg/mL for 30 min at 4 ℃ and then washed with superfluous PBS. Data were recorded using a FACScan instrument (Becton-Dickinson, San Jose, CA, USA) [61
4.10. Scanning Electron Microscopy (SEM)
As described previously by Juba et al., SEM sample preparation was carried out [62
], with modifications. E. coli
ATCC 25922 cells in the mid-log phase in MHB medium under continuous shaking at 200 rpm were resuspended in PBS (pH 7.2) to an OD600
of 0.2. The bacterial cells were subjected to incubator at 37 °C under constant shaking at 200 rpm for 2 h with Cp1 or melittin at 1 × MIC. The control sample lacked the peptide. The bacterial cells were gained by centrifugation and washed with PBS, then fixed with 2.5% glutaraldehyde. Next, the samples were washed with PBS and dehydrated for 15 min in a graded ethanol (50%, 70%, 90%, and 100%). Finally, the bacterial specimens were observed by SEM after lyophilization and gold coating (Hitachi S-4800; Hitachi, Tokyo, Japan).
4.11. Transmission Electron Microscopy (TEM)
The standard procedure for TEM was adopted as described for SEM, TEM sample preparation was conducted [34
]. E. coli
ATCC 25922 cells were treated with 2.5% glutaraldehyde and washed with PBS. The samples were then postfixed with 2% osmium tetroxide for 2 h, washed with PBS, and dehydrated in graded ethanol (50%, 70%, 90%, and 100%). Finally, sections were performed with an ultramicrotome and thoroughly observed with a Hitachi H-7650 TEM.
4.12. Statistical Analysis
Values for peptide analysis were expressed as means ± standard deviations. By using one-way analysis of variance (ANOVA) in SPSS 17.0, the obtained data were analyzed statistically, with a significance level of 5%.